CA2158345A1 - Cloned human alpha 1c adrenergic receptor - Google Patents

Abstract

The human adrenergic receptor of the alpha 1C subtype is cloned and used in an in vitro assay to screen for compounds that specifically bind to the human alpha 1C adrenergic receptor, including compounds effective to reduce symptoms of benign prostatic hypertrophy. The invention includes the assay, the cloned human receptor used in the assay, an isolated human alpha 1C adrenergic receptor free of other human proteins, and compounds identified through the use of this novel, cloned receptor, which selectively bind the human alpha 1C
adrenergic receptor.

Description

21~83~

TITLE OF THE INVENTION
CLONED HUMAN ALPHAlC ADREN~RGIC 2ECEPTOR
.. :
CROSS REFERENCES TO RELATED APPLICATIONS
This is a continll~tion-in-part of U.S. Application serial number 08/032,849, filed on March 15, 1993, pending.

BACKGROUND OF THE ~VENTION

i. Field of the Invention:
This invention relates to a method for defining the potency and selectivity of compounds for use as hllm~n alphalC adrenergic receptor antagonists using cloned hllm~n alpha 1 receptors. The invention also relates to the cloned receptors themselves, to compounds 15 identified according to the method of this invention, and to methods of use of such compounds, alone or in combination with other agents.
Particularly preferred are combinations of compounds identified according to this invention and testosterone 5-alpha reductase inhibitors, to alleviate pathologic conditions, particularly benign prostatic 20 hyperplasia (also known as benign prostatic hypertrophy, BPH).

ii. Background:
Human adrenergic receptors are integral membrane proteins which have been classified into two broad classes, the alpha and 25 the beta adrenergic receptors. Both types mediate the action of the peripheral sympathetic nervous system upon binding of catechol~mines, norepinephrine and epinephrine.
Norepinephrine is produced by adrenergic nerve endings, while epinephrine is produced by the adrenal medulla. The binding 30 affinity of adrenergic receptors for these compounds forms one basis of the classification: alpha receptors bind norepinephrine more strongly than epinephrine and much more strongly than the synthetic compound isoproterenol. The binding affinity of these hormones is reversed for the beta receptors. In many tissues, the functional responses, such as 2 ~ ~ ~ 3 4 5 PCTIUS94tO2609 smooth muscle contract`ion, induced by alpha receptor activation are opposed to responses induced by beta receptor binding.
Subsequently, the functional distinction between alpha and beta receptors was further highlighted and refined by the pharmacological characterization of these receptors from various ~nim~l and tissue sources. As a result, alpha and beta adrenergic receptors were further subdivided into a1, a2, ~1, and ~2 subtypes.
Functional differences between oc1 and a2 receptors have been recognized, and compounds which exhibit selective binding between these two subtypes have been developed. Thus, in WO 92/0073, the selective ability of the R(+) enantiomer of terazosin to selectively bind to adrenergic receptors of the alpha 1 subtype was reported. The al/a2 selectivity of this compound was disclosed as being significant because agonist stim~ tion of the a2 receptors was said to inhibit secretion of epinephrine and norepinephrine, while antagonism of the a2 receptor was said to increase secretion of these ho~nones. Thus, the use of non-selective alpha-adrenergic blockers, such as phenoxyben7~mine and phentol~mine, is limited by their a2 adrenergic receptor mediated induction of increased plasma catecholamine concentration and the attendant physiological sequelae (increased heart rate and smooth muscle contraction).
For a general background on the a-adrenergic receptors, the reader's attention is directed to Robert R. Ruffolo, Jr., ~c-Adrenoreceptors: Molecular Biolo~y~ Biochemistry and Pharmacolo~y.
(Progress in Basic and Clinical Ph~rm~colo~y series, Karger, 1991), wherein the basis of ocl/a2 subclassification, the molecular biology, signal transduction (G-protein interaction and location of the significant site for this and ligand binding activity away from the 3'-terminus of alpha adrenergic receptors), agonist structure-activity relationships, receptor functions, and therapeutic applications for compounds exhibiting a-adrenergic receptor affinity was explored.
The cloning, sequencing and expression of alpha receptor subtypes from ~nim~l tissues has led to the subclassification of the al receptors into a1A, (Lomasney, et al., J. Biol. Chem.. ~:6365-6369 WO 94121660 2 1 5 8 3 ~ ~ PCT/US94/02609 (1991), rat a1A; Bruno et al., BBRC. 179:1485-1490 (1991), hllm~n a1A)~ alg (Cotecchia, et al., PNAS. 85;7159-7163 (1988), hamster alg; Libert, et al., Science. (1989), dog a1g; Ramarao, et al., J. Biol.
Chem.. 267:21936-21945 (1992), hllm~n alB)~ and most recently, in a 5 study using bovine brain, a new alC subtype was proposed (Schwinn, et al.. J. Biol. Chem.. 265:8183-8189, 1990; Hirasawa et al., BBRC
195:902-909 (1993), desc~ibed the cloning, functional expression and tissue distribution of a hllm~n alC adrenergic receptor; Hoehe et al., Human Mol. Genetics 1(5):349 (8/92) noted the existence of a two-allele o Pstl restriction fragment polymorphism in the a1C adrenergic receptor gene; another study suggests that there may even be an alpha-lD
receptor subtype, see Perez et al., Mol. Pharm.., 40:876-883, 1992).
Each al receptor subtype exhibits its own pharmacologic and tissue specificities. Schwinn and coworkers noted that the cloned bovine alC
15 receptor exhibited ph~rm~cological properties proposed for the alA
subtype. Nonetheless, based on its non-expression in tissues where the alA subtype is expressed, and its sensitivity to chloroethylclonidine, the receptor was given a new designation.
The differences in the a-adrenergic receptor subtypes have 20 relevance in pathophysiologic conditions. Benign prostatic hypertrophy, BPH, is an illness typically affecting men over fifty years of age, increasing in severity with increasing age. The symptoms of the condition include, but are not limted to, increased difficulty in urination and sexual dysfunction. These sylllpLollls are induced by enlargement, 25 or hypertrophy, of the prostate gland. As the prostate increases in size, it impinges on free-flow of fluids through the male urethra.
Concommitantly, the increased noradrenergic innervation of the enlarged prostate leads to an increased adrenergic tone of the bladder neck and urethra, further restricting the flow of urine through the 30 urethra.
The mech~ni~m of prostatic hypertrophy is well understood. The male hormone, 5a-dihydrotestosterone has been identified as the principal culprit. The contiml~l production of 5a-dihydrotestosterone by the male testes induces incremental growth of WO 94/21660 ~ 15 8 3 ~ 5 PCT/US94/02609 the prostate gland throughout the life of the male. Beyond the age of about fifty years, in many-men, this enlarged gland begins to obstruct the urethra with the pathologic s~lllptolns noted above.
The elucidation of the mech~ni~m sllmm~rized above has 5 resulted in the recent development of effective agents to control, and in many cases reverse, the pernicious advance of BPH. In the forefront of these agents is Merck & Co., Inc.s' product PROSCAR(~) (finasteride).
The effect of this compound is to inhibit the enzyme testosterone 5-alpha reductase, which converts testosterone into Sa-dihydrotesterone, resulting in a reduced rate of prostatic enlargement, and often reduction in prostatic mass.
The development of such agents as PROSCAR(~ bodes well for the long-teFm control of BPH. However, as may be appreciated from the lengthy development of the syndrome, its reversal also is not 15 immediate. In the interim, those males ~urrt;lillg with BPH continue to suffer, and may in fact lose hope that the agents are working sufficiently rapidly.
In response to this problem, one solution is to identify ph~rm~reutically active compounds which complement slower-acting 20 therapeutics by providing acute relief. Agents which induce relaxation of the urethral smooth muscle, by binding to alpha-1 adrenergic receptors, thus reducing ~e increased adrenergic tone due to the disease, would be good c~n~l1d~tes for this activity. Thus, one such agent is alfuzosin, which is reported in EP 0 204597 to induce urination 25 in cases of prostatic hypertrophy. Likewise, in WO 92/0073, the selective ability of the R(+) enantiomer of terazosin to bind to adrenergic receptors of the al subtype was reported. In addition, in 3, hereby incorporated by reference, combinations of 5-alpha-reductase inhibitory compounds and alphal-adrenergic receptor blockers (terazosin, doxazosin, prazosin, bunazosin, indoramin, 3 alfuzosin) were disclosed. However, no information as to the alA, a1B, or a1C subtype specificity of these compounds was provided as these refinements were not yet available. The instant invention changes this situation by providing a cloned hllm~n a1C adrenergic receptor and 2i583~5 a method for idellliryillg compounds which bind the hllm~n oc1c receptor. ~ `
Typically, identification of active compounds is through use of ~nim~l tissues known to be enriched in adrenergic receptors. Thus, rat tissues have been used to screen for potential adrenergic receptor antagonists. However, bec~llce of species variability, compounds which appear active in ~nim~l tissue may not be active or sufficiently selective in hllm~n~. This results in substantial wastage of time and effort, particularly where high volume compound screening programs are employed. There is also the danger that compounds, which might be highly effective in hllm~nc, would be missed because of their absence of appreciable affinity for the heterologous ~nim~l receptors. In this regard, it has been noted that even single amino acid changes between the sequence of biologically active proteins in one species may give rise to substantial ph~rm~cological differences. Thus, Fong et al., (J. Biol.
Chem.~ 267:25668-25671, 1992) showed that there are 22 divergent amino acid residues between the sequence of the hllm~n neurokinin-l receptor and the homologous rat receptor. They further showed, in studies with mllt~nt receptors, that substitution of only two amino acid residues was both necessary and sufficient to reproduce the rat receptor's antagonist binding ar~ iLy in the hllm~n receptor. Oksenberg et al., (Nature. 360:161-163, 1992) showed that a single amino-acid difference confers major pharmacological variation between the hllm~n and the rodent 5-hydroxytrypt~mine receptors. Likewise, Kuhse et al., (Neuron. 5:867-~73, 1990) showed that a single amino-acid exçh~n~e alters the ph~rm~cology of the neonatal rat glycine receptor subunit.
This difficulty and unpredictability has resulted in a need for a compound screen which will identify compounds that will be active in hllm~n.c.
The in~t~nt inventors have solved these problems by cloning a novel hllm~n adrenergic receptor of the a1C subtype. Their efforts have led to the development of a novel screening assay which enables them to identify compounds which specifically interact with the hllm~n o~lC adrenergic receptor. Marshall et al (Br. J. Pharm., 21583~ ~

107:327 (1992)) speculated that compounds which specifically interact with the alC adrenergic receptor may be responsible for contraction of the hllm~n prostate. The instant invention provides a method for identifying compounds which bind ~e hllm~n alC receptor. In addition, if the compounds are further tested for binding to other 1.
hllm~n alpha 1 receptor subtypes, as well as counterscreened against other types of receptors, the specificity of the compounds for the hllm~n alC adrenergic receptor may be defined.
Compounds identified according to this invention may be used to reduce the acute symptoms of BPH. New agents identified in this m~nnt~.r, or already known agents showing activity in this assay, may now be employed in a novel way to help BPH ~ufrerers contend with the acute symptoms of the syndrome. Thus, this invention is useful to identify compounds which may be used alone or in conjunction with a lS more long-term anti-BPH therapeutics, such as PROSCAR(~. Other uses for ~e invention include identi~lcation of compounds which induce highly tissue-specific, localized alC adrenergic receptor blockade.
Effects of ~is blockade include reduction of intra-ocular pressure, control of cardiac arrhythmias, and possibly a host of alpha-lC receptor mediated central nervous system events. In addition, the cloned alC
receptor can be used for screening of tissue specific expression of ~lC
adrenergic receptors. Effects such as these, induced by or available to analysis with the alC adrenergic receptor also form part of this invention.
2s SUMMARY OF THE INVENTION
The hllm~n adrenergic receptor of the alphalC subtype is cloned and used in an in vitro assay to screen for compounds that bind to the receptor, including compounds which specifically inhibit the activity of the receptor. The invention includes the assay, the cloned receptor used in the assay (cDNA), an isolated hllm~n alphalC
adrenergic receptor, cells expressing the cloned receptor, and compounds identified through the use of this novel, cloned receptor, which selectively bind to the hllm~n alphalC adrenergic receptor, WO 94/21660 2 ~ ~ 8 3 ~ 5 PCT/US94/02609 including specific antagonists of the receptor. One embodiment of this invention is a method of treating benign prostatic hypeplasia (BPH) employing compounds having an afrll~ily for the hllm~n alpha lC
receptor that is at least 12 fold greater than for either the hllm~n alpha s lA or the hllm~n alpha lB receptors.

BRIEF DESCRIPTION OF THE FIGURES

Fig. 1: Sequence of cDNA obtained by PCR of hllm~n heart mRNA, SEQ. ID:4:.

Fig. 2: Comparison of the open reading frame obtained from hllm~n heart, SEQ ID:5:, and the bovine alpha-lC adrenergic receptor sequence, SEQ. ID:6:.

Fig. 3: Sequence of cDNA obtained by screenin~ a hllm~n hippocampus cDNA library using the heart mRNA derived sequence from figure 1, SEQ. ID:7:

20 Fig. 4: Sequence of 3' coding region of hllm~n alpha-lC gene, obtained by PCR amplification of a h~m~n genomic DNA library with oligonucleotides, SEQ. ID:10:.

Fig. 5: Sequence of the ligated portions of hllm~n alpha-lC DNA
25 shown in figures 3 and 4, SEQ. ID:ll:.

Fig. 6: The amino acid sequence of the hllm~n alpha-lC adrenergic receptor, SEQ ID:12:.

30 Fig. 7: The ~li nment of the nucleotide and amino acid sequence of the hllm~n alpha-lC adrenergic receptor, showing the 5'-untranslated region, SEQ. ID:ll: and SEQ. ID:12:.

WO 94121660 21~ 8 3 ~ ~; PCT/US94/02609 Fig. 8: Expression of the hllm~n alpha-lC adrenergic receptor in COS
cells: Binding data using membranes from cells transfected with the expression vector alone and ~e expression vector Cont~inin.~; the hl!m~n alpha-lC adrenergic receptor coding sequences.

Fig. 9: Binding curves of compounds using membranes from COS cells transfected with the hllm~n alpha-lC adrenergic receptor Cont~inin~
expression vector.

o Fig. 10: Nucleotide sequence of the hllm~n alphalA receptor, SEQ.
ID:13:

Fig. 11: Amino acid sequence of the hllm~n alphalA adrenergic receptor, SEQ. ID:14:

Fig. 12: Partial sequence of ~e hllm~n alphalB adrenergic receptor, SEQ. ID:17:

Fig. 13: Partial sequence of the hllm~n alphalB adrenergic receptor, SEQ. ID-20--Pig. 14: Partial sequence of the hllm~n alphalB adrenergic receptor, SEQ. ID:23:

Fig. 15: Composite hllm~n/rat alphalB adrenoreceptror, SEQ. ID:24:

Fig. 16: Amino acid sequence of ~e composite hl-m~n/rat alphalB
adrenergic receptor, SEQ. ID:25:

Fig. 17: Binding curves of compounds using membranes from COS
cells transfected wi~ the hllm~n alphalA, lB, and lC adrenergic receptor expression vectors.

~1~834~

Pig. 18: Sequence of truncated hllm~n alphalC adrenergic receptor, SEQ. ID:26:.

Fig. 19: Nucleotide sequence of the hllm~n a1C adrenergic receptor 5 having a Pstl site, SEQ.ID:27:.

Fig. 20: Amino acid sequence of the hllm~n a1C adrenergic receptor encoded by the Pstl site encoding allele, SEQ.ID:28:.

Fig. 21: Alignment of the nucleotide and amino acid sequences of figures 19 and 20, SEQ.ID:27: and SEQ.ID:28:.

FIG. 22: Nucleotide sequence of the hllm~n a1A adrenergic receptor, Seq.ID:29:.

Fig. 23: Amino acid sequence of the hllm~n a1A adrenergic receptor, SEQ.ID:30:.

Fig. 24: Alignment of the nucleotide and amino acid sequences of 20 figures 22 and 23, SEQ.ID:29: and SEQ.ID:30:.

DETAILED DESCRIPTION OF THE INVENTION
The hllm~n alpha adrenergic receptor of the 1-C subtype was i(lentified, cloned and expressed by the instant inventors. A partial 25 coding region for this receptor was generated by reverse transcriptase-polymerase chain reaction technology, RT-PCR. Accordingly, degenerate oligonucleotides encoding amino acids conserved in the fifth and sixth transmembrane domains of all three al receptor subtypes (A, B, C) were used to prime RT-PCR reactions using hllm~n heart mRNA
30 as template. The predicted sized products were cloned and sequenced.
Tr~n~l~tion of the amplified cDNA yielded an open reading frame encoding a protein 95~o homologous to the bovine alC receptor (Fig.2, SEQ. ID:~: and SEQ. ID:6:). This partial sequence was used to obtain a larger cDNA clone from a hllm~n hippocampus library (Fig. 3, SEQ.

~S~3~ ~

ID:6:). The rem~inin~ coding region was ~btained by PCR
amplification of hllm~n genomic DNA using primers based on the cDNA sequence and the last six amino acids of bovine alC receptor (Fig. 4, SEQ.ID:10:). The complete receptor was then assembled using 5 the partial sequences shown in Fig. 3, SEQ.ID:6: and Fig. 4, SEQ.
ID:10:, to generate the sequence shown in Fig. 5, SEQ. ID:11:. The translation of this sequence is shown in Fig. 6, SEQ. ID:12:, and the ~lignment of the nucleotide and amino acid sequences, and the 5'-untranslated sequences, is shown in Fig. 7, SEQ. ID: 11: and SEQ.

- The 3'-terminal six amino acids of the hllm~n a1C
adrenergic receptor were confirmed by screening a hllm~n genomic library with the radiolabeled 3'-terminal 512 nucleotides of the SEQ.
ID:10: clone previously obtained. A complete hllm~n exon 2 was 15 generated in this manner and sequenced. The nucleotide sequence of this gene is provided in figure 19, SEQ. ID:27: and the amino acid sequence is provided in figure 20, SEQ. ID:28:. We discovered that this clone was identical to the original 3'-terminal portion of the gene, except that:
2 0 1 ) There are five silent nucleotide changes between the new clone and the previously obtained clone (the last f;ve codons, including the stop codon, each have a silent change in the third nucleotide); and 2) At nucleotide position 1636 (amino acid 347), there is a cytosine to thymine base change resul~ng in the formation of a Pstl site at that 25 location and a concommit~n~ single amino acid change of Arg to Cys.
Thus, we have confirmed and localized the site of the two-allele Pstl restriction fragment polymoIphism (RFLP) noted by Hoehe et al., ~Human Mol. Genetics. 1 (5):349 (8/92)]. Through ph~ cological studies using clones of both alleles, we have confirmed that the Arg to 0 Cys change appear to be ph~rm~rologically indistinguishable (see Table II, Example 11, below).
The cloned hl-m~n alC receptor, when expressed in m~mm~ n cell lines (see Fig. 8), is used to discover ligands that bind to ~e receptor and alter its function. In addition, the cloned al C

~1 2158~5 receptor enables quanititation of mRNA levels in hllm~n tissues, including the aorta and prostate, by RNase protection assays. For these purposes, a complete coding sequence of the receptor is provided.
However, as long as the ligand binding and signal transduction segments 5 of the receptor (G-protein interaction) are intact, truncation at the 3' end of the sequence does not affect the functioning of the receptor.
Thus, in addition to the sequence provided in SEQ. ID: 11:, a sequence, truncated at the 3' end, SEQ. ID:26: is disclosed, which consists entirely of human alphalC sequence.
The specificty of binding of compounds showing affinity for the alC receptor is shown by comparing affinity to membranes obtained from COS cells tranfected with the cloned alC receptor and membranes from tissues known to express other types of alpha or beta adrenergic receptors. In addition, the cloned hllm~n alA and a hybrid hllm~n/rat alB (with only the cytoplasmic, carboxy termin~l region being rat sequence) could be used for this purpose, along with the hllm~n alC receptor expressed in COS cells . Expression of the cloned hllm~n alA, alB, and alC receptors and comparison of their binding properties with known selective antagonists provides a rational way for 20 selection of compounds and discovery of new compounds with predictable ph~nn~cological activities.
Once the hllm~n receptor is cloned and expressed in a cell such as COS cells or CHO cells, the receptor is free of other hllm~n proteins. The membranes from cells expressing different hllm~n alpha 25 adrenergic receptor subtypes are then isolated according to methods well known in the art for membrane associated receptor binding assays.
For example, the method of Schwinn, et al., (J. Biol. Chem.. 265:8183-8189, 1990) may be used. A compound of interest is used to compete with the binding of a known, qll~ntifi~ble alpha receptor ligand. Thus, 30 radiolabled prazosin, niguldipine, 5-methyl urapidil, terazosin, dozazosin, phenoxyben7~mine, WB4101, benox~thi~n, HEAT (2-[~-(4-hydroxy-3-iodophenyl)ethylaminomethyl]tetralone, or phentol~mine may be used for this purpose (see, for example, Robert R. Ruffolo, Jr., a-Adrenoreceptors: Molecular Biolo~v. Biochemistry and 2~83~ ~

Pharmacolo~y. (Progress in Basic and Clinical Pharmacolo~y series, Karger, 1991), page 29). Because of the ease of 125Iodine detection, 125I-HEAT may be preferred for ~is purpose. By increasing the amount of unlabeled, test compound, the labeled compound is competed 5 off the receptor. From these experiments, Ic50 values for each test compound and receptor subtype is dele,ll,illed.
Thus, according to this invention, a method is provided for identifying compounds specific for the hllm~n alphalC receptor comprising the following steps:

a. Cloning the hllm~n alphalC adrenerglc receptor;
b. Splicing the the cloned alphalC adrenergic receptor into an expression vector to produce a construct such that the alphalC receptor is operably linked to transcription and translation signals sufficient to 15 induce expression of said receptor upon introduction of said construct into a prokaryotic or eukaryotic cell;
c. Introducing said construct into a prokaryotic or eukaryotic cell which does not express a hllm~n alphalC adrenergic receptor in the absence of said introduced construct;
20 d. Incubating cells or membranes isolated from cells produced in step c.
with a qll~ntifi~ble compound known to bind to hllm~n alpha adrenergic receptors, and subsequently ~lclin~ test compounds at a range of concentrations so as to compete the qll~ntifi~ble compound from the receptor, such that an ICso for the test compound is obtained as the 25 concentration of test compound at which 50% of the qll~ntifi~ble compound becomes displaced from the receptor;
e. Incubating cells or membranes of cells which naturally express or have an introduced, cloned hllm~n alpha adrenregic receptor of a subtype other than the hllm~n alphalC receptor under identical conditions to the incubation conducted in step d, and obt~inin~ the IC50 of the test compound for the non-alphalC receptor; and f. Comparing ~e IC50 for ~e test compound for the alphalC receptor and for the alpha adrenergic receptor of a subtype other than the alphalC to identify compounds having a lower IC50 for the alphalC
receptor.
In addition to providing a sequence for the hllm~n al C
adrenergic receptor, the instant inventors have also discovered a different sequence than that reported by Bruno et al., rBBRC 179:1485-1490 (1991)] for the human alA adrenergic receptor. The new sequence is more homologous to the rat alA adrenergic receptor sequence. Disclosed and claimed herein is the sequence for this new hllm~n alA adrenergic receptor (see Fx~mple 12 and figures 22, 23, and 24, SEQ. ID:29: and SEQ. ID:30:). While no difference in ligand binding has thus far been observed based on the dirre-~l-t amino terminal amino acid sequences between these two receptors, such differences cannot be ruled out except by screening compounds against both clones. Since a new hllm~n sequence is provided herein, compounds illenti~ed according to the method of this invention using the earlier reported hllm~n alA adrenergic receptor sequence can now be collfi-,ned against this clone.
As a result of the cloning, sequencing, expression, and screening efforts described above and further exemplified below, numerous compounds have been tested for their ability to specifically bind to the cloned hllm~n alC receptor with high affinity. Compounds specific for the hllm~n alpha lC adrenergic receptor, that is compounds having an ~îril.ily for the hllm~n alpha lC receptor that is at least 12 fold greater than for either the hllm~n alpha lA or the hllm~n alpha lB
receptors, are identified by this method. Thus, the compounds S(+) niguldipine, (S(+)-1,4-Dihydro-2,6-dimethyl-4-(3-nitrophenyl)-3,5-pyridinedicarboxylic acid 3-(4,4-diphenyl-1-piperidinyl)-propyl methyl ester hydrochloride), and 5-methyl urapidil (5-methyl-6[[3-[4-(2-methoxyphenyl)-l-piperazinyl]propyl]amino]-1,3-dimethyluracil) have been discovered to selectively bind to the hllm~n alC adrenergic receptor. These compounds may be ~lminictered in dosages effective to antagonize the alphalC receptor where such treatment is needed, as in BPH.

~5~3~

Compounds identified according to the method of this invention as being selective h~-rn~n al C adrenergic receptor antagonists may fur~er be defined by counterscreening. This is accomplished according to methods known in the art.using other receptors responsible 5 for mediating diverse biological functions Compounds which are both selective amongst the various h~ n alphal adrenergic receptor subtypes and which have low affinity for other receptors, such as the alpha2 adrenergic receptors, the ~-adrenergic receptors, the muscarinic receptors, the serotonin receptors, and others are particularly preferred. The absence of these non-specific activities may be confirmed by using cloned and expressed receptors in an analogous fashion to the method disclosed herein for identifying compounds which have high affinity for the various hll~m~n alphal adrenergic receptors.
Furthermore, functional biological tests are used to confirm the effects 15 of identified compounds as alphalC adrenergic receptor antagonists.
Compounds identified according to this patent disclosure may be used alone at a~r~liate dosages defined by routine testing in order to obtain optimal inhibition of the human alC adrenergic receptor while minimi7.in~ any potential toxicity. In addition, co-20 ?~lmini~tration or sequential ~lmini~tration of other agents whichalleviate the effects of BPH is desirable. Thus, in one embodiment, this includes ~lmini~tration of compounds identified according to this disclosure and a hllm~n testosterone 5-a reductase inhibitor. Many such compounds are now well known in the art and include such compounds 25 as PROSCAR~, (also lmown as finasteride, a 4-Aza-steroid; see US
Patents 4,377,584 and 4,760,071, for example, hereby incorporated by reference). In addition to PROSCAR~, which is principally active in prostatic tissue due to its selectiv*y for hllm~n 5-a reductase isozyme 2, combinations of compounds which are specifically active in inhibiting 30 isozyme 1 (found particularly in skin) and compounds which act at both of these isozymes, are useful in combination with compounds identified according to this invention.
In the treatment of hyperandrogenic disease conditions, e.g.
benign prostatic hyperplasia (BPH) and/or the prevention and treatment WO 94121660 21 5 8 34 ~ PCT/US94/02609 of prostatic cancer, and the treatment of prostatitis, it would be desirable to have one drug entity which is active against both isozymes to significantly inhibit dihydrotesterone production. It would also be desirable to have one drug entity that is active as a dual inhibitor of both 5 isozymes for the treatment of conditions of the skin and scalp, e.g. acne vulgaris, seborrhea, female hirsutism, and androgenic alopecia.
Additionally, such a dual inhibitor of 5a-reductase 1 and 2 could be used in combination with a 5a-reductase 1 inhibitor or wi~ a 5a-reductase 2 inhibitor, e.g. finasteride (PROSCAR~), for combination therapy in the treatment of hyperandrogenic conditions, in combination with compounds identified according to this inventionas being selective hllm~n alphalC adrenergic receptor antagonists. The dual 5a-reductase isozyme inhibitor could also be used in combination with a potassium channel opener, e.g. minoxidil, for the treatment of male pattern 15 baldness, and such combinations in combination with selective hllm~n alphalC adrenergic receptor antagonists also form part of the in~t~nt invention . Compounds that are active as dual 5a-reductase 1 and 2 inhibitors have been described in W093/23420, EP 0572166; WO
93/23050; W093/23038,; W093/23048; WO93/23041; W093/23040;
20 W093/23039; W093/23376; WO93/23419, EP 0572165; WO93/230~1, each of which is hereby incorporated by reference.
The present invention also has the objective of providing suitable topical, oral, systemic and parenteral ph~rm~ceutical form~ tions for use in the novel methods of treatment of the present invention. The compositions cont~ining compounds identified according to this invention as the active ingredient for use in the specific antagonism of hllm~n alphalC adrenergic receptors can be ~-lmini~tered in a wide variety of therapeutic dosage forms in conventional vehicles for systemic ~lmini~tration. For example, the compounds can be ~-lmini~tered in such oral dosage forms as tablets, capsules (each including timed release and sustained release form~ tions), pills, powders, granules, elixirs, tinctures, solutions, suspensions, syrups and emulsions, or by injection. Likewise, they may also be ~lmini~tered in intravenous (both bolus and infusion), 2~83~ ~

. i .
intraperitoneal, subcutariéous, topical with or without occlusion, or intramuscular form, all using forms well known to those of ordinary skill in the pharmaceutical arts. An effective but non-toxic amount of the compound desired can be employed as an alphalC antagonistic agent.
The daily dosage of the products may be varied over a wide range from 0.01 to 1,000 mg per adult hllm~n/per day. For oral ~lminictration~ the compositions are preferably provided in the form of scored or unscored tablets cont~inin~ 0.01, 0.05, 0.1, 0.5, 1.0, 2.5, 5.0, 10.0, 15.0, 25.0, and 50.0 milligrams of the active ingredient for the sy~ tolllatic adjustment of the dosage to the patient to be treated. An effective amount of ~e drug is ordinarily supplied at a dosage level of from about 0.0002 mg./l~g to about 50 mg.~g. of body weight per day. The range is more par~icularly 15 from about 0.001 mg./kg to 7 mg.~g. of body weight per day. The dosages of the alphalC adrenergic receptor and testosterone 5-alpha reductase inhibitors are adjusted when combined to achieve desired effects. As those skilled in the art will appreciate, less 5-alpha reductase inhibitor may be required when the acute symptoms of 20 BPH are alleviated by treatment with alphalC adrenergic receptor inhibitors has been initi~ted. On the other hand, dosages of these various agents may be independently optimized and combined to achieve a synergistic result wherein the pathology is reduced more than it would be if either agent were used alone.
Advantageously, compounds of the present invention may be ~timini~tered in a single daily dose, or the total daily dosage may be ~lmini~tered in divided doses of two, three or four times daily. Furthermore, compounds for the present invention can be 2~1mini~tered in intranasal form via topical use of suitable intranasal 30 vehicles, or via transdermal routes, using those forms of transdermal skin patches well known to those of ordinary skill in that art. To be ~(lmini~tered in the form of a transdermal delivery system, the dosage ~tlmini~tration will, of course, be continuous rather than intermittent throughout the dosage regimen.

-WO 94/21660 21~ 8 34 5 PCT/US94/02609 For the treatment of acne vulgaris, androgenic alopecia including male pattern baldness, seborrhea, female hirsutism, benign prostatic hyperplasia, prostatitis and the prevention and/or treatment of prostatic cancer, compounds exhibiting at least 12 fold selectivity 5 for inhibition of the alphalC adrenergic receptor can be combined with a therapeutically effective amount of a Sa-reductase 2 inhibitor, such as finasteride, in addition to a Sa-reductase 1 inhibitor, such as 4,7~-dimethyl-4-aza-Sa-cholestan-3-one, in a single oral, systemic, or parenteral pharmaceutical dosage form~ tion. Alternatively, a o combined therapy can be employed wherein the alphalC adrenergic receptor antagonist and the 5a-reductase 1 or 2 inhibitor are ~lmini~tered in separate oral, systemic, or parenteral dosage form~ tions. Also, for the skin and scalp related disorders of acne vulgaris, androgenic alopecia including male pattern baldness, seborrhea, and female hirsutism, the compounds of the instant invention and dual inhibitors of Sa-reductase 1 and 2 could be formnl~ted for topical ~(lmini~tration. For example, niguldipine or S-methyl urapidil and finasteride can be ~lmini~tered in a single oral or topical dosage formlll~tion, or each active agent can be 20 ~lmini~tered in a separate dosage form~ tion, e.g., in separate oral dosage form~ ions, or an oral dosage formlll~tion of finasteride in combination with a topical dosage formlll~tion of a compound exhibiting dual inhibiton of both isozymes of 5a-reductase. See, e.g., U.S. Patent No.'s 4,377,584 and 4,760,û71 which describe 2 dosages and form~ ions for 5a-reductase inhibitors.
Furthermore, since ~lmini~tration of 5a-reductase inhibitors have been found to be useful in combination with a therapeutically effective amount of a potassium ch~nnel opener, such as minoxidil, crom~k~lin, pinacidil, a compound selected from the 0 classes of S-triazine, thiane-1-oxide, benzopyran, and pyridinopyran derivatives or a ph~rm~ceutically acceptable salt thereof, compounds of this invention may also be used in combination therapy for the treatment of androgenic alopecia including male pattern baldness.
The active agents can be ~lmini~tered in a single topical dosage 21583~5~i ~

form~ tion, or each active agent can be ~lmini~tered in a separate dosage formulation, e.g., in separate topical dosage formulations, or an oral dosage formulation of a compound of formula I in combination with a topical dosage fonn~ tion of, e.g., minoxidil.
See, e.g., U.S. Patent No.'s 4,596,812, 4,139,619 and WO 92/02225, published 20 February 1992, for dosages and forrmll~tions of calcium channel openers. ~
For combination tre~trnent with more than one active agent, where the active agents are in separate dosage forml-l~tions, ~e active agents can be ~lmini~tered concurrently, or they each can be ~lmini~tered at separately staggered times.
The dosage regimen lltili7in~ the compounds of the present invention is selected in accordance with a variety of factors including type, species, age, weight, sex and medical condition of the 15 patient; the severity of the condition to be treated; the route of ~lmini~tration; the renal and hepatic function of the patient; and the particular compound thereof employed. A physician or veterinarian of ordinary skill can readily determine and prescribe the effective amount of the drug required to prevent, counter or arrest the 20 progress of the condition. Optimal precision in achieving concentration of drug within the range that yields efficacy without toxicity requires a regimen based on the kinetics of ~e drug's availability to target sites. This involves a consideration of the distribution, equilibrium, and elimin~tion of a drug.
In the methods of the present invention, the compounds herein described in detail can form the active ingredient, and are typically ~c~mini~tered in ~imixlllre with suitable ph~ eutical diluents, excipients or carriers (collectively referred to herein as "carrier" materials) suitably selected with respect to the intended 30 form of ~lrnini~ctration~ that is, oral tablets, capsules, elixirs, syrups and the like, and consistent with conventional pharmaceutical practices.
For instance, for oral ~(lminictration in the form of a tablet or capsule, the active drug component can be combined with WO 94/21660 2 15 ~ 3 ~ 5 PCT/US94/02609 an oral, non-toxic pharmaceutically acceptable inert carrier such as ethanol, glycerol, water and the like. Moreover, when desired or necessary, suitable binders, lubricants, disintegrating agents and coloring agents can also be incorporated into the mixture. Suitable 5 binders include, without limit~tion, starch, gelatin, natural sugars such as glucose or beta-lactose, corn sweeteners, natural and synthetic gums such as acacia, tr~g~c~nth or sodium ~l~in~te, carboxymethylcellulose, polyethylene glycol, waxes and the like.
Lubricants used in these dosage forms include, without limit~tion, sodium oleate, sodium stearate, m~gnesium stearate, sodium benzoate, sodium acetate, sodium chloride and the like.
Disintegrators include, without limit~tion, starch, methyl cellulose, agar, bentonite, x~nth~n gum and ~e like.
The liquid forms in suitably flavored suspending or dispersing agents such as the synthetic and natural gums, for example, tragacanth, acacia, methyl-cellulose and the like. Other dispersing agents which may be employed include glycerin and the like. For parenteral ~lmini~tration~ sterile suspensions and solutions are desired.
Isotonic preparations which generally contain suitable preservatives are employed when intravenous ~lmini~tration is desired.
Topical preparations cont~ining the active drug component can be admixed with a variety of carrier materials well known in the art, such as, e.g., alcohols, aloe vera gel, ~ ntoin, glycerine, vitamin A
and E oils, mineral oil, PPG2 myristyl propionate, and the like, to 2 form, e.g., alcoholic solutions, topical cleansers, cleansing creams, skin gels, skin lotions, and shampoos in cream or gel formnl~tions. See, e.g., EP 0 285 382.
The compounds of the present invention can also be ~clmini~tered in the form of liposome delivery systems, such as small nnil~mellar vesicles, large llnil~mellar vesicles and multilamellar vesicles. Liposomes can be formed from a variety of phospholipids, such as cholesterol, stearylamine or phosphatidylcholines.
Compounds of the present invention may also be delivered by the use of monoclonal antibodies as individual carriers 2~583~5 ~

to which ~e compound molecules are coupled. The compounds of the present invention may alsa b~ coupled wi~ soluble polymers as targetable drug carriers, Such polymers can include polyvinyl-pyrrolidone, pyran copolymer, polyhydroxypropylmethacryl-5 amidephenol, polyhydroxy-ethylaspartamidephenol, or polyethyl-eneoxidepolylysine substituted with palmitoyl residues.
Furthermore, the compounds of the present invention may be coupled to a class of biodegradable polymers useful in achieving controlled release of a drug, for example, polylactic acid, polyepsilon caprolactone, polyhydroxy butyric acid, polyorthoesters, polyacetals, polydihydro-pyrans, polycyanoacrylates and cross-linked or amphipathic block copolymers of hydrogels.

The following examples are provided to further define the 15 invention without, however, limitin~ ~e invention to the particulars of these examples.

~,XAMP~ F 1 20 PCR amplification. cloning and sequencing of phalX:

Based on the amino acid homologies of human alA, rat alB and bovine alC receptors, degenerate oligonucleotides were designed to amplify cDNAs encoding all ~ree receptor subtypes. These oligonucleotides are-WL' (SEQ. ID:1) l l l lCTAGAT TRTTNARRTA NCCNAGCC 28 30 MYC (SEQ. ID:2) TlTACTAGTA TCSTNGTNAT GTAYTG 16WC' (SEQ. ID:3) lTl lCTAGAG AARAANGGNA RCCARC 26 WO 94t21660 2 1 5 ~ 3 ~ 5 PCT/US94/02609 .

Oligonucleotides MYC and WL' were used as primers in a reverse transcription PCR amplification of hllm~n heart mRNA (Clontech) using the RNA PCR kit from Perkin Elmer Cetus. Briefly, 0.5 ug of mRNA was reverse transcribed in a volume of 20 ul using either s random oligonucleotide primers (reaction 1) or oligo dT primer (reaction 2). Reactions 1 and 2 were pooled and served as template for PCR amplification as follows:

PCR Reactions:

Primary reaction (50 ul) 5 ul lOX buffer from Perkin Elmer Cetus GeneAmp Kit 8 ul 1.25 mM each stock of dATP,dCTP,dGTP, and dTTP
3ul first strand cDNA
1 ul 25 pMoles oligo MYC
1 ul 25 pMoles oligo WL' 0.25 ul 1.25 units Amplitaq DNA polymersase 31.75 ul water Reaction conditions; 40 cycles of 94C 1'; 45C 2'; 72C 2' Secondary reaction (100 ul) 9.5 ul lOX buffer from Perkin Elmer Cetus GeneAmp Kit 16ul 1.25 mM each stock of dATP,dCTP,dGTP, and dTTP
5ul first strand cDNA
2 ul 50 pMoles oligo MYC
2 ul 50 pMoles oligo WC' 0.5 ul 2.5 units Amplitaq DNA polymersase 65 ul water Reaction conditions; 40 cycles of 94C 1'; 45C 2; 72C 2' Prep scale tertiary reaction 3 X 200 ul:

3~ ~

19.5 ul lOX buffer 32 ul l.25 mM each stock of dATP, dCTP, dGTP, a~d dl~P
5 ul secondary PCR reaction 4 ul 100 pMoles oligo MYC
4 ul 100 pMoles oligo WC' 1 ul 5 units Amplitaq DNA polymerase 134.5 ul water -Reaction conditions; 30 cycles of 94C 1'; 50C 2'; 72C 2' The PCR product was purified by Qiagen spin columns and digested with restriction endonucleases SpeI and XbaI. The fragment was then ligated into SpeVXbaI cut pGEM9Zf(-). The ligation mix was used to transform E. coli XL-1 blue. Plasmid DNA was isolated from white transform~nts and sequenced by the dideoxy chain termination method. The base sequence obtained is shown in Fig. 1, SEQ. ID:4:.

l~,XAMP~

Isolation of partial alphalC cDNA Clone:

A cDNA library prepared from rnRNA isolated from hllm~n hippocampus (Stratagene) was screened by plaque hybridization using phalX as a probe. Hybridization conditions were as follows:

SXSSC ( lXSSC is O.l5M sodiurn chloride, 0.015M sodium citrate, 50% Form~mide 5X Denhardt's Solution ( 1% Ficoll, 1%
polyvinylpyrrolidone, 1% bovine serum albumin) 0.15 mg/ml salmon sperm DNA
hybridize overnight at 42 C.

* 2158345 Filters were washed 3 times in 2XSSC, 0.1% SDS at room temperature for 5', then 1 time in lXSSC, 0.1% SDS at 50C for 30'.
Positive clones were identified by autoradiography. Phagemid DNA
was rescued from ~e positive plaques and sequenced by the dideoxy chain termin~tion method. The base sequence obtained is shown in Fig.
3, SEQ. ID:7:.

FXAMP~,F 3 PCR amplification~ clonin~ and sequencing of 3'CG of alphalC:

The 3' end of the coding region of hllm~n alphalC adrenergic receptor was amplified from hllm~n genomic DNA using two oligonucleotides:

S3C (SEQ ID:8:) S' 'l~l'l'GAATTCT GAl~CAAGC CCTCTG 3' and 3'C (SEQ ID:9:) 5' TTTGAATTCT TANACYTCYT CNCCRTTYTC 3' as follows:

10 ul lOX buffer from Perkin Elmer Cetus GeneAmp Kit 16 ul 1.25 mM each stock of dATP,dCTP,dGTP, and dTTP

6 ul 1 ug hl-m~n genomic DNA (Promega) 2 ul 50 pMoles oligo S3C
2 ul 50 pMoles oligo 3'C
0.5 ul 2.5 units Amplitaq DNA polymersase 63.5 ul water 2 ~

. . ,;,:
Reaction conditions; 40 cycles of 94C 1'; 50C 2';
72C 2' The PCR product was purified by Qiagen spin columns and digested with restriction endonuclease EcoRI. The fragment was then ligated into EcoRI cut pGEM3Zf(-). The ligation mix was used to transform E. coli XL-l blue. Plasmid DNA was isolated from white transform~nt~ and sequenced by the dideoxy chain termin~tion method.
The base sequence is shown in Fig. 4, SEQ. ID:10:.

F,X AMP~ ,F, 4 Assembly of complete coding region of hllm~n alphalc adrenergic 1 5 receptor:
The complete coding region of hllm~n alphalc adrenergic receptor was assembled by ligating the cDNA clone (see Fx~mple 2, figure 3, SEQ ID:7:) and 3'CG (see Fx~mple 3, figure 4, SEQ ID:10: ) at their 20 common PvuII site ( 1552-1557 of figure 3, SEQ ID:7: and 59-64 of figure 4, SEQ ID:10:). The complete nucleotide sequence is shown in figure 5, SEQ ID: l l :. The amino acid sequence is shown in figure 6, SEQ. ID:12:. Figure 7 shows the structure of the cDNA, including the 5'-untranslated sequences. The very 3' twenty seven nucleotides (6 25 amino acids) shown is the sequence of the PCR primer used to generate the sequence. However, the function of the receptor, both for ligand binding and signal transduction depends on sequences far removed from the carboxy terminll~ of the receptor. A completely hl-m~n sequence is shown in figure 18, SEQ. ID:26: which is truncated at the 3' terimuns.

F,XAMP~,F, ~

Expression of the cloned alphalC adrenergic receptor:

~ 2158345 The complete sequence (SEQ ID:11:) of the human alphalC
adrenergic receptor was subcloned into the eukaryotic expression vector pcDNAI-neo (Invitrogen). The resulting plasmid was transfected into COS-7 cells by electroporation. Cells were harvested after 72 hours 5 and the membranes cont~ining the expressed receptor protein were prepared as described in Schwinn, et al.. J. Biol. Chem.. 265:8183-8189, 1990. Membranes (5-25 ug, see figure 8) prepared from the COS-7 cells transfected with the vector cont~ining the alphalC receptor gene specifically bound the alpha 1 antagonist [125 Il-HEAT;
membranes prepared from the COS-7 cells transfected with the vector alone did not bind the alpha 1 antagonist [125 I]-HEAT (figure 8), proving the expression of the alphalC adrenergic receptor. Binding reactions (total volume = 200 ul) cont~inP~l 50 mM Tris-HCl pH. 7.4, 5 mM EDTA, 150 mM NaCl, 100 pM [125 I]-HEAT, and membranes 15 prepared from COS-7 cells transfected with expression plasmids.
Reactions were incubated at room temperature for one hour with ~h~king. Reactions were filtered onto Wh~tm~n GF/C glass fiber filters with a Brandel cell harvester. Filters were washed three times with ice cold buffer and bound radioactivity was detelmi"ed. Non specific 20 binding was determinP~l in the presence of 10 uM prazosin.

W O 94/21660 2 ~ 5 8 ~ 4 ~ PCTnUS94/02609 ~XAMP~ ~ 6 Screening assay: Alpha 1 C Adrenergic Receptor Binding Membranes prepared from the transfected COS-7 cells may also be used to identify compounds that bind to the hllm~n alphalC adrenergic receptor. These competition binding reactions (total volume = 200 ul) contain 50 mM Tris-HCl pH. 7.4, 5 mM EDTA, 150 mM NaCl, 100 pM
[125 Il-HEAT, membranes prepared from COS-7 cells transfected with the alphalC expression plasmid and increasing amounts of unlabeled ligand.
Reactions are incubated at room temperature for one hour with sh~kin~.
Reactions were filtered onto Wll~tm~n GF/C glass fiber filters with a Brandel cell harvester. Filters were washed three times with ice cold buffer and bound radioactivity was determined. Binding data were analyzed and ICSOs determined by an iterative curve fitting program. Results are shown in Figure 9.

hX ~MP~ F 7 Expression of hllm~n alphalA adrenergic receptor:

The complete coding region for the hllm~n alphalA adrenergic receptor (Bruno, et al., BBRC., 179:1485-1490, (1991); see figure 10, 25 SEQ. ID:13: and figure 11, SEQ. ID:14: herein) was subcloned into the eukaryotic expression vector pcDNAI-neo (Invitrogen). The resulting pl~cmi(l was transfected into COS-7 cells by electroporation. Cells were harvested after 72 hours and the membranes cont~ining the expressed receptor protein were prepared as described in Schwinn, et al.~ J. Biol.
30 Chem.~ 265:8183-8189, 1990. Membranes prepared from the COS-7 cel!s transfected with the vector cont~ining the alphalA receptor gene specifically bound the alpha 1 antagonist [125 Il-HEAT; membranes prepared from the COS-7 cells transfected with the vector alone did not bind the alpha 1 antagonist [125 I]-HEAT.

21~i83~

Bindir.g reactions (total volume = 200 ul) contained 50 mM Tris-HCl pH. 7.4, 5 mM EDTA, 150 mM NaCl, 100 pM [125 I]-HEAT, and membranes prepared from COS-7 cells transfected wi~ expression plasmids. Reactions are incubated at room temperature for one hour 5 with ~h~kin~. Reactions were filtered onto Wh~tm~n GF/C glass fiber filters with a Brandel cell harvester. Filters were washed three times with ice cold buffer and bound radioactivity was determined. Non-specific binding was determined in the presence of 10 uM prazosin.

~,XAMPT,F, 8 Expression of hllm~n alphalB adrenergic receptor:

1. PCR amplification of partial cDNA for human alphalB adrenergic receptor:

Amplification of SXB clones 5XB, SEQ. ID:15: 5' TCT AGA CCA TGA AYC CNG AYC TGG 3' AlB, SEQ. ID:16: 5' 'l l-l GAA TTC ACA TWC CGA CYA CAA
TGC CC 3' Oligonucleotides SXB and AlB were used as primers in a reverse transcription PCR amplification of hl-m~n heart mRNA (Clontech) using the Invitrogen Copy Kit. Briefly, 1.0 ug of mRNA was reverse transcribed in a volume of 20 ul using oligonuleotide WC' as primer.

Primary reaction (50 ul) 5 ul lOX buffer from Perkin Elmer Cetus GeneAmp Kit WO 94/21660 PCT/US94tO2609 ~ ~8~45 8 ul 1.25 mM each stock of dATP,dCTP,dGTP, and dTTP
2.5 ul first strand cDNA
1 ul 25 pMoles oligo SXB
1 ul 25 pMoles oligo AlB
0.25 ul 1.25 units Amplitaq DNA polymersase 32.75 ul water Reaction conditions; 40 cycles of 94C 1'; 58C 2'; 72C 2' The PCR product was directly ligated into pCR vector (Invitrogen) and used to transform E. coli INVaF' (Invitrogen). Plasmid DNA was isolated from white transform~nt~ and sequenced by the dideoxy chain termin~tion method. The base sequence is shown in Fig. 12, SEQ.
ID:17:
2. Amplification of EFK clones EFK, SEQ. ID:18: 5' GAAGGCGCGCTTGAACTC 3' SB1, SEQ. ID:19: 5' AGAGAACCACCAAGAACC 3' Oligonucleotides EFK and SB 1 were used as primers in a reverse transcription PCR amplification of hllm~n aorta mRNA (Clontech) using the Invitrogen Copy Kit. Bnefly, 1.0 ug of mRNA was reverse transcribed in a volume of 20 ul using oligo dT as primer.

Primary reaction (50 ul) 5 ul 10X buffer from Perkin Elmer Cetus GeneAmp Kit 8 ul 1.25 mM each stock of dATP,dCTP,dGTP, and dTTP
2.0 ul first strand cDNA

21S834~i ~; :

1 ul 25 pMoles oligo EF~
1 ul 25 pMoles oligo 5Bl 0.25 ul 1.25 units Amplitaq DNA polymersase 33.25 ul water Reaction conditions; 40 cycles of 94C 1'; 58C 2'; 72C 2' The PCR product was directly ligated into pCR vector (Invitrogen) and used to transform E. coli INVaF' (Invitrogen). Plasmid DNA was O isolated from white transforrn~nt,~ and sequenced by the dideoxy chain termination method. The base sequence is shown in Fig. 13, SEQ.
ID:20:.

3. Assembly of partial cDNA for hllm~n alphalB adrenergic receptor A partial cDNA clone encoding the hllm~n alphalB adrenergic receptor was assembled by joining the SXB sequence (SEQ. ID: 17:) and 20 the EFK sequence (SEQ. ID:20:) at their common BamHI site.

4. Amplification of the 3' end of rat alphalB adrener~ic receptor S4B, SEQ. ID:21: 5' TTT GAA l~C ATG TTC AAG GTG GTG TTC
3' 3'B2, SEQ. ID:22: 5' TTT GAA TTC TAA AASTGN CCN GGN SCC
AGN GGC AT 3' 0 Oligonucleotides S4B and 3'B2 were used as primers in a reverse transcription PCR amplification of rat heart mRNA using the Invitrogen Copy Kit. Briefly, 0.6 ug of mRNA was reverse transcribed in a volume of 20 ul using oligo dT as primer.

2~S~3~5 Primary reaction (50 ul) 5 ul lOX buffer from Perkin Elmer Cetus GeneAmp Kit 8 ul 1.25 mM each stock of dATP,dCTP,dGTP, and dTTP
2.0 ul first strand cDNA
1 ul 25 pMoles oligo EFE~
1 ul 25 pMoles oligo SBl 0.25 ul 1.25 units Amplitaq DNA polymersase 33.25 ul water Reaction conditions; 40 cycles of 94C 1'; 58C 2'; 72C 2' The PCR product was directly ligated into pCR vector (Invitrogen) and used to transform E. coli INVaF' (Invitrogen). Plasmid DNA was isolated from white transform~nt~ and sequenced by the dideoxy chain termin~tion method. The base sequence is shown in Fig. 14, SEQ.
ID:23:.

5. Assembly and expression of a functional h~lm~n/rat hybrid alphalB
adrenergic receptor The partial hllm~n alphalB adrenergic receptor cDNA was joined 25 to the 3' end of the rat alphalB adrenergic receptor cDNA at their common BssHII restriction endonuclease site. This composite sequence is shown in figure 15, SEQ. ID:24:, and the amino acid sequence is shown in Fig. 16, SEQ. ID:25:
The complete coding region for the hllm~n/rat alphalB
30 adrenergic receptor was subcloned into the eukaryotic expression vector pcDNAI-neo (Invitrogen). The resulting plasmid was transfected into COS-7 cells by electroporation. Cells were harvested after 72 hours and the membranes cont~ining the expressed receptor protein were prepared as described in Schwinn, et al.~ J. Biol. Chem., 265:8183-21583~5 8189, 1990. Membranes prepared from the COS-7 cells transfected with the vector collt~ the alphalB receptor gene specifically bour.d the alpha 1 antagonist [125 I]-HEAT; membranes prepared from the COS-7 cells transfected with the vector alone did not bind the alpha 1 antagonist [125 I]-HEAT. Binding reactions (total volume = 200 ul) cont~ine-l 50 mM Tris-HCl pH. 7.4, 5 mM EDTA, 150 mM NaCl, 100 pM [125 I]-HEAT, and membranes prepared from COS-7 cells transfected with expression plasmids. Reactions are incubated at room temperature for one hour with ~hakin~. Reactions were filtered onto o Whatman GF/C glass fiber filters with a Brandel cell harvester. Filters were washed three times with ice cold buffer and bound radioactivity was determined. Non specific binding was determined in the presence of 10 uM prazosin.

~,XAMP~, 9 Selective Binding assays Membranes prepared from COS-7 cells transfected with the hllman alpha 1 receptor subtype expression vectors may also be used to identify compounds that selectively bind to the hllman alphalC adrenergic receptor.
These competition binding reactions (total volume = 200 ul) contain 50 mM Tris-HCl pH. 7.4, 5 mM EDTA, 150 mM NaCl, 100 pM [125 I]-HEAT, membranes prepared from COS-7 cells transfected with the respective alpha 1 subtype expression plasmid and increasing amounts of unlabeled ligand. Reactions are incubated at room temperature for one hour with ~hakin~. Reactions were filtered onto Whatman GF/C glass fiber filters with a Brandel cell harvester. Filters were washed three times with ice cold buffer and bound radioactivity was determined. Binding data were analyzed and IC50s determined by an iterative curve fitting program.
Table I shows the results from such an analysis.

W O 94/21660 PCTnUS94102609 215g34~ ~

IC~o (nM) Compound a1A oc1B alC
prazosin 0.6 ~ 2.4 terazosin 4 5 19 doxazosin 5 2 9 phenoxyben7~minP 6.1 4.3 4.0 benox~thi~n 2.5 68 1.5 phentol~mine 36 650 14 5-methyl urapidil 42 270 3.5 S(+) niguldipine 130 670 1.4 F,XAMP-,F, 10 IDENTIFICATION AND CLONING OF A NEW ALLELE FOR THE
HUMAN ALPHAl-C ADRENERGIC RECEPTOR

Probes:
3'CG: A 525 bps fragment, specific to complete exon.2 of hllm~n alphalc AR, was PCR amplified from hllm~n genomic DNA
3 0 using a sense primer based on the isolated cDNA clone and an antisense primer based on the last six amino acids of bovine alphalc cDNA. This PCR product was subcloned and confirmed by sequencing (see Example 3, SEQ.ID:10:).

~WO 94/21660 2 ~ 5 8 ~ ~ ~ PCT/US94/02609 (~enomic ~,ihr~ry ~creenin~:
Human W138 Fibroblast genomic library synthesized in the Lambda Fix II vector (2 x 106 recombinants; Stratagene,La Jolla, CA) was screened with (3'CG). This probe was labelled with 32P-dCTP
5 (Amersham) by random-primed labelling kit (}3oehringer ~nnheim,Tndi~n~polis,~). A Total 800,000 plaques were screened, using duplicate Hybond-N nylone filters (Amersham,UK). Prior to hybridization, filters were denatured (1.5M NaCl+0.5M NaOH), neutralized (1.SM NaCl+ lM Tris.Cl,pH 8.0) and washed (0.2M Tris.Cl pH 7.5 + 2 SSC), 5' for each. DNA was cross-linked with UV cross-linker (Stratagene,La Jolla,CA). The filters were, then, hybridized in 50% formamide, 5 x SSC(lxSSC= 0.15M NaCl, 0.015M Na citrate, pH7.0), 0.02% polyvinylpyrophosphate, 0.2% Ficoll, 0.2% bovine serurn albumin, 150,ug of sheared & boiled Salmon sperm DNA, 106 cpm of 32P-labelled probe at 42C for 40hrs. Filters were washed in 0.1x SSC +1% SDS solution at 60C for 20'. Two more rounds of screening for 20 "positive" plaques/clones with 3'CG probe confirmed two clones for the alphalC adrenergic receptor, which were named 48.1C and 53.1C. Clone 53.1C was subjected to further 20 aIlalysislinvestigation.
h-clonnin~ of l~xon.~:
53.1C lambda DNA was amplified by plate lysis method and purified with Qiagen midi-lambda kit (Qiagen,Chatsworth,CA). A
25 2.6Kb band excised with EcoRI restriction enzyme was identified by Southern analysis using 3'CG probe. This fr~nent was then subcloned into pGEM3Zf(+) vector.

DNA se~llencin~:
Nucleotide sequence analysis of DNA in both direction was performed by Sanger chain termination method.

W O 94/21660 21~ 8 3 4 5 PCTrUS94/02609 Resl-lt ~nd Oisc-l~sion:
Sequencing analysis of this genomic clone confirmed that clone 53.1c contains sequences for complete exon.2 flanked by an intron at 5'-end. It also revels that there is a nucleotide change from cytosine (C) to thymine(T) at nucleotide position 1636, amino acid position 347.
This change creates a PstI site and changes the codon for arginine (Arg) to cystine (Cys). This data differs from the known/published cDNA
sequen_e of the gene. Southern analysis of hllm~n genomic DNA
confirms the PstI site in the gene/exon.2.

F,XAMPT,F 11 COMPARATIVE PHARMACOLOGY OF ALPHAl-C ~T .T FT FS
We have cloned two gerles for the hllm~n alpha-lc receptor. The coding regions differ by a single nucleotide. The genes 15 encode either Cys or Arg at amino acid 347 near the C terminllc of the receptor. The nucleotide difference lies within a PstI restriction enzyme recognition site thus creating a Restriction Fragment Length Polymorphism (RFLP). The frequency of allele 1 (LRR) is 0.34; allele 2 (LCR) is 0.66 in 83 unrelated individuals (Hoehe et al "A two-allele 20 PstI RFLP for the alpha-lC adrenergic receptor gene" Human Molecular Genetics 1: 349, 1992; Allele I is defined by a 2.1 kb PstI
fragment; allele 2 yields two bands of 1.6 and 0.5 kb). Since the amino acid dirre~ ce occurs within the intracellular tail of the receptor we would not expect any ph~ cological dirre,~;nces between the 25 expressed receptors. To investigate the ph~ cological profiles of the two allelic forms of the hl-m~n alpha-lc adrenergic receptor we ligated the genomic exon 2 fragment of allele 2 to a cDNA clone of allele 1 at a common PvuII restriction site. The two allelic forms were transientl expressed in COS-7 cells using pcDNAI/NEO (Invitrogen) expression vector. Competitive inhibition studies performed in the presence of 125I-HEAT with various antagonists showed no significant difference in their ph~ cological profiles (Table II):

~WO 94/21660 ~ 1 5 8 3 4 ~ PCT/US94/02609 Table II
COMPARATIVE PHARMACOLOGY OF ALPHAl-C ~T T FT FS

I~ (nM) LRR LCR
phentol~mine 15 17 niguldipine 0.8 1.8 prazosin 1.0 0.9 5-me~yl urapidi 3.1 4.3 WB4101 0.9 1.0 ~,X~MP~,F, 1 ~
CLON~G OF A NOVEL ALPHAl-A ADRENERGIC RECEPTOR
A cosmid library cont~inin~ FG293 cell line genomic DNA in the double-cos vector sCos-l was screened as follows: The published hllm~n ala receptor cDNA clone (Bruno et al., BBRC. 179:1485-1490 (1991), and see Fig. 10, SEQ.ID:13:) was cloned into ~e vector 20 pcDNA1 neo to generate the clone pEXala. Filters cont~inin~
approxim~tely 200,000 clones were screened by colony hybridization ([Sambrook, Molecular Cloning, Cold Spring Harbor Laboratory Press, New York, 1989 ]) using a mixed exon 1 probe generated by PCR
corresponding to ala (TMDl-3), alb (TMDl-S) and oc1c (TMDl-5):
25 25 cycles of 95 C 1'; 52 C 30 sec; 72 C 1.5' using 10 ng of pEX
alb, pEX alc or pEX ala and 10 pmoles each of primers 5' MET (5' GAATCCCGACCTGGAC ), SEQ.ID:31:, and 3' BAM
(5'GGATCCTCAGGGTC ), SEQ.ID:32:, for alb, 5' 597 (5' CCATGGTGTTTCTCTCGGG), SEQ.ID:33: and 3' 1219 (5' 30 GACGCGGCAGTACATGAC ), SEQ.ID:34: for alc or 5' 76 (5' GTCATGATGGCTGGGTACTTG ), SEQ.ID:35:, for ala in a 12 ,ul reaction cont~inin~ 1.5 ,uM each unlabelled dNTP and 50 ,uCi 3000 Ci/mmol a-[32P] dCTP. The filters were incubated with 1 x 106 cpm/ml of probe in SX SSC, 35% Formamide, 0.02% SDS, 0.1 %

-W O 94/21660 E~CTnUS94/02609 215~34~

lauroyl sarcosine, 2% blocking buffer (Bohrenger ~nnheim), at 42 C
for 18 hours. The ~llters were washed with 2 liters of 0.5X SSC, 0.1%
SDS, 55 C and exposed to Kodak XAR-5 film. Twelve primary positives were picked from master plates and re-screened using the a1a-specific probe. Cosmid DNA was prepared from second round positiveclones, digested with endonucleases Eco RI or Hind m and subjected to Southern blot analysis: Fragments were resolved by electrophoresis, and transferred to a nitrocellulose membrane (Bohrenger ~nnheim) with 20X SSC (lX SSC = 0.15M Sodium chloride, 0.015M Sodium citrate, pH 7.0) according to the method of Southern ([Southern, 1975 #14]) . The membrane was hybridized, washed and analyzed as described above. Alpha-la, a1b, and a1C receptor clones were identified by co~ alison of restriction patterns with genomic southern blots performed with a1a, a1b, or a1C specific probes. A Cosmid cont~inin~ a1a receptor exon 1 DNA was subjected to restriction digestion by endonuclease Pst I and subjected to southern blot analysis as above using the a1a -specific probe. Two fM~m~nt~ of 2.3 and 1.6 kb were detected and subcloned into the Pst I site of PGEM 3ZF . The presence of the correct S' terminal sequences in the 2.3 kb fr~rne~t was 20 confirrned by sequencing across the junction between inverted repeat and non-repeat sequences. The 5' end of the ala receptor gene was ligated to the cDNA clone at ~eir comrnon PstI site, see figures 22-24, SEQ.ID:29:, and SEQ.ID:30:.

2s F,XAMPT,F 13 EXEMPLARY COUN~RSCREENS

1. A~s~y Title: Doparnine D2,D4 in vitro screen Objective of the Ass~
The objective of this assay is to elimin~te agents which specifically affect binding of [3H] spiperone to cells expressing hllm~n dopamine receptors D2, D3 or D4.

WO 94/21660 PCT/US94tO2609 21~i83g5 Metho~:
Modified from VanTol et al (1991); Nature (Vol 350) Pg 610-613.
Frozen pellets cont~ining specific dopamine receptor subtypes stably expressed in clonal cell lines are lysed in 2 ml lysing buffer (lOmM Tris-HCl/~mM Mg, pH 7.4). Pellets obtained after centrifuging these membranes (15' at 24,450 rpm) are resuspended in 50mM Tris-HCl pH 7.4 co-lt~i-lill~ EDTA, MgCl[2], KCl, NaCl, CaCl[2]
and ascorbate to give a 1 Mg/mL suspension. The assay is initiated by ~lcling 50-75 ~g membranes in a total volume of 500 ,ul cont~ining 0.2 nM [3H]-spiperone. Non-specific binding is defined using 10 ,uM
apomorphine. The assay is tennin~ted after a 2 hour incubation at room temperature by rapid filtration over GF/B filters presoaked in 0.3% PEI, using 50mM Tris-HCl pH 7.4.

. Ass~,y Title: Serotonin SHTla Ohjective of the Assav The objective of this assay is to elimin~te agents which specifically affect binding to cloned hllm~n 5HTla receptor Method:
Modified from Schelegel and Peroutka Biochemical Pharmacology 35: 1943-1949 (1986).
l~mm~ n cells expressing cloned hllm~n SHTla receptors are lysed in ice-cold 5 mM Tris-HCl, 2 mM EDTA (pH 7.4) and homogenized with a polytron homogenizer. The homogenate is centrifuged at 1000Xg for 30', and then the supernatant is centrifuged again at 38,000Xg for 30'. The binding assay contains 0.25 nM [3H]8-OH-DPAT in 50 mM Tris-HCl, 4 mM CaCl2 and lmg/ml ascorbate.
Non-specific binding is defined using 10 ,uM propranolol. The assay is termin~ted after a 1 hour incubation at room temperature by rapid filtration over GF/Cfilters W O 94/21660 PCTrUS94/02609 2~5~3~

F,XAMPT,F, 14 EXEMPLARY FUNCTIONAL ASSAYS
.:
In order to confirm the specificity of compounds for the hllm~n alphalC adrenergic receptor and to define the biological activity of the compounds, the following functional tests may be performed:

. I N V I T R O FU~T , D O G ~D H~nML~N P R O S T A T E
A N D D O G U R E T H R A

Taconic Farms Sprague-Dawley male rats, weighing 250-400 grams are sacrificed by cervical dislocation under anesthesia (methohexital; 50 mg~g, i.p.). An incision is made into the lower abdomen to remove the ventral lobes of the prostate. Each prostate removed from a mongrel dog is cut into 6-8 pieces lon~itlltlin~lly along the Ul`t~ d opening and stored in ice-cold oxygenated Krebs solution overnight before use if necessary. Dog urethra proximal to prostate is cut into approximately 5 mrn rings, the rings are then cut open for contractile measurement of circular muscles. Human prostate chips from transurethral ~ulgely of benign prostate hyperplasia are also stored overnight in ice-cold Krebs solution if needed.
The tissue is placed in a Petri dish co-~t~il,il,g oxygenated Krebs solution [NaCl, 118 mM; KCl, 4.7 mM; CaC12, 2.5 mM;
KH2P04, 1.2 mM; MgS04, 1.2 mM; NaHC03, 2.0 mM; dextrose, 11 mM] warmed to 37C. Excess lipid material and connective tissue are carefully removed. Tissue segments are attached to glass tissue holders with 4-0 surgical silk and placed in a 5 ml jacketed tissue bath cont~ining Krebs buffer at 37C, bubbled with 5% C02/95% 2- The tissues are connected to a St~th~m-Gould force transducer; 1 gram (rat, hllm~n) or 1.5 gram (dog) of tension is applied and the tissues are allowed to equilibrate for one hour. Contractions are recorded on a Hewlett-Packard 7700 series strip chart recorder.
After a single pr.~ g dose of 3 ,uM (for rat), 10 ~M (for dog) and 20 ~M (for hlm~n) of phenylephrine, a cllm~ tive 2~8345 concentration response curve to an agonist is generated; the tissues are washed every 10 minutes for one hour. Vehicle or antagonist is added to the bath and allowed to incubate for one hour, then another cllmlll~tive concentration response curve to the agonist is generated.
EC50 values are calculated for each group using GraphPad Inplot software. pA2 (-log Kb) values were obtained from Schild plot when three or more concentrations were tested. When less than three concentrations of antagonist are tested, Kb values are calculated according to the following formula Kb = rBl, x-l where x is the ratio of EC50 of agonist in the presence and absence of antagonist and [B] is the antagonist concentration.

~. M~A~URli'Mli',NT ()F INTRA-UR}iTHR~, PRF,~URF ~N
AN~THF'~TI7~ ) l)OG~
PURPOSE: Benign prostatic hyperplasia causes decreased urine flow rate that may be produced by both passive physical obstruction of the prostatic uretlll~ from increased prostate mass as well as active 20 obstruction due to prostatic contraction. Alpha adrenergic receptor antagonists such as prazosin and terazosin prevent active prostatic contraction, thus improve urine flow rate and provide symptomatic relief in man. However, these are non-selective alpha-1 receptor antagonists which also have pronounced vascular effects. Because we 25 have identified the alpha-lC receptor subtype as the predominent subtype in the hllm~n prostate, it is now possible to specifically target this receptor to inhibit prostatic contraction without concomitant changes in the vasculature. The following model is used to measure adrenergically mediated changes in intra-urethral pressure and arterial 3 0 pressure in anesthetized dogs in order to evaluate the efficacy and potency of selective alpha adrenergic receptor antagonists. The goals are to: 1) identify the alpha-1 receptor subtypes responsible for prostatic/urethral contraction and vascular responses, and 2) use this model to evaluate novel selective alpha adrenergic antagonists. Novel 215~3~

and standard alpha adrenergic antagonists may be evaluated in this m~nner.

METHODS: Male mongre~ dogs (7-12 kg) are used in this study.
5 The dogs are anesthetized with pentobarbital sodium (35 mg/kg, i.v.
plus 4 mg~g/hr iv infusion). An endotracheal tube is inserted and the ~nim~l ventilated with room air using a Harvard instruments positive displacement large ~nim~l ventilator. Catheters (PE 240 or 260) are placed in the aorta via the femoral artery and vena cava via the femoral veins (2 catheters, one in each vein) for the measurement of arterial pressure and the ~tlmini~kation of drugs, respectively. A supra-pubic incision ~1/2 inch lateral to the penis is made to expose the urethers, bladder and urethra. The urethers are ligated and c~nn~ ted so that urine flows freely into beakers. The dome of the bladder is retracted to facilitate dissection of ~e proximal and distal urethra. Umbilical tape is passed beneath the urethra at the bladder neck and another piece of umbilical tape is placed under the distal urethra approxim~tely 1-2 cm distal to the prostate. The bladder is incised and a Millar micro-tip pressure transducer is advanced into the urethra. The bladder incision 20 iS sutured with 2-0 or 3-0 silk (purse-string suture) to hold the transducer. The tip of the transducer is placed in ~e prostatic urethra and the position of the Millar catheter is verified by gently squeezing the prostate and noting the large change in urethral pressure.
Phenylephrine, an alpha-1 adrenergic agonist, is ~clmini~tered (0.1-100 ug/kg, iv; 0.05 ml/l~g volume) in order to construct dose response curves for changes in intra-urethral and arterial pressure. Following ~tlminictration of increasing doses of an alpha adrenergic antagonist (or vehicle), the effects of phenylephrine on arterial pressure and intra-urethral pressure are re-evaluated. Four or 30 five phenylephrine dose-response curves are generated in each ~nim~l (one control, three or four doses of antagonist or vehicle). The relative antagonist potency on phenylephrine induced changes in arterial and intra-urethral pressure are determined by Schild analysis. The family of averaged curves are fit simultaneously (using ALLFIT software ~' 215~5 package) with a four paramenter logistic equation constraining the slope, .,~i.,i...l..~ response, and ma~hl-ulll response to be constant among curves. The dose ratios for the antagonist doses (rightward shift in the dose-response curves from control) are calculated as the ratio of the EDso's for the respective curves. These dose-ratios are then used to construct a Schild plot and the Kb (expressed as ug/l~g, iv) determined.
The Kb (dose of antagonist c~ ing a 2-fold rightward shift of the phenylephrine dose-response curve) is used to compare the relative potency of the antagonists on inhibiting phenylephrine responses for intra-ulctl,l~l and arterial pressure. The relative selectivity is calculated as the ratio of arterial pressure and intra-urethral pressure Kb's. Effects of the alpha-l antagonists on baseline arterial pressure are also monitored. Comparison of the relative antagonist potency on changes in arterial pressure and intra-urethral pressure provide insight 15 as to whether the alpha receptor subtype responsible for increasing intra-ur~ l pressure is also present in the systemic vasculature.
According to this method, one is able to confirm the selectivity of alphalC adrenergic receptor antagonists that prevent the increase in intra-urethral pressure to phenylephrine without any activity at the vasculature At the end of the experiment, the dogs are killed via an overdose of intravenously ~lmini~tered pentobarbital or saturated KCl.

21~83~S ~

SEQUENCE LISTING

(1) GENERAL INFORMATION:
(i) APPLICANT: Bayne, Marvin L
Clineschmidt, Bradley V
Strader, Catherine D
(ii) TITLE OF INVENTION: CLONED HUMAN ALPHAlC ADRENERGIC RECEPTOR
(iii) NUMBER OF SEQUENCES: 35 (iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: Merck & Co , Inc.
(B) STREET: 126 Lincoln Avenue (C) CITY: Rahway (D) STATE: New Jersey (E) COUNTRY: United States of America (F) ZIP: 07065 (v) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk (B) CONPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: PatentIn Release #1.0, Version #1.25 (vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER:
(B) FILING DATE:
(C) CLASSIFICATION:
(vii) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER: US 08/032,849 (B) FILING DATE: 15-MAR-1993 (viii) ATTORNEY/AGENT INFORMATION:
(A) NAME: Bencen, Gerard H
(B) REGISTRATION NUMBER: 35,746 (C) Kh~KEN~:/DOCKET NUMBER: 18943IA
(ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: (908)594-3901 (B) TELEFAX: (908)594-4720 (C) TELEX: 138825 (2) INFORMATION FOR SEQ ID NO:1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 28 base pairs (B) TYPE: nucleic acid 3 0 ( c ) STRANDEDNESS: both (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA

(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO

21583~

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:

(2) INFORMATION FOR SEQ ID NO:2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 26 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:

(2) INFORMATION FOR SEQ ID NO:3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 26 ba3e pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA

(iii) nY~snr:llCAL: NO
(iv) ANTI-SENSE: NO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:

(2) INFORMATION FOR SEQ ID NO:4:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 235 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: both (D) TOPOLOGY: both (ii) MOLECULE TYPE: cDNA

(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO

wo 94/21660 2 ~ 5 8 3 4 ~ - PCT/US94/02609 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:
GCCGCGTCTA C~l w rGGCC AAGAGGGAGA,G~.~GCCT CAAGTCTGGC CTCAAGACCG 60 ACAAGTCGGA CTCGGAGCAA GTGACGCTCC GCATCCATCG GAAAAACGCC CCGGCAGGAG 120 .

CCCGGGAGAA GAAAGCGGCC AAAACGCTGG GCATCGTGGT ~G~~ C GTCCT 235 (2) INFORMATION FOR SEQ ID NO:5:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 78 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear ~ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:
Arg Val Tyr Val Val Ala Lys Arg Glu Ser Arg Gly Leu Lys Ser Gly Leu Lys Thr Asp Lys Ser Asp Ser Glu Gln Val Thr Leu Arq Ile His Arg Lys Asn Ala Pro Ala Gly GIy Ser Gly Met AIa Ser Ala Lys Thr ~0 45 Lys Thr His Phe Ser Val Arg Leu Leu Lys Phe Ser Arg Glu Lys Lys Ala Ala Lys Thr Leu Gly Ile Val Val Gly Cys Phe Val Leu 25 (2) INFORMATION FOR SEQ ID NO:6:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 93 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (iii) HY~l'H~:l LCAL: NO

(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: internal 21~8345 (xi) SEQUENCE DESCRIPTION: SEQ ID N3:6:
- Leu Val Met Tyr Cys Arq Val Tyr Val Val Ala Lys Arg Glu Ser Arg 1 5 10 15 Gly Leu Lys Ser Gly Leu Lys Thr Asp Lys Ser Asp Ser Glu Gln Val Thr Leu Arg Ile His Arg Lys Asn Ala Gln Val Gly Gly Ser Gly Val Thr Ser Ala Lys Asn Lys Thr His Phe Ser Val Arg Leu Leu Lys Phe Ser Arg Glu Lys Lys Ala Ala Lys Thr Leu Gly Ile Val Val Gly Cys Phe Val Leu Cys Trp Leu Pro Phe Phe Leu Val Met Pro ~2) INFORMATION FOR SEQ ID NO:7:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1601 base pairs (B) TYPE: nucleic acid ( c ) STRANDEDNESS: both (D) TQPOLOGY: both (ii) MOLECULE TYPE: cDNA
(iii) HYHO'l'~:'l'lCAL: NO
(iv) ANTI-SENSE: NO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:
GAATTCCCTC CTAGAAGCTG GAGAGAGCAG GA~C~llCG~ TGGGGCAGCT CAAAATGTAG 60 GTAACTGC wG GCCAGGAGCA rrGcrrArA-T GCCATCGGTC ~l~C~ ~ AGCGTCGACG 120 25 AG M TCGTGA AT~llCC~ AGC~A~ACG M TAAGACAG CGCGGM AAG CAGATTCTCG 240 GGGAGGGAGT ~G W~l~CC~ GCTAGGCCAG CCCGCAGGTG GAGAG w TCC CCGGCAGCCC 360 CGCGCGCCCC TGGCCATGTC TTT MTGCCC 'l~CC~ A l~l~G~ TGAGGGTTCC 420 3 o CAG~rG~ CAGGGTTGTC TCCCACCCGC ~CGC~l~ TCACCCCCAG CCAAACCCAC 480 CTw CAGGGC TCCCTCCAGA AGAGACCTTT TGATTCCCGG ~l~GC~l ~C~C~l~ 540 CGCCAGCCCG GGA wGTGGCC CTGGACAGCC GGACCTCGCC ~G~GC TGGGACCATG 600 ~ ~l CGGGAAATGC TTCCGACAGC TCCAACTGCA CCCAACCGCC GGCACCGGTG 660 AACATTTCCA AGGCCATTCT GCTCGGGGTG ATCTTGGGGG GCCTCATTCT lll~GG w l~ 720 W O 94/21660 PCTnUS94/02609 21~83~

CTGGGTAACA TCCTAGTGAT LL1L1~LL-1A GCCTGTCACC GACACCTGCA CTCAGTCACG 780 ~11L1CCGCCA TCTTCGAGGT CCTAGGCTAC TG~LLLll~ GCAGGGTCTT CTGCAACATC 900 AGGG~1~1CA TGGL1~1GL1 CTGCGTCTGG GCACTCTCCC TGGTCATATC CATTGGACCC 1080 L1~11~G~L1 GGAGGCAGCC ~GCLLLL~AG GACGAGACCA TCTGCCAGAT CAACGAGGAG 1140 CCGGGCTACG TGL'1'L11~'1'L GGL1~ L~GGC TCCTTCTACC T~C~l~lGGC CATCATCCTG 1 2 0 0 GTCATGTACT GCCL~LL1L~1~A CL~1~G~1~G~CC AAGAGGGAGA GCCGGGGCCT CAAGTCTGGC 1 2 6 0 CCGGCAGGAG ~A~Cr.~ T GGCCAGCGCC AAGACCAAGA CGCACTTCTC AGTGAGGCTC 1380 CTCAAGTTCT CCCGGGAGAA GAAAGCGGCC AAAACGCTGG GCATCGTGGT CGGL1~L11C 1440 L~ CL~1~L1GL1 GGL1~LL111 TTTCTTAGTC ATGCCCATTG ~L1L111~11 CCCTGATTTC 1500 AA~LLL1~1~ AAACAGTTTT TAAAATAGTA 1111~L-L1CG GATATCTAAA CAGCTGCATC 1 5 6 0 (2) INFORMATION FOR SEQ ID NO 8 (i) SEQUENCE CHARACTERISTICS
(A) LENGTH 26 base pairs (B) TYPE nucleic acid (C) STRANDEDNESS single (D) TOPOLOGY 1 inear (ii) MOLECULE TYPE CDNA
(iii) ~Y~1~11CAL: NO
(iV) ANTI-SENSE NO

(Xi) SEQUENCE DESCRIPTION SEQ ID NO:8:

( 2 ) INFORMATION FOR SEQ ID NO g:
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH 30 base pairs (B) TYPE nucleic acid (C) STRANDEDNESS single (D) TOPOLOGY linear (ii) MOLECULE TYPE CDNA

(iii) HYPOTHETICAL NO

~WO 94/21660 2 1 5 8 3 4 5 PCT/US94/02609 (iv) ANTI-SENSE: NO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:

(2) INFORMATION FOR SEQ ID NO:10:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 512 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: both (D) TOPOLOGY: both (ii) MOLECULE TYPE: cDNA
(iii) HY~ln~ CAL: NO
(iv) ANTI-SENSE: NO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:

GCTGCATCAA CCCCATCATA TACCCATGCT CCAGCCAAGA GTTCAAAAAG ~'l"l"l'~'AGA 120 ATGTCTTGAG AATCCAGTGT CTCCGCAGAA AGCAGTCTTC CAAACATGCC ~l~G~l~CA 180 TGGGATCAAG AGAGACCTTC TACAGGATCT CCAAGACGGA TGG~lsl~l GAATGGAAAT 300 llllcl~l"l~ CAT~C~l GGATCTGCCA GGATTACAGT GTCCAAAGAC CAATCCTCCT 360 GTACCACAGC CCGGGTGAGA AGTAAAAGCT TTTTGCAGGT ~lG~i~ GTAGGGCCCT 420 (2) INFORMATION FOR SEQ ID NO:11:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 2004 base pairs (B) TYPE: nucleic acid (C~ STRANDEDNESS: both (D) TOPOLOGY: both (ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:

WO 94/21660 2 1 5 ~ 3 ~ 5 PCT/US94/02609 GTAACTGCGG GCCAGGAGCA GCGCCCAGAT GCCATCGGTC LL~l~CLl..G AGCGTCGACG 120 AGAATCGTGA A1~ LCCCC AGCCAGGACG AATAAGACAG CGCGGAAAAG CAGATTCTCG 240 TAATTCTGGA ATTGCATGTT GCAAGGAGTC TCCTGGATCT TCGCACCCAG ~l.CGGLlAC 300 rrrrrrrrrr TGGCCATGTC TTTAATGCCC I~CLLL.l~A ~l~.~GLL.l~ TGAGGGTTCC 420 CA wLL.GGL CA~..~l~ TCCCACCCGC GCGCLCLL-lC TCACCCCCAG CCAAACCCAC 480 CTGGCAGGGC TCCCTCCAGA AGAGACCTTT TGATTCCCGG L.~LCGLLL. LLLLCLl~CG 540 CGCCAGCCCG GGAGGTGGCC CTGGACAGCC GGACCTCGCC CG~LLLLG~ TGGGACCATG 600 Ll~lll.~ CGGGAAATGC TTCCGACAGC TCCAACTGCA CCCAACCGCC GGCACCGGTG 660 AACATTTCCA AGGCCATTCT LLlC w G~l~ ATCTTGGGGG GCCTCATTCT .-.LGG W lG 720 CACTACTACA TCGTCAACCT GGCGGTGGCC GACL.CL.GL TCACCTCCAC w lGLlLCCC 840 -LlCCGCCA TCTTCGAGGT CCTAGGCTAC T~CL~lCG GCAGGGTCTT CTGCAACATC 900 TGrGrGGrAr. TGGATGTGCT GTGCTGCACC GCGTCCATCA TGGGC~l--~ CATCATCTCC 960 AGGLL.~lLA TGGCTCTGCT CTGCGTCTGG GCACTCTCCC TGGTCATATC CATTGGACCC 1080 CCGGGCTACG ~l~L~ L GGLlLl~GC TCCTTCTACC I~CLlL-GGL CATCATCCTG 1200 GTCATGTACT GCCGCGTCTA CL1~L1~L-L'C AAGAGGGAGA GCCGGGGCCT CAAGTCTGGC 1260 L~l~LL.LlGL. GGLl~CL-1. TTTCTTAGTC ATGCCCATTG ~ CCCTGATTTC 1500 AAGCCCTCTG AAACAGTTTT TAAAATAGTA ...GG~lLG GATATCTAAA CAGCTGCATC 1560 3 o AACCCCATCA TATACCCATG CTCCAGCCAA GAGTTCAAAA AGGCCTTTCA GAATGTCTTG 1620 AGAATCCAGT ~.~lLC~AG AAAGCAGTCT TCCAAACATG CCCTGGGCTA CACCCTGCAC 1680 AGAGAGACCT TCTACAGGAT CTCCAAGACG GATGGCGTTT GTGAATGGAA A-~ L1 1800 ~WO 94/21660 PCTIUS94/02609 G~G~l~A GAAGTAAAAG -llll~CAG ~ 'rG~l GTGTAGGGCC CTCAACCCCC 1920 (2) INFORMATION FOR SEQ ID NO:12:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 466 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (iii) HYPOTHETICAL: NO
(iv~ ANTI-SENSE: NO
(v) FRAGMENT TYPE: N-terminal (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:
Met Val Phe Leu Ser Gly Asn Ala Ser Asp Ser Ser Asn Cys Thr Gln Pro Pro Ala Pro Val Asn Ile Ser Lys Ala Ile Leu Leu Gly Val Ile Leu Gly Gly Leu Ile Leu Phe Gly Val Leu Gly Asn Ile Leu Val Ile ~0 ~5 Leu Ser Val Ala Cys His Arg His Leu His Ser Val Thr His Tyr Tyr Ile Val Asn Leu Ala Val Ala Asp Leu Leu Leu Thr Ser Thr Val Leu Pro Phe Ser Ala Ile Phe Glu Val Leu Gly Tyr Trp Ala Phe Gly Arg Val Phe Cys Asn Ile Trp Ala Ala Val Asp Val Leu Cys Cys Thr Ala Ser Ile Met Gly Leu Cys Ile Ile Ser Ile Asp Arg Tyr Ile Gly Val Ser Tyr Pro Leu Arg Tyr Pro Thr Ile Val Thr Gln Arg Arg Gly Leu 1~0 1~5 140 Met Ala Leu Leu Cys Val Trp Ala Leu Ser Leu Val Ile Ser Ile Gly Pro Leu Phe Gly Trp Arg Gln Pro Ala Pro Glu Asp Glu Thr Ile Cys Gln Ile Asn Glu Glu Pro Gly Tyr Val Leu Phe Ser Ala Leu Gly Ser ~158345 Phe Tyr Leu Pro Leu Ai~,a Ile Ile Leu Val Met Tyr Cys .brg Va] Tyr Val Val Ala Lys Arg Glu Ser Arg Gly Leu Lys Ser Gly Leu Lys Thr Asp Lys Ser Asp Ser Glu Gln Val Thr Leu Arg Ile His Arg Lys Asn Ala Pro Ala Gly Gly Ser Gly Met Ala Ser Ala Lys Thr Lys Thr His Phe Ser Val Arg Leu Leu Lys Phe Ser Arg Glu Lys Lys Ala Ala Lys Thr Leu Gly Ile Val Val Gly Cys Phe Val Leu Cys Trp Leu Pro Phe 0 275 . 280 285 Phe Leu Val Met Pro Ile Gly Ser Phe Phe Pro Asp Phe Lys Pro Ser Glu Thr Val Phe Lys Ile Val Phe TrP Leu Gly Tyr Leu Asn Ser Cys Ile Asn Pro Ile Ile Tyr Pro Cys Ser Ser Gln Glu Phe Lys Lys Ala Phe Gln Asn Val Leu Arg Ile Gln Cys Leu Arg Arg Lys Gln Ser Ser Lys His Ala Leu Gly Tyr Thr Leu His Pro Pro Ser Gln Ala Val Glu Gly Gln His Lys Asp Met Val Arg Ile Pro Val Gly Ser Arg Glu Thr Phe Tyr Arg Ile Ser Lys Thr Asp Gly Val Cys Glu Trp Lys Phe Phe 385 390 3g5 400 Ser Ser Met Pro Arg Gly Ser Ala Arg Ile Thr Val Ser Lys Asp Gln 405 ~10 415 Ser Ser Cys Thr Thr Ala Arg Val Arg Ser Lys Ser Phe Leu Gln Val Cys Cys Cys Val Gly Pro Ser Thr Pro Ser Leu Asp Lys Asn His Gln Val Pro Thr Ile Lys Val His Thr Ile Ser Leu Ser Glu Asn Gly Glu Glu Val (2) INFORMATION FOR SEQ ID NO:13:
( i ) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1621 base pairs (B) TYPE: nucieic acid (C) STRANDEDNESS: double (D) TOPOLOGY: both _WO 94/21660 PCTtUS94tO2609 21S83~5 (ii) MOLECULE TYPE cDNA
- (iii) HYPOTHETICAL NO
(iv) ANTI-SENSE NO

(xi) SEQ~ENCE DESCRIPTION SEQ ID NO 13 ~G~ OE ~l. GGGGGACTGG TGGTGAGCGC GCAGG OE GTG OE CGT WGCG I~llC~l~ OE 240 AGCCTTCATC CTTATGGCCG TGGCAGGTAA OE lG~ll~lC ATCCTCTCAG l~GCLl~AA 300 CCGCCACCTG CAGACCGTCA CCAACTATTT CATCGTGAAC ~lG~C~l~ CCGACCTGCT 360 GCTGAGCGCC ACCGTACTGC OE ll~lC~ CACCATGGAG ~ll~lG~ OE l T.lGGGC~ll 420 TGGCCGC OE~ TTCTGCGACG TATGGGCCGC CGTGGACGTG ~l~lG~ A CGGCCTCCAT 480 CCCAGCCATC ATGACCGAGC GCAAGGCGGC CGCCATCCTG ~ l W ~lC~lAGC 600 CCTGGTGGTG TCCGTAGGGC ~l~cl~GG CTGGAAGGAG COE ~l~C CTGACGAGCG 660 ~'l"l~'l'~C~l ATCACCGAGG AG OE ~lA C OE l~l~llC I~l OE~l~l OE l~ll~lA 720 2 0 CCTGCCCATG GCGGTCATCG TGGTCATGTA CTGCC OE GTG TA~l W lCG CGCGCAGCAC 780 CACGCGCAGC CTCGAGGCAG GCGTCAAGCG CGAGCGAGGC AAGGCCTCCG A~l~ 840 GCGCATCCAC `T~l~ CGGCCACGGG CGCCGACGGG OE GCACGGCA TGCGCAGCGC 900 CAAGGGCCAC A OE ll.C~A G~lC OE l~l~ ~lGC~l~ CTCAAGTTCT CCCGTGAGAA 960 GAAAGCGGCC AAGACTCTGG CCATCGTCGT GG~l~l~ll~ ~1 OE l~l~l G~llCC~lll 1020 2 5~ 1 1 . 1 1 1 ~ 1 . CTGCCGCTCG G~l OE ll~ll CCCGCAGCTG AAGCCATCGG AGG~l~ll 1080 TTCCAGCC OE GAGTTCAAGC GC~C~ll.~l CC~l~l~lG CGCTGCCAGT ~C~lC~l~G 1200 CC OE CGCC OE ~'l'~'l'~l GGC~l~l~lA CGGCCACCAC T~ OE ~l CCACCA OE GG 1260 3 o CCTGCGCCAG GACTGCGCCC CGAGTTCGGG CGACGCGCCC CCCGGAGCGC CGCTGGCCCT 1320 CGCCAGCCGT CGAAGCCACC CAGC OE CTTC CGCGAGTGGA GOE l~lGGG ~ OE ~llCCG~ 1440 WO 94/21660 PCT/US94/02609 ~
2~ ~3~

A , 1621 2) INFORMATION FOR SEQ ID NO:14-(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 501 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (iii) ~Y~u~ CAL: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: N-terminal (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:
Met Ala Ala Ala Leu Arg Ser Val Met Met Ala Gly Tyr Leu Ser Glu Trp Arg Thr Pro Thr Tyr Arg Ser Thr Glu Met Val Gln Arg Leu Arg Met Glu Ala Val Gln His Ser Thr Ser Thr Ala Ala Val Gly Gly Leu Val Val Ser Ala Gln Gly Val Gly Val Gly Val Phe Leu Ala Ala Phe Ile Leu Met Ala Val Ala Gly Asn Leu Leu Val Ile Leu Ser Val Ala Cys Asn Arg His Leu Gln Thr Val Thr Asn Tyr Phe Ile Val Asn Leu Ala Val Ala Asp Leu Leu Leu Ser Ala Thr Val Leu Pro Phe Ser Ala loo 105 110 Thr Met Glu Val Leu Gly Phe Trp Ala Phe Gly Arg Ala Phe Cys Asp Val Trp Ala Ala Val Asp Val Leu Cys Cys Thr Ala Ser Ile Leu Ser 130 1~5 140 Leu Cys Thr Ile Ser Val Asp Arg Tyr Val Gly Val Arg His Ser Leu Lys Tyr Pro Ala Ile Met Thr Glu Arg Lys Ala Ala Ala Ile Leu Ala Leu Leu Trp Val Val Ala Leu Val Val Ser Val Gly Pro Leu Leu Gly Trp Lys Glu Pro Val Pro Pro Asp Glu Arg Phe Cys Gly Ile Thr Glu 2158~5 Glu Ala Gly Tyr Ala Val Phe Ser Ser Val Cy8 Ser Phe Tyr Leu Pr-Met Ala Val Ile Val Val Met Tyr Cys Arg Val Tyr Val Val Ala Arq Ser Thr Thr Arg Ser Leu Glu Ala Gly Val Lys Arg Glu Arg Gly Lys Ala Ser Glu Val Val Leu Arg Ile His Cys Arg Gly Ala Ala Thr Gly Ala Asp Gly Ala His Gly Met Arg Ser Ala Lys Gly His Thr Phe Arg 0 Ser Ser Leu Ser Val Arg Leu Leu Lys Phe Ser Arg Glu Lys Lys Ala Ala Lys Thr Leu Ala Ile Val Val Gly Val Phe Val Leu Cys Trp Phe ~05 310 315 320 Pro Phe Phe Phe Val Leu Pro Leu Gly Ser Leu Phe Pro Gln Leu Lys Pro Ser GlU Gly Val Phe Lys Val Ile Phe Trp Leu Gly Tyr Phe Asn Ser Cys Val Asn Pro Leu Ile Tyr Pro Cys Ser Ser Arg Glu Phe Lys Arg Ala Phe Leu Arg Leu Leu Arg Cys Gln Cys Arg Arg Arg Arg Arg Arg Arg Pro Leu Trp Arg Val Tyr Gly Hi~ His Trp Arg Ala Ser Thr Ser Gly Leu Arg Gln Asp Cys Ala Pro Ser Ser Gly Asp Ala Pro Pro Gly Ala Pro Leu Ala Leu Thr Ala Leu Pro Asp Pro Asp Pro Glu Pro Pro Gly Thr Pro Glu Met Gln Ala Pro Val Ala Ser An~ Arg Ser His Pro Ala Pro Ser Ala Ser Gly Gly Cys Trp Gly Arg Ser Gly Asp Pro Arg Pro Ser Cys Ala Pro Lys Ser Pro Ala Cys Arg Thr Arg Ser Pro 3 o Pro Gly Ala Arg Ser Ala Gln Arg Gln Arg Ala Pro Ser Ala Gln Arg Trp Arg Leu Cys Pro (2) INFORMATION FOR SEQ ID NO:15:
( i ) SEQUENC E CHARACTERI ST I CS:

2~583~ ~

(A) LENGTH: 24 base pairs (B) TYPE: nucleic aci.d (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(iii) ~Y~u~i~n~ CAL: NO
(iv) ANTI-SENSE: NO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:

(2) INFORMATION FOR SEQ ID NO:16:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 29 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(ii~) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:

(2) INFORMATION FOR SEQ ID NO:17:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 921 base pairs (B) TYPE: nucleic acid (C) STF~NDEDNESS: double (D) TOPOLOGY: both (ii) MOLECULE TYPE: cDNA
(iii) ~Y~ul~llCAL: NO
(iv) ANTI-SENSE: NO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:

CCCAGCTGGA CATCACCAGG GCCATCTCTG TGGGCCTGGT GcTGGGcGcr TTCATCCTCT 180 ~wo 94nl660 2 1 5 ~ 3 ~ ~ PCT/US94/02609 CGCCCACCAA CTACTTCATT GTCAACCTGG CCATGGCCGA C~lG~l~.lG AGCTTCACCG 300 s TC~L~CCCL1 CTCAGCGGCC CTAGAGGTGC TCGGCTACTG G~.~c.~GGG CGGATCTTCT 360 GTGACATCTG GGCAGCCGTG GAT~l~.~l GCTGCACAGC GTCCATTCTG A~l~-~C~ 420 CCCGGAGGAA GGCCATCTTG GCC~lG~-CA ~-~.c.GG~l CTTGTCCACC GTCATCTCCA 540 TC~G~.cl C~.,~ AAGGAGCCGG CACCCAACGA TGACAAGGAG TGCGGGGTCA 600 CCGAAGAACC CTTCTATGCC ~----.lCCT CTCTGGGCTC CTTCTACATC C~lclG~CGG 660 .. ~L~l~G~ TATGTGAATT C 921 (2) INFORMATION FOR SEQ ID NO:18:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 18 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (Dl TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:

(2) INFORMATION FOR SEQ ID NO:19:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 18 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO

(ivl ANTI-SENSE: NO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:

2 ~ ~ 83~ 5 PCT/US94/02609 - 56 ~

(2) INFORMATION FOR SEQ ID NO:20:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 389 base pairs (B) TYPE: nucleic acid (c) STRANDEDNESS: both (D) TOPOLOGY: both (ii) MOLECULE TYPE: cDNA
(iii) HY~u~rH~ CAL: NO
(iv) ANTI-SENSE: NO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:

CTGACCCTGA GGATCCATTC CAAGAACTTT CACGAGwACA CCCTTAGCAG TACCAAGwCC 120 AAAGCAGCTA AGACGTTGGG CATTGTGGTC GGTATGTTCA ~l~-l~l~rG GCTACCCTTC 240 TTCATCGCTC TAC~ullGG ~rC~ll~l-c TCCACCCTGA AGCCCCCCGA ~CC~r~ll. 300 AA W rG~l~l Tul~ ~ CTACTTCAAC A~l~C~luA ACCCCATCAT CTACCCATGC 360 TCCAGCAAGG AGTTCAAGCG CG~lll~l 389 (2) INFORMATION FOR SEQ ID NO:21:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 27 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: ~ingle (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(iii) ~Y~OlHkllCAL: NO

(iv) ANTI-SENSE: NO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:

(2) INFORMATION FOR SEQ ID NO:22:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 35 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear W O 94/21660 8 ~ 5 PCT~US94/02609 ~ 5 7 (ii) MOLECULE TYPE: cDNA
(iii~ ~YPus~ CAL: NO
(iv) ANTI-SENSE: NO

(Xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:

(2) INFORMATION FOR SEQ ID NO:23:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 582 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: both (D) TOPOLOGY: both (ii) MOLECULE TYPE: cDNA
(iii) ~YPOl~ CAL: NO
(iv) ANTI-SENSE: NO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:23:
GAATTCATGA TTCAAGGTGG T~.l~l~G~. GGGCTACTTC AACAGCTGCC TCAATCCCAT 60 CATCTACCCG TGCTCCAGCA AGGAGTTCAA ~C~CG~llC ATGCGTATCC I-lG~ CA 120 ~l~CC~G~l GGCCGCCGCC GrcGrrrrcG IC~C~ .A ~GCGC~lGCG CTTACACCTA 180 CC~C~l~ ACCCGCGGCG ~lC~TGGA GAGATCACAG TCGCGGAAGG A~l~l~l~GA 240 TGACAGCGGC AGCTGCATGA GCGGCCAGAA GAGGACCCTG CC~.C w C~ CGCCCAGCCC 300 GGGCTACCTG GGTCGAGGAA CGCAGCCACC CGTGGAGCTG TGCGC~--CC CCGAGTGGAA 360 ACCCGGGGCG CTGCTCAGCT TGCCAGAGCC .~ G~CG~ ~ G~ .C TCGACTCTGG 420 CAACATGCCC ~l~G~C~ GCCACTTTTA AAAGCCGAAT TC 582 (2) INFORMATION FOR SEQ ID No:24:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1567 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: both ~ (D) TOPOLOGY: both (ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO

3~5 (iv) ANTI-SENSE NO

(xi) SEQUENCE DESCRIPTION SEQ ID-~0 24 TTGCCATCGT GGGCAACATC CTAGTCATCT I~ ~l~lGGC CTGCAACCGG CACCTGCGGA 240 c~rr~A~A~ CTACTTCATT GTCAACCTGG CCATGGCCGA ~lG~l~ ~ AGCTTCACCG 300 GTGACATCTG GGCAGCCGTG GAI~l~l~l GCTGCACAGC GTCCATTCTG AGCCTGTGCG 420 CCATCTCCAT CGATCGCTAC AT~G w ~ ~ GCTACTCTCT GCAGTATCCC A ~lG~l~A 480 CCCGGAGGAA GGCCATCTTG GCCCTGCTCA ~l~'l'~'l'~ CTTGTCCACC GTCATCTCCA 540 TCGGGCCTCT C~'l"l~'l'~ AAGGAGCCGG CACCCAACGA TGACAAGGAG TGCGGGGTCA 600 CCGAAGAACC CTTCTATGCC ~l~ll~lC~l ~ ~l~ CTTCTACATC ~l~lG~GG 660 l.~.G~lC w TATGTTCATC l"l~l~"lG~C TACCCTTCTT CATCGCTCTA ~G~ll~G~'l' 960 C~ l~ CACCCTGAAG CCCCCCGACG ~ ~llCAA W'l'~1~11~ T~G~l~ W~'l' 1020 CCTTCATGCG TATCCTTGGG TGCCAGTGCC GCGGTGGCCG ~ C~GC W~ 1140 25 GTCTAGGCGC ~lG~G~l AC ACCTACCGGC CGTGGACCCG CGGCGGCTCG CTGGAGAGAT 1200 CCCTGCCCTC GGG~lCGCCC AGCCCGGGCT AC~lG w lCG AGGAACGCAG CCACCCGTGG 1320 3 o GCCGCCGCGG CCGTCTCGAC TCTGGGCCAC TCTTCACCTT CAAGCTCCTG wCGATCCTG 1440 AGAGCCCG w AACCGAAGCG ACAGCCAGCA ACGGGGGCTG CGACACCACG ACCGACCTGG 1500 (2) INFORMATION FOR SEQ ID NO 25 ~WO 94/21660 PCT/US94/02609 2~583~

ti) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 515 amino acids ~ lB) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (iii) HY~OIHrllCAL: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: N-terminal (xi) SEQUENCE DESCRIPTION: SEQ ID No 25 Met Asn Pro Asp Leu Asp Thr Gly His Asn Thr Ser Ala Pro Ala His Trp Gly Glu Leu Lys Asn Ala Asn Phe Thr Gly Pro Asn Gln Thr Ser Ser Asn Ser Thr Leu Pro Gln Leu Asp Ile Thr Arg Ala Ile Ser Val Gly Leu Val Leu Gly Ala Phe Ile Leu Phe Ala Ile Val Gly Asn Ile Leu Val Ile Leu Ser Val Ala Cys Asn Arg His Leu Arq Thr Pro Thr Asn Tyr Phe Ile Val Asn Leu Ala Met Ala Asp Leu Leu Leu Ser Phe Thr Val Leu Pro Phe Ser Ala Ala Leu Glu Val Leu Gly Tyr Trp Val Leu Gly Arg Ile Phe Cys Asp Ile Trp Ala Ala Val Asp Val Leu Cys Cys Thr Ala Ser Ile Leu Ser Leu Cys Ala Ile Ser Ile Asp Arg Tyr Ile Gly Val Arg Tyr Ser Leu Gln Tyr Pro Thr Leu Val Thr Arq Arg Lys Ala Ile Leu Ala Leu Leu Ser Val Trp Val Leu Ser Thr Val Ile Ser Ile Gly Pro Leu Leu Gly Trp Lys Glu Pro Ala Pro Asn Asp Asp Lys Glu Cys Gly Val Thr Glu Glu Pro Phe Tyr Ala Leu Phe Ser Ser Leu Gly Ser Phe Tyr Ile Pro Leu Ala Val Ile Leu Val Met Tyr Cys Arg Val Tyr Ile Val Ala Lys Arg Thr Thr Lys Asn Leu Glu Ala Gly 2~8~4~

Val Met Lys Glu Met Ser Asn Ser Lys ~lu Leu Thr Leu Arg Ile His Ser Lys Asn Phe His Glu Asp Thr Leu Ser Ser Thr Lys Ala Lys Gly S His Asn Pro Arg Ser Ser Ile Ala Val Lys Leu Phe Lys Phe Ser Arg 275 280 ~.- 285 Glu Lys Lys Ala Ala Lys Thr Leu Gly Ile Val Val Gly Met Phe Ile Leu Cys Trp Leu Pro Phe Phe Ile Ala Leu Pro Leu Gly Ser Leu Phe 0 Ser Thr Leu Lys Pro Pro Asp Ala Val Phe Lys Val Val Phe Trp Leu Gly Tyr Phe Asn Ser Cys Leu Asn Pro Ile Ile Tyr Pro Cys Ser Ser Lys Glu Phe Lys Arg Ala Phe Met Arg Ile Leu Gly Cys Gln Cys Arg Gly Gly Arg Arg Arg Arg Arg Arg Arg Arg Leu Gly Ala Cys Ala Tyr Thr Tyr Arg Pro Trp Thr Arg Gly Gly Ser Leu Glu Arg Ser Gln Ser Arg Lys Asp Ser Leu Asp Asp Ser Gly Ser Cys Met Ser Gly Cln Lys 2 0 Arg Thr Leu Pro Ser Ala Ser Pro Ser Pro Gly Tyr Leu Gly Arg Gly Thr Gln Pro Pro Val Glu Leu Cys Ala Phe Pro Glu Trp Lys Pro Gly Ala Leu Leu Ser Leu Pro Glu Pro Pro Gly Arg Arg Gly Arg Leu Asp 2 5 Ser Gly Pro Leu Phe Thr Phe Lys Leu Leu Gly Asp Pro Glu Ser Pro Gly Thr Glu Ala Thr Ala Ser Asn Gly Gly Cys Asp Thr Thr Thr Asp Leu Ala Asn Gly Gln Pro Gly Phe Lys Ser Asn Met Pro Leu Gly Pro 3 oGly His Phe (2) INFORMATION FOR SEQ ID NO:26:

( i ) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1987 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: both ~WO 94nl660 PCT/US94tO2609 2~834~

(D) TOPOLOGY both (ii) MOLECULE TYPE cDNA
(iii) HYPOTHETICAL NO
(iv) ANTI-SENSE NO

(xi) SEQUENCE DESCRIPTION SEQ ID NO 26 GAATTCCCTC CTAGAAGCTG G~r~A~Ar~rAG GAGCCTTCGG TGGGGCAGCT CAAAATGTAG 60 GTAACTGCGG GCCAGGAGCA GcGccrArAT GCCATCGGTC CCTGCCTTTG AGCGTCGACG 120 TAATTCTGGA ATTGCATGTT GCAAGGAGTC TCCTGGATCT TCGCACCCAG ~llCGG~ AC 300 GGGAGGGAGT CCG~l~ GCTAGGCCAG CCCGCAGGTG GAGAGGGTCC CCGGCAGCCC 360 l 5 CAGGGCTGGC CAG~ C TCCCACCCGC GCG~l. TCACCCCCAG CCAAACCCAC 480 CTGGCAGGGC TCCCTCCAGA AGAGACCTTT TGATTCCCGG ~lCCCGCG~l ~CGC~l~C~ 540 CGCCAGCCCG GGAGGTGGCC CTGGACAGCC GGACCTCGCC ~(Gr~ l;l;r TGGGACCATG 600 ~l~l,l~l~l CGGGAAATGC TTCCGACAGC TCCAACTGCA CCCAACCGCC GGCACCGGTG 660 AACATTTCCA AGGCCATTCT GCTCGGGGTG AT~llGGG~ GCCTCATTCT TTTCGGGGTG 720 CTGGGTAACA TCCTAGTGAT C~l~lCC~lA GCCTGTCACC GACACCTGCA CTCAGTCACG 780 TG~~ ~ CGCCATCTTC GAGGTCCTAG GCTACTGGGC CTTCGGCAGG ~l~ll~lGCA 900 ACATCTGGGC GGCAGTGGAT ~lG~l~lG~l GrArrr~rGTc CATCATGGGC CTCTGCATCA 960 GA~l~ll CGGCTGGAGG CAGC~ CC CCGAGGACGA GACCATCTGC CAGATCAACG 1140 AGGAGCCGGG CTACGTGCTC .l~l~G~lC l~G~l~l~l CTACCTGCCT CTGGCCATCA 1200 ~lG~C~l~AA GACCGACAAG TCGGACTCGG AGCAAGTGAC GCTCCGCATC CATCGGAAAA 1320 A~CCCCG~ AGGAGGCAGC GGGATGGCCA GCGCCAAGAC CAAGACGCAC TTCTCAGTGA 1380 GG~ CAA GTTCTCCCGG GAGAAGAAAG CGGCCAAAAC GCTGGGCATC ~l~lC~G~l 1440 G~~ CCT CTGCTGGCTG C~~TAGTCATGCC CATTGGGTCT l~ll~lG 1500 ~1~83~5 TCTTGAGAAT CCAGTGTCTC CGCAGAAAGC AGTCGCTAGT TCCAAACATG CC~-~GIi-lA 1680 A1llll~ TCCATGCCCC GTGGATCTGC CAGGATTACA GTGTCCAAAG ACCAATCCTC 1860 CTGTACCACA GCC~Ii~l~A GAAGTAAAAG CTTTTTGCAG GTCTGCTGCT GTGTAGGGCC 1920 (2) INFORMATION FOR SEQ ID N3:27:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1997 base pairs ~B) TYPE: nucleic acid (C) STRANDEDNESS: both (D) TOPOLOGY: both (ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO

(xi) SEQUENCE DESCRIPTION: SEQ ID No:27:
AA-lCC~ TAGAAGCTGG AGAGAGCAGG AGC~-~ GGGGCAGCTC AAAATGTAGG 60 TAACTGCGGG CCAGGAGCAG CGCCCAGATG CCATCGGTCC ~lG~LllGA GCGTCGACGG 120 GAATCGTGAA ~ CC~A Gc~Ac~ArGA ATAAGACAGC GCGGAAAAGC AGATTCTC~T 240 GC~GC~- GGCCATGTCT TTAATGCCCT GCCCCTTCAT ~lGGC~ll~l GAGG~ll~C~ 420 AG~G~lGGCC AGGGTTGTCT CCCACCCGCG CGCG~C~l-l CACCCCCAGC CAAACCCACC 480 GCCAGCCCGG GAGGTGGCCC TGGACAGCCG GAC~l~GCC~ iCCC(I.GI_l GGGACCATGG 600 Tlj~1"1"1~rL~1~ GGGAAATGCT TCCGACAGCT CCAACTGCAC CCAACCGCCG GCACCGGTGA 660 ACATTTCCAA GGCCATTCTG CTCGGGGTGA '~ li~G CCTCATTCTT l~GGG~lGC 720 TGGGTAACAT CCTAGTGATC ~ CC~ AG CCTGTCACCG ACACCTGCAC TCAGTCACGC 780 ~WO 94121660 PCT/US94/02609 2~8~

ACTACTACAT CGTCAACCTG ~l~CCG ACCTCCTGCT CACCTCCACG GTGCTGCCCT 840 T~ GCCAT CTTCGAGGTC CTAGGCTACT GGGC~l CGG CAGGGTCTTC TGCAACATCT 900 GGGCGGCAGT GGATGTGCTG TGCTGCACCG CGTCCATCAT G~ C ATCATCTCCA 960 TCGACCGCTA CATCGGC~liG AGCTACCCGC TGCGCTACCC AACCATCGTC ACCCAGAGGA 1020 5 GGGGTCTCAT GG~l~rG~lC TGC~l~lGG~ CA~l~lCC~l GGTCATATCC ATTGGACCCC 1080 TCTTCGGCTG GAGGCAGCCG GCCCCC~A~G ACGAGACCAT CTGCCAGATC AACGAGGAGC 1140 CGGGCTACGT ~l~ll~l~ GCTCTGGGCT CCTTCTACCT ~-'~l~l~GCC ATCATCCTGG 1200 TCATGTACTG C~G~ lAC ~lG~l~GCCA AGAGGGAGAG ~r~GGGccTc AAGTCTGGCC 1260 TCAAGTTCTC CCGGGAGAAG AAAGCGGCCA AAACGCTGGG CATC~l~l~ G~lG-llCG 1440 TC~l~lG~l~ G~r~l,,, TTCTTAGTCA TGCCCATTGG ~~ CCTGATTTCA 1500 AGC~ lGA AACAGTTTTT AAAATAGTAT TTTGGCTCGG ATATCTAAAC AGCTGCATCA 1560 GAATCCAGTG TCTCTGCAGA AAGCAGTCTT CCAAACATGC ~l~G~lAC ACCCTGCACC 1680 CGCCCAGCCA G~ AA GGGCAACACA AGGACATGGT GCGCATCCCC GTGGGATCAA 1740 GAGAGACCTT CTACAGGATC TCCAAGACGG AT~lll~ TGAATGGAAA llllr~l~ll 1800 CC~G~lGAG AAGTAAAAGC TTTTTGCAGG T.l~ lG TGTAGGGCCC TCAACCCCCA 1920 t2) INFORMATION FOR SEQ ID NO 28 2 5 ~ i ) SEQUENCE CHARACTERISTICS
(A) LENGTH 466 amino acids (B) TYPE amino acid (C) STRANu~N~SS single (D) TOPOLOGY linear (ii) MOLEC~LE TYPE protein (iii) HYPOTHETICAL NO

(v) FRAGMENT TYPE N-terminal (xi) SEQ~ENCE DESCRIPTION SEQ ID NO 28 Met Val Phe Leu Ser Gly Asn Ala Ser Asp Ser Ser Asn Cys Thr Gln W O 94/21660 PCTrUS94/02609 21~3~5 Pro Pro Ala Pro Val Asn Ile Ser Lys Ala Ile Leu Leu Gly Val Ile Leu Gly Gly Leu Ile Leu Phe Gly Val l~eu Gly Asn Ile Leu Val Ile ' 45 Leu Ser Val Ala Cys His Arg His Leu His Ser Val Thr His Tyr Tyr Ile Val Asn Leu Ala Val Ala Asp Leu Leu Leu Thr Ser Thr Val Leu Pro Phe Ser Ala Ile Phe Glu Val Leu Gly Tyr Trp Ala Phe Gly Arg Val Phe Cys Asn Ile Trp Ala Ala Val Asp Val Leu Cys Cys Thr Ala Ser Ile Met Gly Leu Cys Ile Ile Ser Ile Asp Arq Tyr Ile Gly Val Ser Tyr Pro Leu Arg Tyr Pro Thr Ile Val Thr Gln Arg Arg Gly Leu Met Ala Leu Leu Cys Val Trp Ala Leu Ser Leu Val Ile Ser Ile Gly Pro Leu Phe Gly Trp Arg Gln Pro Ala Pro Glu Asp Glu Thr Ile Cys Gln Ile Asn Glu G1U Pro Gly Tyr Val Leu Phe Ser Ala Leu Gly Ser Phe Tyr Leu Pro Leu Ala Ile Ile Leu Val Met Tyr Cys Arg Val Tyr Val Val Ala Lys Arg Glu Ser Arq Gly Leu Lys Ser Gly Leu Lys Thr Asp Lys Ser Asp Ser Glu Gln Val Thr Leu Arg Ile His Arg Lys Asn 225 230 235 2~0 Ala Pro Ala Gly Gly Ser Gly Met Ala Ser Ala Lys Thr Lys Thr His Phe Ser Val Arg Leu Leu Lys Phe Ser Arg Glu Lys Lys Ala Ala Lys Thr Leu Gly Ile Val Val Gly Cys Phe Val Leu Cys Trp Leu Pro Phe Phe Leu Val Met Pro Ile Gly Ser Phe Phe Pro Asp Phe Lys Pro Ser Glu Thr Val Phe Lys Ile Val Phe Trp Leu Gly Tyr Leu Asn Ser Cys Ile Asn Pro Ile Ile Tyr Pro Cys Ser Ser Gln Glu Phe Lys Lys Ala Phe Gln Asn Val Leu Arg Ile Gln Cys Leu Cys Arg Lys Gln Ser Ser W0 94/21660 PCTtUS94tO2609 2~8345 Lys His Ala Leu Gly Tyr Thr Leu His Pro Pro Ser Gln Ala Val Glu ~ 355 360 365 Gly Gln His Lys Asp Met Val Arg Ile Pro Val Gly Ser Arq Glu Thr Phe Tyr Arg Ile Ser Lys Thr Asp Gly Val Cys Glu Trp Lys Phe Phe Ser Ser Met Pro Arg Gly Ser Ala Arq Ile Thr Val Ser Lys Asp Gln Ser Ser Cys Thr Thr Ala Arg Val Arg Ser Lys Ser Phe Leu Gln Val Cys Cys Cys Val Gly Pro Ser Thr Pro Ser Leu Asp Lys Asn His Gln Val Pro Thr Ile Lys Val His Thr Ile Ser Leu Ser Glu Asn Gly Glu Glu Val (2) INFORMATION FOR SEQ ID NO:29:
(i~ SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1776 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: both (D) TOPOLOGY: both (ii) MOLECULE TYPE: cDNA
(iii) ~Y~OTn~ CAL: NO
(iv) ANTI-SENSE: NO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:29:
2 5 CTCCCTGCCG G~r~~ r~l~ GCC~GCCAC CGACGGCCGG CGTTGAGATG 60 GGCTCCAGCG CGG~GCGG CGGG~C ~C~GC~ ~ C(:u~ ~ GGAGGGCCCG 180 ~1~ ~l~C~ ~ '~G~ GG~G~ ~C~l~l~ CGCAGGCAGC 240 GGCGAGGACA ACCGGAGCTC fGe~'~x:~ G CCGGGGAGCG ~G CGGCGACGTG 300 AATGGCACGG CGGCCGTCGG GGGACTGGTG GTGAGCGCGC A~ lGGG ~lGGG~lC 360 ll~l~AG CCTTCATCCT TATG~C~lG GCAGGTAACC l~8l~lCAT CCTCTCAGTG 420 GCCTGCAACC GCCACCTGCA GACCGTCACC AACTATTTCA TCGTGAACCT ~CC~l~CC 480 GA~ TGAGCGCCAC CGTACTGCCC TTCTCGGCCA CCATGGAGGT T~8G~-llC 540 2~ ~3~ ~

TGG~C~IlL~ ~CC~GC~l~ CTGCGACGTA TGGGCCGCCG TGGACGTGCT GTGCTGCACG 600 CTCAAGTACC CAGCCATCAT GACCGAGCGC AA~C w C~ CCATCCTGGC ~ s~G 720 GTCGTAGCCC TG~l~s~lC CGTAGGGCCC CTGCTGGGCT GGAAGGAGCC CGTGCCCCCT 780 GACGAGCGCT TCTGCGGTAT CACCGAGGAG GCGGGCTACG ~l~l~ll~lC CTCCGTGTGC 840 T~ll~lAcc TGCCCATGGC GGTCATCGTG GTCATGTACT GCCGCGTGTA CGTGGTCGCG 900 ~l~lG~s~ GCATCCACTG Trrrr~rrrr GCCACGGGCG CCGACGGGGC GCACGGCATG 1020 CGCAGCGCCA AGGGCCACAC CTTCCGCAGC TCGCTCTCCG I~CGC~lGCT CAAGTTCTCC 1080 CGTGAGAAGA AAGrrrrrAA GACTCTGGCC ATC~l~l~ ~l~l~ll~l G~ l~G 1140 ll~lll~l T~lll~lC~l GCCGCTCGGC TC~ll~llCC CGCAGCTGAA GCCATCGGAG 1200 G~C~lcllCA AGGTCATCTT CTGGCTCGGC TACTTCAACA GCTGCGTGAA CCCGCTCATC 1260 TACCCCTGTT CCAGCCGCGA GTTCAAGCGC GC~ ~l~C ~l.lC~l~CG CTGCCAGTGC 1320 CGTCGTCGCC GGCGCCGCCG ~C~ lGG CGTGTCTACG GrrA-rrArTG GCGGGCCTCC 1380 ACCAGCGGCC TGCGCCAGGA CTGCGCCCCG AGTTCGGGCG A(~GcG(~ rr CGGAGCGCCG 1440 GCTCCGGTCG CCAGCCGTCG AAAGCCACCC AGCGCCTTCC GCGAGTGGAG G~~ G~G 1560 ~G~ w GCGCGCAGCG CGCAGAGGCA G~lG~CCC AGCGCTCAGA GGTGGAGGCT 1680 ~l~l~lAG GCGTCCCACA CGAGGTGGCC GAGGGCGCCA CCTGCCAGGC CTACGAATTG 1740 ~ 2) INFORMATION FOR SEQ ID NO:30:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 572 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (iii) HYPOTHETICAL: NO

(iv) ANTI-SENSE: NO

(v) FRAGMENT TYPE: N-terminal (xi) SEQUENCE DESCRIPTION: SEQ ID No:30:

~WO 94121660 PCT/US94/02609 21~8345 Met Thr Phe Arg Asp Leu Leu Ser Val Ser Phe Glu Gly Pro Arg Pro Asp Ser Ser Ala Gly Gly Ser Ser Ala Gly Gly Gly Gly Gly Gly Ala Gly Gly Ala Ala Pro Ser Glu Gly Pro Ala Val Gly Gly Val Pro Gly Gly Ala Gly Gly Gly Gly Gly Val Val Gly Ala Gly Ser Gly Glu Asp Asn Arg Ser Ser Ala Gly Glu Pro Gly Ser Ala Gly Ala Gly Gly Asp Val Asn Gly Thr Ala Ala Val Gly Gly Leu Val Val Ser Ala Gln Gly Val Gly Val Gly Val Phe Leu Ala Ala Phe Ile Leu Met Ala Val Ala Gly Asn Leu Leu Val Ile Leu Ser Val Ala Cys Asn Arg His Leu Gln Thr Val Thr Asn Tyr Phe Ile Val Asn Leu Ala Val Ala Asp Leu Leu Leu Ser Ala Thr Val Leu Pro Phe Ser Ala Thr Met Glu Val Leu Gly Phe Trp Ala Phe Gly Arg Ala Phe Cys Asp Val Trp Ala Ala Val Asp Val Leu Cys Cys Thr Ala Ser Ile Leu Ser Leu Cys Thr Ile Ser Val Asp Arg Tyr Val Gly Val Arg His Ser Leu Lys Tyr Pro Ala Ile Met Thr Glu Arg Lys Ala Ala Ala Ile Leu Ala Leu Leu Trp Val Val Ala Leu Val Val Ser Val Gly Pro Leu Leu Gly Trp Lys Glu Pro Val Pro Pro Asp Glu Arg Phe Cys Gly Ile Thr Glu Glu Ala Gly Tyr Ala Val Phe Ser Ser Val Cys Ser Phe Tyr Leu Pro Met Ala Val Ile Val Val Met Tyr Cys Arg Val Tyr Val Val Ala Arg Ser Thr Thr Arg Ser Leu Glu Ala Gly Val Lys Arg Glu Arg Gly Lys Ala Ser Glu Val Val Leu Arg Ile His Cys Arg Gly Ala Ala Thr Gly Ala Asp Gly Ala His Gly Met Arg Ser Ala Lys Gly His Thr Phe Arg Ser Ser Leu Ser Val Arg wo 94~21660 2 ~. S 8 3 ~ ~ PCT/US94/02609 Leu Leu Lys Phe Ser Arg Glu Lys Lys Ala. Ala Lys Thr Leu Ala Ile Val Val Gly Val Phe Val Leu Cys Tr,p Phe Pro Phe Phe Phe Val Leu 355 360:. 365 Pro Leu Gly Ser Leu Phe Pro Gln Leu Lys Pro Ser Glu Gly Val Phe Lys Val Ile Phe Trp Leu Gly Tyr Phe Asn Ser Cys Val Asn Pro Leu Ile Tyr Pro Cys Ser Ser Arg Glu Phe Lys Arg Ala Phe Leu Arg Leu Leu Arg Cys Gln Cys Arg Arg Arg Arg Arg Arg Arg Pro Leu Trp Arg Val Tyr Gly His His Trp Arg Ala Ser Thr Ser Gly Leu Arg Gln Asp Cys Ala Pro Ser Ser Gly Asp Ala Pro Pro Gly Ala Pro Leu Ala Leu Thr Ala Leu Pro Asp Pro Asp Pro Glu Pro Pro Gly Thr Pro Glu Met Gln Ala Pro Val Ala Ser Arg Ars Lys Pro Pro Ser Ala Phe ArSI Glu Trp Arg Leu Leu Gly Pro Phe Arg Arq Pro Thr Thr Gln Leu Arg Ala Lys Val Ser Ser Leu Ser His Lys Ile Arg Ala Gly Gly Ala Gln Arg Ala Glu Ala Ala Cys Ala Gln Arg Ser Glu Val Glu Ala Val Ser Leu Gly Val Pro His Glu Val AIa Glu Gly Ala Thr Cys Gln Ala Tyr Glu Leu Ala Asp Tyr Ser Asn Leu Arg Glu Thr Asp Ile (2) INFORMATION FOR SEQ ID NO:31:
( i ) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 16 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: ~oth ( D ) TOPOLOGY: l inear (ii) MOLECULE TYPE: cDN~

(iii) hY~o~ cAL: NO
(iv) ANTI-SENSE: NO

2~583~

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:31:

(2) INFORMATION FOR SEQ ID No:32:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 14 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: both (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO

(xi) SEQUENCE DESCRIPTION: SEQ ID No:32:

(2) INFORMATION FOR SEQ ID NO:33:
(i) SEQUENCE C~ARACTERISTICS:
(A) LENGTH: 19 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: both (D) TQPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO

(xi) SEQUENCE DESCRIPTION: SEQ ID No 33 CCATGGTGTT I~ CG~ 19 25 (2) INFORMATION FOR SEQ ID NO:34:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 18 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: both (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:34:

3~

(2) INFORMATION FOR SEQ ID No 35 (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: both (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO 35

Claims (24)

WHAT IS CLAIMED IS:

1. A DNA which consists essentially of a DNA which codes for a human adrenergic receptor of the alpha 1-C subtype.

2. The DNA of Claim 1, wherein the DNA coding for the receptor is operably linked to regulatory sequences such that the receptor may be expressed upon introduction into a prokaryotic or eukaryotic cell.

3. The DNA of Claim 2, wherein the receptor has the nucleic acid sequence SEQ ID: 11:, figure 5, a variant thereof which, through redundancy of the genetic code, encodes a human alpha 1C
receptor, or a truncation thereof, which continues to encode a functional human alpha 1C adrenergic receptor with intact G-protein and ligand binding sites.

4. The DNA of Claim 3 having the sequence, SEQ.
ID: 11:, figure 5; SEQ. ID:26:; figure 18; or SEQ.ID:27, figure 19.

5. A human alpha 1C adrenergic receptor expressed by a cell which contains a cloned nucleic acid construct encoding said receptor.

6. A cell expressing a cloned human alpha 1C adrenergic receptor.

7. A method for identifying compounds which selectively bind to the human alpha 1C adrenergic receptor with an affinity greater than twelve fold higher for said alpha 1C adrenergic receptor than the affinity for a human alpha adrenergic receptor other than the alpha 1C adrenergic receptor which comprises the steps of:
a. Cloning the human alpha 1C adrenergic receptor;
b. Splicing the the cloned alpha 1C adrenergic receptor into an expression vector to produce a construct such that the alpha 1C receptor is operably linked to transcription and translation signals sufficient to induce expression of said receptor upon introduction of said construct into a prokaryotic or eukaryotic cell;
c. Introducing said construct into a prokaryotic or eukaryotic cell which does not express a human alpha 1C adrenergic receptor in the absence of said introduced construct;

d. Incubating cells or membranes isolated from cells produced in step c.
with a quantifiable compound known to bind to human alpha adrenergic receptors, and subsequently adding test compounds at a range of concentrations so as to compete the quantifiable compound from the receptor, such that an IC50 for the test compound is obtained as the concentration of test compound at which 50% of the quantifiable compound becomes displaced from the receptor;
e. Incubating cells or membranes of cells which naturally express or have an introduced, cloned human alpha adrenergic receptor of a subtype other than the human alpha 1C receptor under identical conditions to the incubation conducted in step d, and obtaining the IC50 of the test compound for the non-alpha 1C receptor, and f. Comparing the IC50 for the test compound for the alpha 1C receptor and for the alpha adrenergic receptor of a subtype other than the alpha 1C to identify compounds having a 12-fold lower IC50 for the alpha 1C receptor.

8. A method of alleviating the effects of BPH which comprises administering a pharmaceutically effective amount of a compound which specifically binds to the human alpha 1C adrenergic receptor.

9. The method of Claim 8 wherein the compound is S(+)-niguldipine, (S(+)-1-4-Dihydro-2,6-dimethyl-4-(3-nitrophenyl) 3,5-pyridinedicarboxylic acid 3-(4,4-diphenyl-1-piperidinyl)-propyl methyl ester hydrochloride), or 5-methyl urapidil, 5-methyl-6[[3-[4-(2-methoxyphenyl)-1-piperazinyl]propyl]amino]-1,3-dimethyluracil.

10. The method of Claim 8 wherein said compound is administered in conjunction with a compound effective to inhibit human testosterone 5-alpha reductase.

11. The method of Claim 10 wherein the compound effictive to inhibit human testosterone 5-alpha reductase is a 5-alpha reductase isozyme 1 inhibitor.

12. The method fo Claim 10 wherein the 5-alpha reductase inhibitor is finasteride.

13. The method of Claim 10 wherein the compound effective to inhibit human testosterone 5-alpha reductase is a dual 5-alpha reductase isozyme 1 and an isozyme 2 inhibitor.

14. A method of alleviating the effects of BPH which comprises administering a pharmaceutically effective amount of finasteride and S(+)-niguldipine or 5-methyl urapidil.

15. A DNA which consists essentially of a DNA which codes for an entirely human adrenergic receptor of the alpha 1-A
subtype.

16. The DNA of Claim 15, wherein the DNA coding for the receptor is operably linked to regulatory sequences such that the receptor may be expressed upon introduction into a prokaryotic or eukaryotic cell.

17. The DNA of Claim 16, wherein the receptor has the nucleic acid sequence SEQ ID:29:, figure 22, a variant thereof which, through redundancy of the genetic code, encodes a human alpha 1A
adrenergic receptor, or a truncation thereof, which continues to encode a functional human alpha 1A adrenergic receptor with intact G-protein and ligand binding sites.

18. A human alpha 1A adrenergic receptor expressed by a cell which contains a cloned nucleic acid construct encoding said receptor.

19. A cell expressing a cloned, human alpha 1A
adrenergic receptor having a sequence greater than 95% homologous with the sequence of SEQ.ID:29:.

20. A method treating BPH which comprises administering to a patient in need of such treatment an inhibitorily effective amount of compound having at least 12 fold specificity for the human alpha 1C adrenergic receptor as compared with the human alpha 1A and alpha 1B receptor.

21. A composition comprising an inhibitorily effective amount of compound having at least 12 fold specificity for the human alpha 1C adrenergic receptor as compared with the human alpha 1A and alpha 1B receptor and an inhibitorily effective amount of a human testosterone 5-alpha reductase inhibitor.

22. A composition comprising an inhibitorily effective amount of a compound having at least 12 fold specificity for human alpha 1C adrenergic receptor as compared with the human alpha 1A and alpha 1B receptor and an inhibitorily effective amount of a human testosterone 5-alpha reductase type 1 selective inhibitor, a type 2 selective inhibitor, or a type 1 and a type 2 selective inhibitor.

23. A method of treating benign prostatic hyperplasia in a subject which comprises administering to the subject a therapeutically effective amount of a compound which binds to a human .alpha. 1C
adrenergic receptor with a binding affinity greater than twelve-fold higher than the binding affinity with which the compound binds to a human .alpha. 1A adrenergic receptor or a human .alpha. 1B adrenergic receptor.

24. The method of Claim 23 wherein said compound is administered in combination with an inhibitorily effective amount of a human testosterone 5-alpha reductase type 1 selective inhibitor, a type 2 selective inhibitor, or a type 1 and a type 2 selective inhibitor.