CA2250129A1 - Candida albicans tata-binding protein, nucleic acid and assays - Google Patents

Abstract

The invention encompasses a novel transcription factor from Candida albicans, TBP, a nucleic acid sequence encoding TBP, and methods of screening for inhibitors of Candida albicans growth by targeting TBP.

Description

W O 97/36925 PCTrUS97/06170 CANDlDA ALB~CANS TATA-BINDING PROTEIN. NUCLETC ACTD AND ASSAYS

ABSTRACT OF THE DISCLOSURE
The invention ellco.,.l,a~ses a novel Lldl~seliption factor from Candida albicans, TBP, a nucleic acid seq~lenre encoding TBP, and methods of sc~ ,nillg for inhibitors of Candida albicans growth by targeting TBP.
The invention relates in general to lr~nsc~ ion factors and to methods for screening for allLirul.gal agents.
The invention was made in part using gove,.. ~-t funds, NIH grant no.
GM46498, and therefore the U.S. go~ lllent has certain rights in the invention.

BACKGROUND OF THE INVENTION
The yeast Candida albicans ~C. albicans) is one of the most pervasive 25 funga} pathogens in hllmqn.c. It has the capacily to o~o,~ irqlly infect a diverse ~yecLnlnl of colllpiolllised hosts, and to invade many diverse tissues in the human body.
It can in many ill~c~ es evade antibiotic tl~ and the immlm~ system. Although Candida albicans is a member of the normal flora of the mucous membranes in the respiratory, gastroint~stinql~ and female genital tracts, in such locations, it may gain 30 dominqn~e and be associated with pathologic conditions. Som~timps it producesprogressive systemic disease in debilitated or immlmosuppressed patients, particularly if cell-m~iq-ted i~"~ ily is impaired. Sepsis may occur in patients with colll~lolllised cellular i------~ y, e.g., those undergoing cancer chemotherapy or those with lymphoma, AIDS, or other conditions. Candida may produce bloodstream invasion, CA 022~0129 1998-09-30 W O 97/36925 PCTrUS97/06170 thrombophlebitis, endocarditis, or infection of the eyes and virtually any organ or tissue when introduced intravenously, e.g., via tubing, needles, narcotics abuse, etc.
Candida albicans has been shown to be diploid with b~l~nre~ lethals, and therefore probably does not go through a sexual phase or meiotic cycle. This yeast S appears to be able to syollL~lleously and reversibly switch at high frequency between at least seven general phenotypes. Switching has been shown to occur not only in standard laboratory strains, but also in strains isolated from the mouths of healthy individuals.
Nystatin, ketoconazole, and amphotericin B are drugs which have been used to treat oral and systemic Candida infections. However, orally ~(lminictered nystatin is 10 limited to tre~tm~nt within the gut and is not applicable to systemic tre~tm~-nt. Some systemic infections are susceptible to tre~tment with ketoconazole or amphotericin B, but these drugs may not be effective in such L,~ enl unless combined with additional drugs.
AmphoL~lic,ll B has a relatively narrow the,ay~uLic index and ~lu~lerous undesireable side effects and toxicities occur even at thcla~ulic col~ce"L~aLions. While ketoconazole and other azole a~irungals exhibit .si~nifir~ntly lower toxicity, their m~c~nicm of action, inactivation of cyLochroll~e P4soyluSll.r~;r group in certain el~y~es, some of which are found in h-lm~n.c, precludes use in p~ti~ntc that are sim~ usly receiving other drugs that are metabolized by the body's cyloch.ullle P4so el~y~lles. In addition, les;~ re to these compounds is emerging and may pose a serious problem in the future.
There is a need in the art for an effective tre~m~ont of opportunistic infections caused by Candida albicans. Therefore, one object of the invention is to provide scleellillg assays for idellliryillg potential inhibitors of Candida albicans growth.
Another object of the invention is to provide scle~nil,g assays and to identify potential inhibitors of Candida albicans growth that are based on inhibition of ~ seliytion in this organism.
Synthesis of mRNA in eukaryotes requires RNA polymerase II and accessory Llansc,iytion factors, some of which are general and act at most, if not all, promoters, and others of which confer ~ecificily and control. Five general factors, a, b, d, e, and g, have been purified to homogeneity from the yeast S. cerevisiae, and have 30 been identified as c~ulll~lparts of human or rat factors, TFIIE, TFIIH, TFIID, TFIIB and TFIIF, respectively. These factors assemble at a promoter in a complex with RNA
polymerase II to initiate l~a~lsc~ Lion. Binding studies have shown that the order of assembly of the initiation complex on promoter DNA begins with factor d (TFIID), is followed by factor e (TFIIB), and then by polymerase and the rem-q-ining factors. Factors b (TFIIH), e (TFIIB) and g (TFIIF), however, bind directly to polymerase II, and as many as four of the five factors may assemble with the polymerase in a holoenzyme before promoter binding. The functional ~igni~l( Anre of interactions revealed by binding 5 studies is not clear in that only a few percent of initiation complexes may give rise to ansclipls.
Many aspects of llansc~ ion by RNA polymerase II are conserved between yeast and higher eukaryotes. For example, there is extens}ve amino acid sequencesimilarity among the largest subunits of the yeast, Drosphila and mqmmqli~n polymerases.
10 Other components of the transcription apparatus, such as TATA-binding and enh~nrer binding factors, are in some i~xlAnres i~rcl-~nge~kle between yeast and mqmmAliqn in vitro binding or ll~sclil.tion systems. There are, nonPth~l~s~, signific,qnt dirre,c,~ces be~ the two ~y~llls. TATA el~rntontc are located from 40 to 120 or more base pairs u~Ll~alll of the inithAti-~n site of an S. cerevisiae promoter, and where these el~PrnPnt~
15 occur, they are required for gene exl,lession. The fact that C. albicans genes function in S. cerevisiae suggests that it also uses the 40 to 120 base pair spacing bcl~een the TATA elPnnpnt and initiation site. In cOllLla~ ..s.,,,"qliqn (as well as S. pombe)TATA
elernPntc and ~ , ;plion start sites are only 25 to 30 bp apart, and deletion of a TATA
elem~nt does not always reduce the frequency of ~1~ scli~lion initiqti~m, although it may 20 alter the jnititZltiOn site. There are also varying degrees of homology bcl~,e.l llAi-!i' - ;l-lion factor sequenres from yeast and ~ qli~n sources. Some of the mllltic~kunit factors, such as RNA polymerase II, TFIIF, and TFIID, contain different l~ul~lb.,.~ of subunits in hl-mAn~ and yeast. The molecular weights of corresponding polypeptides differ b~lwcell hllmq-n~ and yeast, with seql)e~rçs being found in a given 25 yeast factor not being found in its human coullte.~ll and vice versa.
TATA-binding protein (TBP) is the central initiation factor for lldllscli~lion by all three nuclear RNA polymerases, and is highly conserved throughout the eukaryotic kingdoln The 180 amino acid carboxy-~ninql core domain is s~1fflri~nt for TATA
element binding, for all e~e~ l fimrtion~ in S. cerevisiae. and is 80% i-1Pntirql belwee 30 S. cerevisiae and hllmqn~. In vitro, yeast and human TBPs can functionally replace one another in terms of basal RNA polymerase II llal}sc~iption, and they display nearly i(ientirql DNA seq~lenre requirements for TATA elements. However, TBP exhibits species-specific behavior in vivo. For example, human and yeast TBP's are not species PCTrUS97/06170 hlLercl1angeable in supporting cell growth (Gill and Tjian, Cell 65:333-340, (1991);
Cormack et al., Cell 65:341-348 (1991)). Human and S. cerevisiae TFIIB's have 50-60%
amino acid seql~nre identity, and also are not species interchangeable in supporting cell growth.
S Operative s~lbsti~ltion of the same l.ansc~ tion factor in transcription systems of dirr~,~el~l yeast species is not predictable. This is true despite a high degree of amino acid sequence identity among some llanscli~tion factors from different yeast species. For example, the ability of a given t,anscliplion factor to support efficient and accurate llansc,iption in a heterologous yeast species is not predictable. Li et al. (1994, Science 263:805) tested the i~llc~ g~bility of S. cerevisiae and S. pombe llallScliption factors in vitro, and report that many S. cerevisiae components cannot substitute individually for S. pombe RNA l~lsc~ ion factors a, e, or polymerase II, but some combinations of these col"pol~e"~s were effective. In one in~t~nre, active lla,-sc,illlion could not be l.,co~-c~ cl when S. cerevisiae-derived TFIIB was the sole substitution into a TFIIB-~lepleted set of factors from S. pombe. A TFIIB-RNA polymerase II combination from S. cerevisiae was able to ~lilule, inrli~ ~ting that the r~mcliollal interaction of these two co,l,l)o,l~ s is not only unlJo~ but also that the activity may be dependent on species-s~ecir,c d~ that cannot be complemPntPd by either component derived from a different org~nicm The unpredictability in making ~sLilulions of a given factor among dirrel~,.,l yeast species is also evident in that such s~lbstit ltions are not reciprocal;
that is, substitutions of S. pombe fractions into an S. cerevisiae ll~.;,il,lion system are less effective than the reverse s~bstitltions (Li et al., supra).
The yeast Candida albicans differs from most yeast strains in that it does not use the same genetic code that most Ol~g~ , wllelher .. ~.. ~li~n or yeast, utilize.
25 Santos et al. (1995, Nucleic Acids Research, 23:1481) report that the codon CUG, which in the universal code is read as a leucine, is ~l~co~ as a serine in Candida. Therefore, any CUG codon .that is Secoded in Candida albicans as a serine, would be decoded as a leucine in the transformed S. cerevisiae. Any gene cont~ining a CUG codon would thtlcrole be tr~n~l~te~l as different amino acid sequences in Candida albicans and S.
30 cerevisiae. Such mistranslation may produce an inactive protein, since the amino acids serine and leucine have m~rkrrlly different cl~mir~l l,rop~ ies and serine is known to be an essenti~l residue in the active site of some el~y,lles. Repl~re~-.c..l of leucine by serine W O 97/36925 PCT~US97/06170 s at CUG encoded residues is a serious problem in the use of many l~oller systems (e.g.
~-g~lqrtosi~-q-ce, Chlorqmrhrnirol acetyllral~r,,l~se, Flux) in Candida albicans. Previous experiments have shown that tranclation by Candida of CUG as serine instead of leucine often resulted in the production of inactive l~,pOlL,~ proteins.
Another object of the invention is to provide an assay for screening for selective inhibition of Candida albicans growth and/or viability.
Yet another object of the invention is to provide a molecular target for inhibition of Candida albicans llanSCli~liOn or l.dl~scli~lion initiation.

SUMMARY OF THE INVENTION
The invention el~co~ ac.sPs a recombinant nucleic acid cou~l"isillg a nucleic acid seql.enre enr.otling Candida albicans TBP.
The invention also encomracses a vector colllplisillg a nucleic acid seql~enre çnr.o-ling Candida albicans TBP, and a transformed host cell contAining a nucleic acid sequence encoding Candida albicans TBP.
The invention also ~ o~nl~Ac~s~s a method for producing recombinant Candida albicans TBP, COll~li~illg cultunng a host cell transformed with a nucleic acid encoding Candida albicans TBP under conditions ~llrr~ .t to permit e~lession of the nucleic acid enrotling Candida albicans TBP, and isolating Candida albicans TBP.The "l~ulion also e~o~ qcses a scl~,n~llg method for idenlirying an inhibitor of Candida albicans growth, COlllp~i~illg dçtecting inhibition of mRNAllansclilllion in an in vitro llallSClillliOn assay col"~lising a DNA template, RNA
polymerase II, l~,cOlllbillallt Candida albicans TBP, and a c~n~ qt~ inhibitor, wherein production of an mRNA transcript compl~prnpntqry to the DNA template occurs in the q~bsenre if the c-qn.li~l-qte inhibitor.
The invention also enromrqc.ces a seleel~illg method for identifying an inhibitor of Candida albicans growth, comprising d~PIeC~ g in the l,r~sellce of a cqn~ qte inhibitor inhibition of formation of a complex COllll~liS~l~g a DNA template andrecombinant Candida albicans TBP, wll~ciu in the absence of the cqnf~ qte inhibitor, ~ 30 formation of the complex occurs. The method also may be pelro~ ed in the ple3e.1ce of additional factors, such as TFIIB, RNA polyln~ se II and TFIIF.
The invention also encomraCses a S~;lccllmg method for identifying an inhibitor of Candida albicans growth, co~ lisil~g ~letecting in the pl~sellce of a c~n-lid~te inhibitor, irlhibition of formation of a complex comprising Candida albicans TFIIB and Candida albicans TBP, whereill in the absence of the c~n~ te inhibitor formation of the complex occurs. Preferably, the complex will include a DNA template.
S The invention also e.~ro~ csP~s a scll,~nillg mPthnd for identifying an inhibitor of Candida albicans growth, colll~)lisillg ~leL~cl;l~g in the ~lesence of a c~nt~ te inhibitor inhibition of formation of a complex com~,lisillg RNA polymerase II, Candida albicans TBP, and Candida albicans TFIIB, wherein in the absence of the c~n~ t~
inhibitor formation of the complex occurs. Preferably, the complex will include a DNA
template and the RNA polymerase II from C. albicans.
In the above-described s~ el~lg methods, detection may be performed in the plesellce of a plurality of c~n~ te inhibitors. In screening methods of the invention which involve SCl~.Cllillg of a plurality of c~n~ te inhibitors, the plurality of inhibitors may be screened together in a single assay or individually using multiple .Cim~llt~nPous individual ~letPcting steps.
The invention also enco-~ cs~s a method of ~ ell~hlg Candida albicans growth in culture, comprising cont~rting the culture with an inhibitor that selectively inhibits the biological activity of Candida albicans TBP.
The invention also el~co~r~cses a method of pl~,~ellling Candida albicans growth in a m~mm~l, Co~ ;sillg ~mini~tpring to a ~---------~1 a thel~cl"ir~lly effective amount of an inhibitor that inhibits the biological activity of Candida albicans TBP.
As used herein, "inhibition" refers to a reduction in the parameter being measured, whether it be Candida albicans growth or viability, Candida albicans TBP-mP~ tP(~ ,nscli~lion, or formation of a Candida albicans TBP lldnscli~Lion complex.
The amount of such reduction is measured relative to a sL~ndar~ (control). Rec~nse of the multiple interactions of Candida albicans TBP in tl~nsc;li~tion initiation, the target product for ~etection varies with respect to the particular SCl~ illg assay employed.
Three llrer~.led detection products p~scllled in this disclosure are; a) newly lldnsclibed mRNA, b) a DNA-TBP complex, and c) a TBP-TFIIB-RNA polylllc.~se II complex.
"Reduction" is ~e~lnPfl herein as a decrease of at least 25% relative to a control, preferably of at least 50%, and most preferably of at least 75%.
As used herein, "growth" refers to the no~nal growth pattern of Candida W O 97/36925 PCTrUS97/06170 albicans, i.e., to a cell doubling time of 60 - 90 mimlt~. "Viability" refers to the ability of Candida albicans to survive in culture for 48 hours.
"Biological activity" refers to the ability of TBP to form a L~nsc~ ion complex with a DNA template or other ynoteil~- of the ~lansc-il lion complex, or to S interact with other llans~ lion compollcllls so as to permit initiation of L-~nscliylion.
"DNA template~ refers to double stranded DNA and, where inrlic~tçd by the particular binding assay to single stranded DNA, at least 10 nucleotides in length, that may be lleg~Livcly supercoiled if double-stran-lP~l, possesses a promoter region, and contains a yeast TATA conce~ C region. DNA templates useful herein preferably will 10 contain a TATA sequenre that is located from 40 to 120 or more base pairs upstream of the inhit~tion site (~ict~nre measured from the first T of the TATA element to the 5'-most initiation site). An especially efficient DNA template for use in methods of the invention involving ~lanscliy~ion is devoid of ~ O~ f residues, and thelefo~ a "G-minus" or "G-less" cassette is yrer~lcd~
"mRNA l~ refers to a full-length ~ s~;liyl as well as to truncated ,al scliyts, oligonucleotide lla~lSCliyLS and ~inllrlçotide RNAs.
"Formation of a complex~ refers to the binding of TBP to other ll~nscli~lion factors (i.e., protein-protein binding) as well as to binding of TBP to a DNA
template; such binding will, of course, be a non-covalent ~Ccoci~tion Other fe,dlul~es and advantages of the invention will be aypalclll from the description, ylcfc~l~,d embo~ thereof, the dl~wing~, and from the claims.

BRIEF DESCRIPTION OF THE DRAWINGS
Fig. 1 pl~se.lls the nucleotide and amino acid sequences of the Candida 2~ albicans k~scliylion factor TBP.
Fig. 2 yl~se~ nucleotide and amino acid sequenre of the Candida albicans transcription factor TFIIB.

DESCRIPTION
The invention is based on the discovery of a novel protein, Candida albicans TBP, and on the isolation of recon.bin~..l DNA encoding Candida albicans transcription factor TBP. Rec~llse TBP is esse~ti~l for viability of the cell, a compound that blocks the biological activity of the protein is expecte~ to have fimgiri~l yioy~ies~

PCTrUS97/06170 Therefore, the invention is also based on the development of assays for screening from inhibitors of TBP.
Isolation and Chala~;Lcl;G~Iion of the Candida albicans TBP Gene Given the unpredictability with respect to operative substitutions of a given S transcription factor among dirr~,lel~l yeast strains, one cannot assume that strategies for cloning of the gene encoding a given transcription factor which are based on factor function, such as genetic compleme~t~tion, will work Other cloning strategies, which do not require f~nrtion~l comple~ ion, such as those based on homology at the nucleic acid level, may be utilized in an attempt to cil~;unlvent a requirement for factor 10 filnrtion For example, Southern hybridization of specific sequPn~ec to a library carried in E coli and PCR amplification of potentially highly homologous regions of a gene are two strategies that have been s~cc~r~llly used to clone homologous genes from dirÇer~
Ol~a~ ."~, The approach used to clone the Candida albicans homolog of TBP
15 involved genetic colll~ on of mutant S. cerevisiae strains. A library of Candida albicans genomic seq.~Pnres was introduced into a strain of S cerevisiae that contained a m-lt~ted TBP gene (sptlS) This mutant strain was capable of growth at 30~ C, but was non-viable at 37~ C, due to a le~pel~lu,e se.~i~ive mllt~tion in the TBP gene Following transform~tio~ of the library into the strain, the cells were grown at 37~ C, and the 20 colonies which grew at this non-p~ ;.ve le~ alu~ were further studied as potentially carrying a Candida albicans homolog of the d~f~;live gene. This a~ oach will only work if a Candida albicans homolog is able to substi~l1te functionally in vivo for the d~Çe~;Li~e gene.
After c~n~ clones were i~ol~ted by growth at the lonpell,lissive 25 t~llpelalul." the library plasmid DNA was recovered from the cell and retested to confirm that the C. albicans sequen~ es on the pl~mirl were sub~ for the S. cerivisiae gene.
Subclones of the C. albicans seql~ent~çs were constructed by ~ndard cloning methods, and the minim~l Candida DNA sequen~es that substituted were sequenced using standard methods.
The nucleotide sequenre encoding Candida albicans TBP and the predicted amino acid sequence of the encoded protein are p.~l,se~ed in Fig 1 (SEQ ID NOS: 1 and 2). I~e nucleotide seqllenre encoding Candida albicans TFIIB and the predicted amino W O 97/36925 PCTrUS97/06170 acid sequPnce of the çnco~ed protein are l)lesell~ed in Fig. 2 (SEQ ID NOS: 3 and 4).

Methods For Screening Potential Inhibitors of Candida albicans Growth and/or Viability Because TBP initiation factor is es~Pnti~l for lldnscliL)lion initiation, the S recombinant Candida albicans TBP gene and recombinant protein e~oded by this gene may be utilized in scl.,el~ing assays for inhibitors of Candida albicans growth and viability. The screening assays of this invention detect inhibition of the Candida albicans TBP-mPdi~e~l component of Iranscli~lion initiation, either by llleas~ g inhibition of lldncli~lion, ll~s~ lion initation, or initiation complex formation, or by assaying 10 formation of a protein/DNA or a protein/protein complex.

Scleening for Inhibitors of TldnscliyLion a) Tldnsclil,lion Assay Components.
An in vitro ~dnscli~lion assay con.~istin~ of the minim~l COlll~Ol~lltS
~IPcess~ y to synthesi7P an mRNA Lldns~;LipL from a DNA template can be used to screen for inhibition of mRNA production. The cl~ ; of such an assay consist of; a) a DNA
template, b) RNA polyl,lc.~.sc II, c) 1cccjlllbilldnL Candida albicans TBP, and d) a TFIIB
which is preferably Candida albicans TFIIB. In order to increase the efficiency of 20 lldnsc~ ioll, additional components of the lldnscliplion complex may be included, as desired; e.g., TFIIE, TFIIF, TFIIH, etc.
Parvin and Sharp (Cell 73, 533-540, 1993) have reco~ d gene lldl~cli~tion in vitro with a minim~l reaction cont~ining a DNA template, RNA
polymerase II, TFIIB, and TBP. For efficient Ll~scli~lion under minim~l conditions, the 25 DNA template (a) is supercoiled, and (b) possesses a promoter region cont~ining a TATA
consel~us region. Additionally, Lue et al. (Science 246, 661-664, 1989) have detellllil~ed that ll~nsclil~tion may be ~etected most efficiently with a DNA template devoid of gl.~nosin~ residues (a G-minus or G-less cassette ). Promoter depe~Pnre is demonstrated by the loss of signal when a plasmid lacking promoter sequences is utilized as a template.
30 Correct initiation is demonstrated by the production of a band with a mobility consistent with the size of the expected product on del~luring polyacrylamide electrophoresis gels.
As stated above, Candida albicans TBP forms a ll~ns~ lion initiation complex with RNA polymerase II. Therefore, it is desired that an in vitro transcription W O 97/3692~

assay according to the invention contain RNA polymerase II. Although it is possible to perform an inhibitor ~cl~,e~ ,g assay using RNA polymerase II from a yeast strain other than Candida albicans, e.g., S. cerevisiae, it is most desirable to use a homologous assay in which the transcription complex components are from Candida albicans .
S A method for S. ccr~isiae RNA polII purification is described in Edwards et al. (Proc. Natl. Acad. Sci. USA 87: 2122-2126 (1990)). Alternatively, highly purified RNA polymerase Il from Candida albicans was provided as follows.
RNA polymerase II activity was measured in reactions cont~ining 50 mM
Tris-C1, pH 7.9 (4~ C), 50 mM (NH4)2 SO4, 2.5 mM MnC12, 0.1 mM EDTA, 5 mM
DTT, 100 ~g/ml BSA, 0.6 mM ATP, CTP and GTP, 25 ~M UTP (2.5 ~Ci) ~a32P~ UTP
and 100 ~g/ml heat-denaLured calf thymus DNA in a final volume of 50 ~1. Reactions were inrl-b~t~ri for 60 min. at 30~ C and le~ d by addition of 50 ~l 15% (w/v) trichloroacetic acid. Acid-insoluble radioactivity was collected by filtration through glass fiber filters and ql-~ntifiP~ by liquid scintillation ~c.,L,ophotometry. One unit of RNA
polymerase activity catalyzes the ulco~ tion of 1 pmol of UTP into acid-insoluble material in 60 min. under the conditions described above.
Candida albicans was obtained from the American Type Culture Collection (ATCC 10231) and cultured in YPD ~ (Current Protocols in Molecular Biology, Vol. 2, 13, Suppl. 19 (1989)) at 30~ C with vigorous agitation and aeration. Allprocedures were carried out at 4~ C using 18 liter cultures. Cells were harvested by centrifugation (5000 rpm, 10 min., Sorvall H6000 rotor), washed once with--11 ice-cold deionized water and repelleted as above. The cell pellet (200-300 g wet weight) was thoroughly resuspended in a volume of Buffer A (50 mM Tris-HCl, pH 7.9, 4~ C, 10%
glycerol, 1 mM EDTA, S mM MgCI2, and pfotease inhibitor) co-~ 300 mM (NH4)2 S04 equivalent to the packed volume of cells (~letermin~d by weight ~c.$~1ming a density of 1 g/ml cells). RP-sll~pended cells were either processed im m~ tely as described below or frozen by pipetting into liquid N2 and stored at -80 C. Frozen cells were thawed on ice prior to proceeding. Following the addition of NP-40 to a final concentration of 0.1%, cells were disrupted by glh~diulg with 1 ml acid-washed glass beads/ml cell suspension (Sigma, 400-625 ~M) using 12 bursts of 30 sec. each in a Bead Beater (BioSpec). Glass beads were allowed to settle out and the :iU~Je~llal~lnt was centrifuged at 30,000 x g for 40 min. Solid (NH4)2 S04 was slowly added to a final collcellLlation of 0.4 glml sup~ and the resl~ltin~ cipl~te was pelleted by centrifugation at 100,000 x g for 30 min. The pe}let was resuspended with a volume of Buffer A sufficient to yield a conductivity equivalent to Buffer A cont~inin~ 75 mM (NH4)2 SO4.
Following centrifugation of the res~lsp~ ion at 10,000 x g for 10 min, this ~ .e...~ 1. 5 mg protein/ml) was loaded onto a 300 ml DE-52 DEAE-cellulose5 column equilibrated with Buffer A cont~ining 75 mM(NH4)2 SO4. After washing with S
column volumes Buffer A cont~ining 75 rnM (NH4)2 SO4, and 5 column volumes Buffer A cont~ining 0.15 M (NH4)2 SO4, RNA polymerase II was eluted with 5 column volumes Buffer A cont~ining 0.4 M (NH4)2 SO4. Fractions were collected cont~ining the peak of protein, determin~d by absorbance at 280 nm and pooled. The pool was dialyzed against 10 Buffer A co..l~ 20% glycerol for 3 hr. at 4~ C.
The 0.4 M (NH4)2 SO4 eluate from DEAE-cellulose (261 mg protein, 290 ml) was diluted with sufficient Buffer A to lower the conductivity to the equivalent of Buffer A co..l;.i..;l~ 0.15 M (NH4)2 SO4, centrifuged at 10,000 x g for 10 min. and the ~,Jl,c~ ..l was loaded at a flow rate of 30 ml/hr onto an 30 rnl DEAE-cell~llose column 15 equilibrated with Buffer A co~ ini~ 0.15 M (NH4)2 SO4. After ~Sl~lllg with 3 column volumes of Buffer A COI~t~ 0.15 M (NH4)2 SO4, the column was developed with a 200 ml linear gradient of 0.15 - 0.4 M(NH4)2 SO4 in Buffer A at a flow rate of 45 ml/hr.
Fractions from the single peak of ~ -sensiLive RNA polymerase activity, eluting around 0.22 M (NH4)2 SO4, were pooled (21.1 mg protein, 45 ml) and loaded directly 20 onto a 5 ml Heparin agarose column equilibrated with Buffer A co..~ g 0.2 M (NH4)2 SO4. The column was washed with 3 column volumes of Buffer A cont~inin~ 0.2 M
(NH4)2 SO4 and developed with an 80 ml linear gradient of 0.2 - 0.6 M (NH4)2 SO4 in Buffer A. The active fractions, which eluted at ~ro~in~lely 0.42 M (NH4)2 SO4 were pooled (2.0 mg protein, 15 ml), frozen in 300 ~l ~li a~lots in liquid N2, and stored at -80~
25 C where activity was stable for at least 6 months.
P- t;rr~ion of protein initiation factors used in the assay is accomplished by standard meth~3s known in the art (e.g., phosphocellulose chromatography followed by gel filtration), as described in (Nature 346, 387-390 (1990)).
To screen for Candida albicans TBP-mP~ ted l~ lion inhibition, a 30 ~ sclil,lion assay is l~,con~ l using recombinant Candida albicans TBP.
Supercoiled plasmid DNA cont~ining the CYC1 promoter linked to the G-less cassette described by Lue et al. (Science 246, 661-664 (1989)), is purified by standard methods for purification of supercoiled circular DNA (Current Protocols in Molecular Biology, W O 97/36925 PCTrUS97/06170 Vol. 2, 13, Suppl. 19 (1989)). 10 - 100 ng of Candida albicans TFIIB, 10 - 100 ng of Candida albicans TBP, 10 - 100 ng Candida albicans RNA polymerase II and 1 ,ug plasmid DNA are added to 50 ~l reaction ~ ul~s cont~ining 50 rnM HEPES, pH 7.5, 10% glycerol, 90 mM pot~cci-lm ghlt~m~te, 0.75% polyethylene glycol (molecular weight 5 3350), 10 mM m~ s;l~ acetate, 5 rnM EGTA, 5 mM DTT, 0.4 mM ATP, 0.4 mM
CTP, 10 ~4M [~-32P]UTP, 0.2 mM 3'-O-methyl-GTP, and cont~ining or lacking a ç~n-litl~t~. inhibitor molecule. Reactions are inr~lb~ted at 30~ C for 30 - 60 min. and RNA
synthesis is ~letecte~l as described below.
b) Detection of Tla~sclibed RNA.
The detection of newly transcribed RNA is achieved by standard methods (Current Protocols in Molecular Biology, Vol. 1, 4.10, Suppl 24 (1989)). As one example, RNA ~yll~lesis can be detectPd as h~col~olation of a radioactively or fluorescelllly labeled nucleotide into higher molecular weight RNA products, de~ .il,Pd by one of the following m~tho-ls: 1) acid-insoluble labeled material qll~ntit~tPd by the 15 approl,liate method (e.g. scintill~tion cu~ for ra~ioactive pre~,ul~ul~, fluorometry for flUOl~SCell~ ,UI~jO1S); 2) labeled reaction product that llyblidi~es to oligonucleotides compl~ml~nt~ y to the coll~,elly i~ lrd LlallSClipl (i.e., l~l~llclll blot analysis); 3) the ~sellce of a labeled band with the a~pr~lid~ mobility detcoct~d by autoradiography, on delu~ulillg polyacrylamide elecL opholcsis gels: 4) any other method that discrimin~tes 20 mononucleotides from polynucleotides, where polynucleotides are the desired RNA
product. Such m~tho-1c may utilize one or more well known techniques of molecular biology (Current Protocols in Molecular Biology, Vol. 2, 13, Suppl. 19 (1989)), for example; UV analysis; affinity ~y~ s (e.g., affinity chromatography, nitrocellulose filtration, biotin/s~ Lavidin systems, imm-lno~ffinity,) (Current Protocols in Molecular 25 Biology, Vol. 2, 13, Suppl. 19 (1989)); and high pe.rolllldnce liquid chromatography.
The inclusion of an inhibitor molecule that il~lclr l- s with Candida albicans TBP biological activity inhibits lldnscli~>Lion. In this assay inhibition is measured as a reduction in the amount of mRNA Llalls~lipt produced relative to the amount of mRNA
transcript produced in the absence of the inhibitor (the positive control). A decrease in 30 ~mollnt of mRNA Lransclipl is indicative of an inhibitor. The ~G~ ti-)n of effective levels of mRNA Llanscli~L inhibition is described below.

W O 97/3692S PCTrUS97/06170 Screening for Inhibition of DNA-Protein Complex Formation A DNA-protein binding assay consisting of the minim~l components nPcessqry to permit DNA-Candida albicans TBP binding to occur can be used to screen 5 for inhibition of the formation of the DNA-Candida albicans TBP complex duringllans, li~Lion initiation. The ess~ntiql elçrnent~ of such an assay consist of; a) a DNA
template, b) recombinant Candida albicans TBP, and optionally c) a cq-n~ qte Candida albicans TBP i~hibilor.
The inclusion of an inhibitor molecule that hlle~ ,s with the interaction 10 b~lweel~ the Candida albicans TBP and the DNA template inhibits Ll~nsclil,Lion initiation.
The inhibitor may interact directly with the Candida albicans TBP protein, and/or it may interact with the DNA template at the DNA site of Candida albicans TBP binding. In this assay inhibition is measured as a reduction in the amount of DNA- Candida albicans TBP
complex produced relative to the amount of DNA- Candida albicans TBP complex 15 produced in the absenre of the inhibitor (the positive control). A decrease in the amount of DNA- Candida albicans TBP compleY is indicative of an inhibitor. Deterrninqtion of erreclive levels of DNA- Candida albicansTBP inhibition is described below.
One DNA binding assay is co~ ,d as follows. 10 - 100 ng Candida albicans TBP, e~lessed in and pulirled from E. Coli as desclibed above, is inrubatçd 20 with 0.5 ng labeled (e.g. rv lioactively or fluoresc.,l,~ly labeled) oligonucleotide contqining a TATA el~mP~t such as the one ~scrihe(l by B~ldtow~ki et al. (Cell 56, 549-561 (1989)) in reactifm.c co~ g 10 - 20 mM HEPES (or equivalent), pH 7.5 - 8.0, 5 mM MgCI2, 12% glycerol, 10 mM dill"o~ e;lol (DTT), 100 ,ug/ml BSA, 5 - 20 ~g/rnl poly (dG-dC):(dG-dC) and a c-q-n~ qt~- inhibitor of complex formation. Reactions are in-~lbqte~l at 25 30~ C for 30-60 min.
Formation of a DNA-TBP complex may be detecte~l as retention of labeled DNA (the label being cletect~d by an appl~pliate mPtho~ology such as scintillq-tion counting for radiolabeled DNA or fluoro"Rtly for fluu,esce"lly labeled DNA) utili7.ing known affinity m~thf (ls for protein immobilization (e.g., biotin/streptavidin, nitrocellulose 30 filtration, affinity chromatography, immllnnqfflnity). No~rl tention of labeled DNA due to the failure of Candida albicans TBP-DNA complex formation is indicative of aneffective inhibitor.

W O 97/36925 PCTrUS97/06170 Complex formation may also be detected as retention of labeled Candida albicans TBP (e.g. radioactively, fluo.~scelllly) ~1tili7ing known methods for immobilizing DNA. Nonle~ellLion of labeled Candida albicans TBP due to the failure of Candidaalbicans TBP-DNA complex formation is indicative of an effective inhibitor. These S mf th~-1c are suitable for high-throughput ch~mi~ ~l compound library screening applications such as those commonly used in drug discovery.
A third example of detectin~ DNA/protein complex formation involves detection of an electrophroretic mobility shift of labeled DNA on 4% polyacrylamide gels cont~ining 5% (V/V) glycerol, 25 mM Tris, 100 mM glycine, lmM EDTA, 5 mM MgCl2, 10 pH 8.3 in the presence of Candida albicans TBP. The position of the labeled oligonucleotide is ~etected by approl)liate methods (e.g., autoradiography for radioactive oligonucleotide). The ~hslo.nte or deviation of the eYpecte~ mobility shift due to DNA-Candida albicans TBP complex formation is indicative of an effective inhibitor.
Finally, other methods for ~et~cting or separating DNA-protein complexes 15 may be used, inrllltlin~ W crosslinking analysis, high ~elrulll~a~ce liquid chromatography, phage display technology (U.S. Patent No. 5,403,484. Viruses E~res~ g Chimeric Binding PloLeins), floulesence polarization, and surface plasmon .esona.~ce (Biacore, Pl.~ r;~ Biosensor, North America) as described below.

Screenin,~ for Inhibition of DNA-Protein Complex Formation A DNA-protein binding assay consisting of the minim~l components n~cess~ry to permit DNA-Candida albicans TBP a~soci~tion to occur can be used toscreen for inhibition of the formation of the DNA-TBP-Candida albicans TFIIB complex 25 during ll~nscli~tiorf initi~ion. The components of such an assay include: a) a DNA
temrl~te, b) recombinant Candida albicans TBP, c) TFIIB, preferably from C. albicans, and optionally d) a canrlid~tP Candida albicans TBP inhibitor.
The inclusion of an inhibitor molecule that ill~.r~ with the interaction between the Candida albicans TBP and the DNA template inhibits ll~nscli~Lion initiation.
30 The inhibitor may interact directly with the Candida albicans TBP protein, and/or it may interact with TFIIB and/or with the DNA template at the site of TFIIB/TBP binding. In this assay inhibition is measured as a reduction in the amount of DNA-TBP-TFIIB
complex produced relative to the amount of DNA-TBP-TFIIB complex produced in the W O 97/36925 PCTrUS97/06170 absence of the inhibitor (the positive control). A decrease in the amount of DNA-TBP-TFIIB complex is indicative of an inhibitor. De~ on of effective levels of DNA-TBP-TFIIB inhibition is described below.
One DNA binding assay is constructed as follows. 10 - 100 ng Candida 5 albicans TBP, expressed in and purified from E. Coli as described above, is incubated with 0.5 ng labeled (e.g. radioactively or fluorescently labeled) oligonucleotide cont~inin~
a TATA elem~nt such as the one described by Bu~tow~ki et al. (Cell 56, 549-561 (1989) and 10 - 100 ng Candida albicans TFIIB in reactions cont~ining 10 - 20 mM HEPES (or equivalent), pH 7.5 - B.0, 5 mM MgCI2, 12% glycerol, 10 mM dithiothreitol (DTT), 100 10 ~4g/ml BSA, 5 - 20 ~g/ml poly (dG-dC):(dG-dC) and a c~n~ te inhibitor of complex formation. Reactions are inruhat~d at 30~ C for 30-60 min.
Formation of a DNA-TBP-TFIIB complex may be detecte(l as retention of labeled DNA (the label being ~etected by an app.ul)lialc methodology such as scintill~tion cc,ul.lhlg for radiolabeled DNA or fluorùnlc~ for fluorcsccl~lly labeled DNA) ntili~ing 15 known affinity m~th~l.c for protein immobilization (e.g., biotin/streptavidin, nitrocelllllose filtration, affinity cl~oll~atography, immllno~fflnity). No-ll~,tel-lion of labeled DNA due to the failure of Candida albicans TFIB-TBP-DNA complex form~tion is indicative of an effective inhibitor.
Complex form~tion may also be ~etec.te~l as l.,t~ ion of labeled Candida 20 albicans TBP (e.g. r; ~ioactively~ fluo~cenLly) lltili7ing known m~thotl~ for immobilizing DNA. Nolllcknlion of labeled Candida albicans TBP due to the failure of Candida albicans TFIIB-TBP-DNA complex formation is indicative of an effective inhibitor. The preceding two mPth-)tlc are suitable for high-throughput ch~miral compound library sclccl~ing applications such as those commonly used in drug discovery.
A third example of detectin~ DNA/protein complex formation involves ~etection of an electrophoretic mobility shift of labeled DNA on 4% polyacrylamide gels cont~ining 5% (v/v) glycerol, 25 mM Tris, 100 mM glycine, lmM EDTA, 5 rnM MgCI2,pH 8.3 in the presence of Candida albicans TFIIB and TBP. The position of the labeled oligonucleotide is ~letected by approl)l;ate methoflc (e.g., autoradiography for radioactive oligonucleotide). The absence or deviation of the e~e~;led mobility shift due to DNA-Candida albicans TBP complex formation is indicative of an effective inhibitor.
Finally, other m~th~-~e for clet~cting or s~:~a~ lg DNA-protein complexes may be used, including UV cros~lin~ing analysis, high performance liquid W 097t36925 PCT~US97106170 chromatography, phage display technology (U.S. Patent No. 5,403,484. Viruses E,~ essillg Chimeric Binding Proteins), and surface plasmon r~sondllce (Biacore,Pharmacia Biosensor, North America) as described below.

Screenin~ for Inhibition of Protein-Protein Complex Formation A protein-protein binding assay CO.~ g of the minim~l components n~ces.c~. ~ to permit Candida albicans TBP-Candida albicans TFIIB binding to occur can be used to screen for inhibition of the formation of the Candida albicans TBP-Candida albicans TFIIB complex during ll~lsclil"ion initiation. The elem~ontc of such an assay consist of; a) recombinant Candida albicans TBP, b) TFIIB, preferably a recombmant Candida albicans TFIIB, and optionally c) a c2n~ te inhibitor of binding.
The inrlll5ion of an inhibitor molçcl-le that hl~r~,.cs with the hlteld.;~ion be~weell the Candida albicans TBP and Candida albicans TFIIB inhibits ~ sc~ ion 15 initiation. The inhibitor may interact with the Candida albicans TBP or TFIIB protein and thus induce a conrol,national change which ~levell~ binding, or it may directly inhibit the clion of Candida albicans TFIIB and TBP plote~s. In this assay, inhibition is meas~ d as a reduction in the amount of Candida albicans TBP-TFIIB complex produced relative to the ~m~ nt of Candida albicans TBP-TFIIB complex produced in the absence 20 of the inhibitor (the ~osili~e control). A decrease in the amount of TFIIB-TBP complex is indicative of an inhibitor. Detc ...;..-'ion of effective levels of inhibition of Candida albicans TBP-TFIIB binding is described below.
One assay for fo.~ tio., of Candida albicans TBP-TFIIB complex is provided as follows. 10 - 100 ng Candida albicans TFIIB and 10 - 100 ng Candida 25 albicans TBP are t:Apl~ssed in and purified from E. coli as described above, and are added to re~ctionc con~ g 10 - 20 mM HEPES (or equivalent), pH 7.5 - 8.0, 5 mMMgCl2, 12% glycerol, 10 mM dithiothreitol (DTT) 100 ~g/ml BSA, and a c~m inhibitor. The Illi~lUlC iS then in~ b~d at 30~ C for 30 - 60 min.
Pormation of a complex comprising Candida albicans TBP and Candida 30 albicans TFIIB may be ~l~tected by an electrophoretic mobility shift of labeled (e.g.
radioactive or fluor~,scenl) TBP or TFIIB on 4% polyacrylamide gels cont~ining 5% (v/v) glycerol, 25 mM Tris, 100 mM glycine, lmM EDTA, 5 mM MgCl2, pH 8.3 in the CA 02250l29 l998-09-30 W O 97/36925 PCTrUS97/06170 ~,csence of the llnl~behpd partner. The position of the labeled partner is detPcte~ by apploplidLe metho-lc (e.g., autoradiography for radioactive oligonucleotide). The ~hsen~e or deviation of the eYpectPd mobility shift due to Candida albicans TFIIB-TBP complex formation is indicative of an effective inhibitor.
S Formation of a complex col~ illg Candida albicans TBP and Candida albicans TFIIB may be AetPctPd as retention of labeled TBP utili7.ing known affinity metho-lc for irnmobilizing the Candida albicans TFIIB protein (e.g., biotin/streptavidin, nitroce~ lose filtration, affinity chrol"atography, immlmoaf~lnity). The failure of formation of the Candida albicans TFIIB-TBP complex is indicative of inhibition, and is 10 in(lic~tP,d by llomete~ on of labeled TBP. Alternatively, the immobilized elem~rt may be Candida albicans TBP and the labeled partner Candida albicans TFIIB.
In the above example, a stronger signal may be co~ c;d in the presence of both TBP and TFIIB and, in addition, a DNA template cont~ining a TATA ehP~nPnt The complex is then .~ ed by lntor~iography~ Phos~holi~llager technology, or 15 scintill~tion CUUllLillg for ra~ y labeled factors, fluoro~ h~ for fluol~sc.,.llly labeled factors, l.. ;.,(,.. rtry for factors labeled with ligands that are detected using chPmil~ sc~l or phnsl~hoJ~ ce~ll pl~g mPth-dologies, or other similar ~lPtectionmPtho~s or materials labeled as described above that are s~lldard in the art.
Other mPth-~Ac for ~lPL~,ti"g or s~aratillg protein-protein complexes may 20 be used, inrh~rling W crosslinking analysis, high pe~ro~ nre liquid chromatography, phage display technology, and surface plasmon lesonallce as described herein.

Assa~ for Formation of TBP-TFIIB-RNA Poly,llcl~se II-DNA Complex Formation of a TBP, TFIIB, RNA polymerase II, DNA complex is known to be n~rkP~lly stim~ te~ by the ad~lition of another factor, TFIIF. Previous data inrlic~tto.s that TFIIF from S. cerevisiae can function in species as distantly related as Schiznsaccharom~ces po~e and hl-m~n.c, sL~on~ly su~ge~Lmg that this factor can functionally replace its C. albicans homolog. Accordingly, this factor is purified from 5. cerevisiae by published methods (Sayre, 1992,J. Biol. Chem. 267:23383) and used to reconstitute formation of a complex cont~ining C. albicans TBP, TFIIB, RNA polymerase II and promoter cont~ining DNA such as that described for reconstitution of the TFIIB-TBP-DNA complex.

PCTrUS97/06170 Complex formation is carried out in reactions cont~ining~ for example, 10 -100 ng Candida albicans TBP, 10 - 100 ng Candida albicans TFIIB, 10 - 100 ng Candida albicans RNA polymerase II, 10 - 100 ng S. cerevisiae TFIIF, 0.5 ng double-stranded TATA element cont~ining-oligonucleotide (same as that used for TFIIB-TBP-5 DNA complex assay), 10 - 20 mM HEPES (or equivalent), pH 7.5 - 8.0, 5 mM MgCl2, 12% glycerol, 10 mM dithio~.~eilol (DTT), 100 ~ug/ml BSA, 5 - 20 ~g/ml poly (dG-dC);
(dG-dC) and compound(s) to be tested for inhibitory activity. Following inrllb~tinn at 30~
C for 30 - 60 min, complexes are detected by one of the metho~s described above for the TBP-TFIIB-DNA complex. The TBP-TFIIB-RNA polymerase II-DNA complex has a 10 slower electrophoretic mobility than the TBP-TFIIB-DNA complex i~lentifi~d by using the elecLIùphol~ic m~thod In addition, complex formation can be ~letected as TBP, TFIIB-depen-lent retention of RNA polymerase II activity (lllea;,ul~,d by incoly ,l~Lion of labeled nucleotide pl.,CUl~Ol~ into acid-insoluble product using the assay for RNA
polyll-e~ase activity desclil)ed in the RNA polymerase II purifi~tion protocol above) on 15 a matrix with bound TATA el~ ~..e .l co..~ g DNA. The IC50 of inl~ilo,~ colllyuui~ds will be ~el~ ...i..~ by titration into reaclions recol.~ c~ as described above. The ICso of these colllpoullds against re~~ on~ l~col.~ d with human TBP, TPIIB and RNA
polylllelase II will also be d~ ~.. in~d by the same mPth~ Human RNA pOlyl~c.asc II
and TFIIF are l,u,irled as described pl~ iuusly (Flores et al., 1990, J. Biol. Chem.
20 265:5629-5634; Reinberg et al., J. Biol. Chem 262:3310-3321). Those compounds whose ICso against re~ction~ cont~inin~ C. albicans factors is < 1/5 of their ICso against reactions l~co~ ed with human factors will be tested for their ability to inhibit C.
albicans growth as described below.

Phage Display Inhibitor Sc~ ,nillg In addition to the above mentioned ~ dard tec~ni~ es of the art, other technologies for molecular identifir~tion can be employed in the i~entifi-~tion of inhibitor molecules. One of these technologies is phage display technology (U.S. Patent 30 No. 5,403,484. Viruses E~l,les~ing Chimeric Binding ~lcins). Phage display permits identifir~tion of a binding protein against a chosen target. Phage display is a protocol of molecular SCl~,e,ning which utilizes recolllbh~n~ bacteriophage. The technology involves transforming bacteriophage with a gene that encodes an applopliate ligand (in this case, W O 97/36925 PCTrUS97/06170 a c~n~ te inhibitor) capable of binding to the target molecule of interest. For the purposes of this disclosure, the target molecule may be Candida albicans TBP, or a DNA-protein or protein-protein complex formed using TBP and/or TFIIB, as described herein.
The transformed bacteriophage (which preferably is tethered to a solid support) express S the c~n~ te inhibitor and display it on their phage coat. The cells or viruses bearing the c~n~ t~ inhibitor which recogmze the target molecule are isolated and amplified. The successful inhibitors are then characterized.
Phage display technology has advantages over standard affinity ligand scl~e~ g technologies. The phage surface displays the microplot~ ligand in a three 10 r~imPn~ional conformation, more closely resembling its naturally occl~rring conro~ ation.
This allows for more specific and higher affinity binding for scl,,.,ni~lg purposes.

Biospecific Interaction Analvsis A second relatively new sclee~ lg technology which may be applied to the inhibitor scleenillg assays of this invention is biospecific interaction analysis (BIAcore, ph~rm~ria Bios~n~or AB, Uppsala, Sweden). This technology is described in detail by Jonsson et al. (Bioterhniq~les 11:5, 620-627 (1991)). Biospecific ~lt~,~dclion analysis utilizes surface plasmon lesol~lce (SPR) to monitor t_e adsorption of biomolecular 20 complexes on a sensor chip. SPR measules the changes in l~;rlac~ e index of a polarized light directed at the surface of the sensor chip.
Specific ligands (i.e., c~n~ late inhibitors) capable of binding to the target molecule of interest (i.e., Candida albicans TBP or a protein-protein or protein-DNA
complex co~t~ining TBP) are immobilized to the sensor chip. In the ~les~"~ce of the 25 target molecule, specific binding to the immobilized ligand occurs. The nascent immobilized ligand-target molecule complex causes a change in the refractive index of the polarized light and is ~etected on a diode array. Biospecific ill~.aclion analysis provides the advantages of; 1) allowing for label-free studies of molecular complex formation; 2) studying molecular interactions in real time as the assay is passed over the sensor chip;
30 3) d~l~ti.-g surface concentrations down to 10 pg/mm2; d~tecting interactions between two or more molecules; and 4) being fully aulolllated (Biotçcllniq~lPs 11:5, 620-627 (1991)).

W O 97/36925 PCTrUS97/06170 Hi~h Throu~hput Screenin~ of Potential Inhibitors It is con~e~ lated according to the invention that the ~ ,.,.ling methods disclosed herein encompass s.;leenillg of multiple sa-m--ples .simllltqnPously, also referred 5 to herein as 'high throughput' scret;ning. For example, in high throughput screel~ing, from several h.~ ,d to several thousand c~ntli~qte inhibitors may be screened in a single assay. Several examples of high throughput screening assays useful according to the invention are as follows.
A protein A (pA)-C. albicans TBP fusion protein is ge~ dt~ by inserting 10 the coding sequen~e of TBP in fr~ne dow~ ~ll of the pA coding sequence of theplasmid pRIT2T (Phqrmqriq Biotech). The fusion construct is intl~ced, and the res~ltq-nt recombinant protein is e~ aclcd and purified according to the m~nllfactllrer's recommPn~lp~d con~ition~. This plocedurc can also be carried out for the pl~,~aldlion of a pA-Candida albicans TFIIB fusion protein except that the dow,.sl,~a." coding sequence 15 is that of TFIIB protein; all other steps would remain the same.
A Dynatech Microlite 2 IlPicl. lil~,r plate or equivalent high protein-binding capacity plate is coated with 1 ~g/well human IgG by inrllb~ing 300 ~l 3.33 ~4g/ml human IgG (Sigma) in coating buffer (0.2 M sodium call,onate, pH 9.4) in the well for 4-12 hr at 4~C. The coating buffer is then ~l~c~ and the wells are washed five times 20 with 300 ~1 PBS. 300 ,ul blocking buffer (SuperBlock~ blocking buffer; Pierce) cont~ining 3.33 ~g/ml pA-TBP or pA-TFIIB are added and the plate is inruhate~l for 4 or more hours at 4~C. The plates may be stored in this form at 4~C until ready for use.
When ready for use the plates are washed five times with 300 ,ul PBS. Test compound at a final concellllation of 20-200 ~M, labeled TBP or TFIIB (i.e., nonfusion protein), 25 whichever is not added during the coating step, and 10 - 1000 fmol plulllotel-cont~ining oligonucleotides are s~lspen~ in HEG buffer cont~ining 200 ~g/m~ BSA in a total volurne of 150 ~l and are added and the reaction is inrubated at room telll~J.,lalulc with gentle agitation for 60 min. The plate is then washed five times with PBS using a Dynatech plate washer or equivalent. Bound labeled protein is qn~ntit~terl by adding 250 30 ~l Miclusci~lt (Packard) per well and is counted in a microtiter plate-compatible seintill~tion ~pe~ ophotometer.
As an ~ltern~tive, the protein A fusion and the second, non-fusion protein can be ine~lb~t~ in the presence of test compound in polypropylene microtiter plates W O 97/36925 PCTrUS97/06170 under the same buffer and in~ub~tion conditions described above. The reaction mix is then transferred to the wells of a microtiter plate coated with human IgG (which is prepared as described above, and is stored in blocking buffer and is washed five times with 300 ~1 PBS immPfli~tely before use) and is inr'lb?~d for 60 min at room tc.n~eldture with gentle S agitation. RPt~ntion of radioactive protein is q~l~nti~lP(~ as above.
Interaction of TBP and TFIIB, which is measured as retention of rar~ioa~tivity on the plate, is d~pe ~-ient on human IgG coating the plate and wild-type Candida albicans TBP or TFIIB, one of which must be fused to pA. (~n~ te inhibitors or extracts that inhibit retention of r~ion~tivity by more than 30% are i-lPIltifi~d 10 and the inhibiloly activity is further purified if nrces~.y.
I~lhil,ilols i~PntifiP~l as deccri~ed above are then tested for their ability toinhibit Candida albicans TBP-dependent L-~ulscl;~lion in an in vitro ~ sclil,tion system as described herein, and also may be tested for their ability to inhibit Candida albicans growth.
~ther fusion or modified protein ~ S that are contemplated include, but are not limited to, glutathione-S-l~ lase, maltose binding protein, infllnPn7~ virus hPm~ tinin, FLAGn~ and hPY~ ti~linP fusions to Candida albicans TBP or Candida albicans TFIIB which are pl~arcd, eA~.es~ed, and purified by published mPtho~c or biotinylated Candida albicans TBP or TFIIB which are pl~aled using reactive biotin 20 precursors available co.. lfl,,ially. The ~ulirled fusion or ml tlifiPd protein is immobilized on a microtiter plate cont~inin~ the a~,r~l,liale ligand for each fusion protein (e.g. gll~t~thione~ amylose, CA157 antibody, etc., respectively) and the assay is carried out and the results evaluated in esse .~;~lly the same ~ r as described above.

C~ndid~te Inhibitors A "c~n~ te inhibitor," as used herein, is any compound with a potential to inhibit Candida albicans TBP-m~ ted llal~scliplion initiation or complex formation.
A c~n~ tP inhibitor is tested in a concenlIdtion range that d~p~n~1s upon the molecular 30 weight of the molecule and the type of assay. For example, for inhibition of protein/protein or protein/DNA complex formation or llai~s~;liplion initi~tion, small molecules (as defined below) may be tested in a concelll àlion range of lpg - 100 ug/ml, preferably at about 100 pg - 10 ng/ml; large molecules, e.g., peptides, may be tested in W 097/36925 PCT~US97/06170 the range of 10 ng - 100 ug/ml, preferably 100 ng - 10 ug/ml.
Inhibitors of Candida albicans growth or viability may target the novel transcription factor described herein, TBP, or it may target a protein or nucleic acid that hlLe,ac~ with the novel transcription factor so as to prevent the natural biological 5 interaction that occurs in vivo and leads to tldnsclipLion initiation in Candida. Thus, an inhibitor idPntifiPd as described herein will possess two ~r~clLies: 1) at some concentration it will inhibit Candida albicans growth or viability; and 2) at the same concellLlàtion, it will not signfir~ntly affect the growth of ~ n, particularly human, cells.
~n~ te inhibitors will include peptide and polypeptide inhibitors having an amino acid sequence based upon the novel TBP sequences described herein. For PY~mplP, a fragment of TBP may act as a co...pe~;~ive inhibitor with respect to TBP
binding to other protems involved in Candida L~ scli~lion, e.g., RNA polymerase II, TFIIB, or with respect to binding of the lldnscli~ion complex to the DNA template.
C~n~1id~tP inhibitor colllpoullds from large libraries of synthetic or natural compounds can be scl~,ned. Numerous means are ~;u~ ly used for random and directed synthesis of saccharide, peptide, and nucleic acid based compounds. Synthetic compound libraries are commercially available from a llulll~l of co~.pAniPS inr!-ltlin~ Maybridge ChPmir~l Co. (Trevillet, Cornwall, UK), Co~lge.le~ lc.,lon, NJ), Brandon Associates 20 (Me~im~r~ NH), and Miclùsoulce (New Milford, CT). A rare ch~mir~l library is available from Aldrich (Milwaukee, WI). ColllbindL~l;al libraries are available and can be prepared. ~Itc~ ely, libraries of natural culll~uunds in the form of bactPri~l1 fungal, plant and animal extracts are available from e.g., Pan Laboldtolies (Bothell, WA) or MycoSearch (NC), or are readily produceable. Additionally, natural and synthPtir~lly 25 produced libraries and compounds are readily mo-lifi~d through conventional chr~llir~l, physical, and bioch~.~.ir~l means.
Useful compounds may be found within lwlllelous çhPmical classes, though typically they are organic compounds, and preferably small organic compounds. Small organic compounds have a molecular weight of more than 50 yet less than about 2,500 30 daltons, preferably less than about 750, more preferably less than about 350 daltons.
Exemplary classes include heterocycles, peptides, saccharides, steroids, and the like. The colllpuullds may be mo(lifiPd to e.~h~i~reefficacy, stability, ph~ celltir~l compatibility, and the like. Structural id~ rlr~lion of an agent may be used to identify, generate, or .. . . ....

W O 97/36925 PCTrUS97/06170 screen additional agents. For example, where peptide agents are i~lentifipd~ they may be modified in a variety of ways to ~ernh~nre their stability, such as using an ululaluldl amino acid, such as a D-amino acid, particularly D-alanine, by functionqli7ing the amino or carboxylic termim~, e.g. for the amino group, acylation or alkylation, and for the 5 carboxyl group, e~ .cdtion or qmi-lifi-~tion, or the like. Other methods of stabilization may include e~ ..lqtion, for example, in liposomes, etc.

Measurement for effective inhibition The amount of inhibition by a cqndid-q-te inhibitor is qu-q-ntified using the following formula, which describes reactions n~co~ çcl with a ra~lioartively labeled moiety.

(CPMposijve Con~rol ~ CPMsamplc) Percent Inhibition = - x 100 (CPMPosilive Conlrol) where CPMPoilivc Conool iS the a~ gc of the cpm in complexes or RNA molecules formed in reactions that lack the cqn~ qt~ inhibitor, and CPMs~ p~c is the cpm in complexes 20 formed in reactions co~tqinin~ the cqn~iid-qte inhibitor. C-q-n-lidq-te inhibitors for which the percent inhibition is 50% are titrated into re~ction~ contqining either Candida albicans TBP or hlunan TBP (eA~l~,s~d in and purified from E. coli using e~i.cting recombinant clones (P~telsoll et al., Science 248, 1625-1630, 1990; Kao et al., Science 248, 1646-1650, 1990; Hoffman, et al., Nature 346, 387-390, 1990, and assayed as described above) 25 and their IC50 with respect to human and Candida albicans TBP de~e~ninPd from graphs of compound colr~ ion vs. % inhibition. The IC50 is defined as the conce~ dLion that results in 50% inhibition. C~n~1id~te inhibitors for which the IC50 against Candida albicans TBP-co~ g reactions is less than or equal to 1/5 the IC50 against human TBP-cont~ining reactions are further tested for their ability to inhibit the growth of Candida 30 albicans in culture as described below.

W O 97136925 PCTrUS97/06170 Measurement for inhibition of Candida albicans ~rowth in culture Once an inhibitor is identified in one or more of the binding or transcription assays described herein, it may be desirable to Cletf~ f' the effect of the inhibitor on the S growth and/or viability of Candida albicans in culture. A cq-n~il1qtp~ i.~il,i~or is tested for its ability to inhibit growth of Candida albicans cells in culture as follows. Methods for pelro~ ing tests on growth inhibition in culture are well-known in the art. Once such yruce lul~ is based on the NCCLS M27P method (The National Committ~Ae for Clinical Laboratoly Standards, Refe.~,~ce Method for Broth Dilution ~..lir~ gdl Su~ce~libility 10 Testing of Yeasts; proposed ~ndd~-l, 1992), as follows. Serial dilutions (two- or three-fold steps starting from a mqxim--m concellll~tion of 100 - 200 ~g/ml) of cqn~ qte inhibitor are ç,l~,all d using RPMI-1640 .~ as diluent and an aliquot of 100 ,ul of each dilution is added to the wells of a 96-well poly~ ne lllicrotil, . plate. Five Candida albicans colonies, picked from a Sabo~laud De~ use Agar plate in~Clllqtpd 14-20 15 hr previously with the test Candida albicans strain (Cat,alog ..w~e. 10231 from the ican Type Culture CollPction Yeast Catalog), are resuspended in RPMI-1640 m~dillm such that the density of cells is 10,000 - 30,000 cells/ml. 100 ~1 of the cell SUSpen~iQn is added to each of the wells of the 96-well lnicluli~. plate co..~i..;..g diluted cqn~ A inhibitor and mP~Ihlm control. Cultures are mixed by agitation and inruba~e~ at 20 35~ C for 48 hr. without agita~iQn~ after which cell growth is monitored by visual eclion for the formqtion of turbidity and/or myce!ial colonies. The minimllm concentration of cqndi~lqt~ inhibitor at which no cell growth is det~Fcte~l by this method is defined as the minimllm inhibitory co.~F .~.~(ion (MIC) for that co~ uul~d. Examples of MICs for known anlirul~l colll~uui~ds obtained using this techniq~le are 0.125 - 0.5 25 ~g/ml for flllronq7ole and 0.25 - 1.0 ~g/ml for ~n~~h~ icin B (The National Co~ Pe for Clinical Laboratory Standards, ReÇt~ c~ Method for Broth Dilution Anlir~ al Susceptibility Testing of Yeasts; l)loposesd s~ldard, 1992). An inhibitor identified by the mP~hnfl~ described herein, will have MIC which is equivalent to or less than the MICs for fluconq7Ole or a nphotericin B.

CA 022~0129 1998-09-30 W O 97/36925 PCTrUS97/06170 Tlanscllplion Inhibition Coul~lers~;.cen Using Human TBP
A compound identified as an inhibitor of Candida albicans according to one or more of ehe assays described herein may be tested further in order to determine its 5 effect on the host organism. In the development of useful antifungal compounds for human thc.dpcuLics, it is desirable that such compounds act as effective agents in inhibiting the viability of the fungal pathogen while not .signifir~ntly inhibiting human cell systems. Specifically, inhibitors of Candida albicans ide~tifip(l in any one of the above described assays may be c~ u~ ened for inhibition of human TBP.
Recombinant human TBP can be obtained from existing sources and purified by published methods (for example, see Peterson et al., Kao et al., and Hoffman et al., supra) and contacted with the c~n-lid~te inhibitor in assays such as those described above but using a human system. The effectiveness of a Candida albicans TBP inhibitor as a human th,l~y~uLic is determinP~ as one which exhibits a low level of inhibition 15 against human TBP relative to the level of inhibition with respect to Candida albicans TBP. For example, it is yl~çelled that the amount of inhibition by a given inhibitor of human TBP in a human system be no more than 20% with respect to the amount of inhibition of Candida albicans TBP/TF~B in a Candida system when tested in any of the assays described above.
Dosage and Pharrn~re~tir~l Formulations For thel~yculic uses, inhibitors idPntifiPd as described herein may be mini.stered in a ph~ e.~lic~lly acceptable/biologically colllpalible formulation, for example, in the form of a cream, ointm~nt, lotion or spray for topical use, or in a 25 physiological solution, such as a salt solution, for intPrn~l ~flmini~tration. The amount of inhibitor ~mini~tered will be determined according to the degree of pathogenic infection and whether the infection is systemic or loc~li7ed, and will typcially be in the range of about lug - 100 mg/kg body weight. Where the inhibitor is a peptide or polypeptide, it will be a~mini.~tered in the range of about 100 - 500 ug/ml per dose. A
30 single dose of inhibitor or multiple doses, daily, weekly, or intermittently, is contemplated according to the invention.
The route of a~mini~tration will be chosen by the physician, and may be topical, oral, transdermal, nasal, rectal, intravenous, intr~mnsc~ r~ or subcutaneous.

CA 022~0l29 l998-09-30 W O 97/36925 PCT~US97/06170 Budapest Treaty Deposit E. coli transformed with a plasmid cont~ining the gene encoding Candida albicans TBP has been deposited in an international depository, the A.T.C.C., Roclcville, MD, under the accession number 69900, on September 15, 1995. E. coli transformedS with a plasmid cu"~ g the gene encoding Candida albicans TFIIB has been deposited in an international depository, the A.T.C.C., Rockville, MD, under the accession number 69899, on September 15, 1995. A.T.C.C. Nos. 69900 and 69899 will be available to the public upon the grant of a patent which discloses the accession l~ulllbtl~ in conjull.;lion with the invention described herein. The deposits were made under the Budapest Treaty, 10 will be available beyond the enrolceable life of the patent for which the deposit is made, and will be m~int~in~d for a period of at least 30 years from the time of deposit and at least 5 years after the most recent request for the r"".;~ of a sample of the deposit is received by the A. T. C. C. It is to be understood that the availability of these deposits does not COl~lilul~ a license to practice the subject invention in derogation of patent rights 15 granted for the subject invention by gO~"l...llr..l;~l action.
OTHER EMBODIMENTS
The foregoing examples demona~lale experiments pelro~ ed and co..lP~-,pl~t~d by the present hlvt~ ls in making and call~illg out the invention. It is believed that these examples include a disclosure of tt~chniqlles which sene to both apprise 20 the art of the practice of the invention and to demol~llate its usefulness. It will be appreciated by those of skill in the art that the techniques and embo~im~ntc disclosed herein are preferred embo~imPnt~ only that in general llulllel~us equivlaent methods and terhniquec may be employed to achieve the same result.
All of the references identifi-od hereinabove, are hereby expressly 25 hlcoll,olaled herein by reference to the extent that they describe, set forth, provide a basis for or enable compositions and/or methods which may be hll~ol~nl to the practice of one or more embo~im~onts of the present inventions.

~ . . . ..

CA 022~0129 1998-09-30 W O 97/3692S PCTrUs97/06170 SEQUENCE LISTING

(i) GENERAL INFORMATION
(i) APPLICANT: SCRIPTGEN PHARMACEUTICALS, INC.
(ii) TITLE OF THE INVENTION: NOVEL TATA-BINDING PROTEIN FROM CANDIDA
ALBICANS, NUCLEIC ACID SEQUENCE CODING THEREFORE, AND METHODS OF SCREENING
FOR INHIBITORS OF CAMDIDA ALBICANS GROWTH
(iii) NUMBER OF ~QU~N~S: 4 (iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: DARBY & DARBY P.C.
(B) STREET: 805 Third Avenue (C) CITY: New York (D) STATE: New York (E) COUN1~Y: United States of America (F) ZIP: 10022-7513 (v) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Diskette (B) COMPUTER: IBM Compatible (C) OPERATING SYSTEM: DOS
(D) SOFTWARE: FastSEQ Version 1.5 (vi) CURRENT APPLICATION DATA:
~A) APPLICATION NUMBER:
(B) FILING DATE:
~C) CLASSIFICATION:
(vii) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER: 08/626,309 (B) FILING DATE: 01-APR-1996 4û

(viii) AllORN~Y/AGENT INFORMATION:
(A) NAME: S. PETER LUDWIG, ESQ.
(B) REGISTRATION NUMBER: 25,351 (C) REFERENCE/DOCKET NUMBER: 0342/2C488-WO
(ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: (212)527-7700 5û (B) TELEFAX: (212)753-6237 (C) TELEX:

(2) INFORMATION FOR SEQ ID NO:l:
(i) S~YU~N~ CHARACTERISTICS:
(A) LENGTH: 219 amino acids (B) TYPE: amino acid (C) STR~Nn~nN~S: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: NO
~ (iv) ANTISENSE: NO
(v) FRAGMENT TYPE: internal (vi) ORIGINAL SOURCE:
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:

CA 022~0l29 l998-09-30 W O 97l36925 PCTrUS97/06170 ~8 Met Lys Ser Ile Glu Glu Asp Glu Lys Asn Lys Ala Glu Asp Leu Asp Ile Ile Lys ~ys Glu Asp Ile Asp Glu Pro Lys Gln Glu Asp Thr Thr Asp Ser Asn Gly Gly Gly Gly Ile Gly Ile Val Pro Thr Leu Gln Asn Ile Val Ala Thr Val Asn Leu Asp Cys Arg Leu Asp Leu Lys Thr Ile Ala Leu His Ala Arg Asn Ala Glu Tyr Asn Pro Lys Arg Phe Ala Ala Val Ile Met Arg Ile Arg Asp Pro Lys Thr Thr Ala Leu Ile Phe Ala Ser Gly Lys Met Val Val Thr Gly Ala Lys Ser Glu Asp Asp Ser Lys Leu Ala Ser Arg Lys Tyr Ala Arg Ile Ile Gln Lys Leu Gly Phe Asn Ala Lys Phe Cys Asp Phe Lys Ile Gln Asn Ile Val Gly Ser Thr Asp Val Lys Phe Ala Ile Arg Leu Glu Gly Leu Ala Phe Ala His Gly Thr Phe Ser Ser Tyr Glu Pro Glu Leu Pro Pro Gly Leu Ile Tyr Arg Met Val Lys Pro Lys Ile Val Leu Leu Ile Phe Val Ser Gly Lys Ile Val Leu Thr Gly Ala Lys Lys Arg Glu Glu Ile Tyr Asp Ala Phe Glu Ser Ile Tyr Pro Val Leu Asn Glu Phe Arg Lys Asn (2) INFORMATION FOR SEQ ID NO:2:
( i ) S~UU~N~h' CHARACTERISTICS:
(A) LENGTH: 344 amino acids (B) TYPE: amino acid tC) STRA~n~n~S: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (iii) ~Y~Ol~-llCAL: NO
(iv) ANTISENSE: NO
(v) FRAGMENT TYPE: internal (vi) ORIGINAL SOURCE:
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:
Met Ser Pro Ser Thr Ser Thr Ala Val Gln Glu Tyr Ile Gly Pro Asn Leu Asn Val Thr Leu Thr Cys Pro Glu Cys Lys Ile Phe Pro Pro Asp Leu Val Glu Arg Phe Ser Glu Gly Asp Ile Val Cys Gly Ser Cys Gly Leu Val Leu Ser Asp Arg Val Val Asp Thr Arg Ser Glu Trp Arg Thr Phe Ser Asn ASp Asp Gln Asn Gly Asp Asp Pro Ser Arg Val Gly Asp Ala Gly Asn Pro Leu Leu Asp Thr Glu Asp Leu Ser Thr Met Ile Ser Tyr Ala Pro Asp Ser Thr Lys Ala Gly Arg Glu Leu Ser Arg Ala Gln Ser Lys Ser Leu Val Asp Lys Lys Asp Asn Ala Leu Ala Ala Ala Tyr Ile Lys Ile Ser Gln Met Cys Asp Gly Tyr Gln Leu Pro Lys Ile Val Ser Asp Gly Ala Lys Glu Val Tyr Lys Met Val Tyr Asp Glu Lys Pro .. , . _ . . ..

CA 022~0129 1998-09-30 W O 97/36925 PCTrUS97/06170 Leu Arg Gly Lys Ser Gln Glu Ser Ile Met Ala Ala Ser Ile Phe Ile Gly Cys Arg Lys Ala Asn Val Ala Arg Ser Phe Lys Glu Ile Trp Ala Lys Thr Asn Val Pro Arg Lys Glu Ile Gly Lys Val Phe Lys Ile Met Asp Lys Ile Ile Arg Glu Lys Asn Ala Ala Asn Pro Asn Ala Ala Tyr Tyr Gly Gln Asp Ser Ile Gln Thr Thr Gln Thr Ser Ala Glu Asp Leu Ile Arg Arg Phe Cys Ser His Leu Gly Val Asn Thr Gln Val Thr Asn Gly Ala Glu Tyr Ile Ala Arg Arg Cys Lys Glu Val Gly Val Leu Ala Gly Arg Ser Pro Thr Thr Ile Ala Ala Thr Val Ile Tyr Met Ala Ser Leu Val Phe Gly Phe Asp Leu Pro Pro Ser Lys Ile Ser Asp Lys Thr Gly Val Ser Asp Gly Thr Ile Lys Thr Ser Tyr Lys Tyr Met Tyr Glu Glu Lys Glu Gln Leu Ile Asp Pro Ser Trp Ile Glu Ser Gly Lys Val Lys Leu Glu Lys ~le Pro Lys Asn ~2) INFORMATION FOR SEQ ID NO:3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 657 base pairs (B) TYPE: nucleic acid (C) sTRA~n~s single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTISENSE: NO
(v) FRAGMENT TYPE:
(vi) ORIGINAL SOURCE:
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:

TCTTCATATG AACCAGAATT AlllCLlGGG TTAATTTATA GAATGGTGAA ACCAAAAATT 540 GTTTTACTTA TAll~L-lllC TGGGAAAATT GTTTTGACGG GTGCCAAAAA GACAGAAGAA 600 (2) INFORMATION FOR SEQ ID NO:4:
(i) SEQu~NL~ CHARACTERISTICS:
(A) LENGTH: 1095 base pairs (B) TYPE: nucleic acid (C) sTR~Nn~n~Fss (D) TOPOLOGY:

CA 022~0l29 l998-09-30 (1i) MOLECULE TYPE:
(iii~ HYPOTHETICAL: NO
(iv) ANTISENSE: NO
(v) ERAGMENT TYPE:
(vi) ORIGINAL SOURCE:
(xi) S~uU~ DESCRIPTION: SEQ ID NO:4:

ATTGGACCAA ACTTGAATGT TACATTAACA l~lC~lGAGT GTAAGATATT TCCACCTGAT 120 TTGGTAGAGA GGTTCAGCGA AGGTGACATT ~l~l~lGGCA GTTGTGGGCT AGTATTGAGT 180 GAlC~l~llG TGGATACGAG ATCAGAATGG AGAACTTTCA GTAACGATGA CCAAAATGGT 240 GATGATCCTT ClC~l~llGG TGATGCAGGT AAcc~lllAT TAGACACAGA GGACTTGTCC 300 TCTAAATCTC TAGTCGATAA AAAAGAcAAT GCATTGGCTG CAGCATATAT CAAGATTTCT 420 AAGACTAATG TACCTCGTAA GGAAATTGGT AAA~l~ll~A AGATCATGGA CAAGATCATT 660 ACCCAAACTT CGGCCGAGGA TTTGATTAGA AGA1l~l~ll CTCACTTGGG TGTTAACACA 780 GGTAGATCGC CAACTACAAT TGCTGCAACT GTAATTTACA TGGCTTCACT A~l~lllGGA 900

Claims (15)

1. A recombinant nucleic acid comprising a nucleic acid sequence encoding Candida albicans TBP.

2. A vector comprising a nucleic acid sequence encoding Candida albicans TBP.

3. A transformed host cell containing a nucleic acid sequence encoding Candida albicans TBP.

4. A recombinant polypeptide comprising Candida albicans TBP.

5. A fragment of Candida albicans TBP, said fragment being characterized in that it inhibits the biological activity of Candida albicans TBP in transcription initiation.

6. A fragment of Candida albicans TBP, said fragment being characterized in that it prevents the growth of Candida albicans.

7. A method for producing recombinant Candida albicans TBP, comprising culturing the host cell of claim 3 under conditions sufficient to permit expression of the nucleic acid encoding Candida albicans TBP, and isolating said Candida albicans TBP.

8. A screening method for identifying an inhibitor of Candida albicans growth, comprising detecting inhibition of mRNA transcription in an in vitro transcription assay comprising a DNA template, RNA polymerase II, recombinant Candida albicansTBP, and a candidate inhibitor, wherein production of an mRNA transcript from said DNA template occurs in the absence of said candidate inhibitor.

9. A screening method for identifying an inhibitor of Candida albicans growth, comprising detecting in the presence of a candidate inhibitor inhibition of formation of a complex comprising a DNA template and recombinant Candida albicans TBP, wherein in the absence of said candidate inhibitor, formation of said complex occurs.

10. A screening method for identifying an inhibitor of Candida albicans growth, comprising detecting in the presence of a candidate inhibitor inhibition of formation of a complex comprising Candida albicans TFIIB and Candida albicans TBP, wherein in the absence of said candidate inhibitor formation of said complex occurs.

11. A screening method for identifying an inhibitor of Candida albicans growth, comprising detecting in the presence of a candidate inhibitor inhibition of formation of a complex comprising RNA polymerase II, Candida albicans TBP, and Candida albicans TFIIB, wherein in the absence of said candidate inhibitor formation of said complex occurs.

12. The screening method of claim 8, 9, 10 or 11, wherein said detecting is performed in the presence of a plurality of candidate inhibitors such that said inhibition is indicative of inhibition by a said candidate inhibitor of said plurality.

13. The screening method of claim 8, 9, 10, or 11, wherein multiple detecting steps are performed simultaneously using a plurality of candidate inhibitors, wherein detection of inhibition by any one candidate inhibitor is detectable independently of said plurality.

14. A method of preventing Candida albicans growth in culture, comprising contacting said culture with an inhibitor that selectively inhibits the biological activity of Candida albicans TBP.

15. A method of preventing Candida albicans growth in a mammal, comprising administering to said mammal a therapeutically effective amount of aninhibitor that inhibits the biological activity of Candida albicans TBP.