MXPA97010239A - Anti-obesi proteins - Google Patents

Abstract

A process for the preparation of (+) - 2 amino bicyclo (3.1.0.) - hexan-2-6-dicarboxylic acid or a pharmaceutically acceptable salt thereof comprising the hydrolyzation of (-) - 2-spiro-5'- acid hydantoinbicyclo (3.1.0.) - hexan-6-carboxylic acid or a salt thereof and optionally forming a pharmaceutically acceptable salt. Intermediaries are also exposed in the process

Description

ANTIOBESITY PROTEINS FIELD OF THE INVENTION The present invention is in the field of human medicine, particularly in the treatment of obesity and disorders related to obesity. More specifically, the invention relates to anti-obesity proteins which, when administered to a patient, regulate tissues * fatty. BACKGROUND OF THE INVENTION Obesity, and especially obesity of the upper body, is a common and very serious public health problem in the United States and throughout the world. According to recent statistics, more than 25% of the population of the United States and more than 27% of the population of Canada are overweight [R. J. Kuczmarski, A er. J. of Clin. Nutr. , 55, 495S-502S (1992); B. A. Reeder et al. , Can. Med. Assoc. J., 146, 2009-1019 (1992)]. Obesity 20 of the upper body is the most important risk factor for cardiovascular diseases and cancer. The latest estimates of the medical cost of obesity are $ 150,000,000,000 worldwide. The problem has become so serious that the general surgeon has taken the initiative to combat the ever increasing adiposity in American society.

REF: 26392 Much of this pathology induced by obesity can be attributed to the intense association with dyslipidemia, hypertension and insulin resistance. Many studies have shown that reducing obesity by diet and exercise 5 surprisingly reduces these risk factors. Unfortunately, these treatments are largely unsuccessful with a failure rate that reaches 95%. This failure may be due to the fact that the condition is strongly associated with genetic inherited factors that contribute to an increased appetite, preferably for high-calorie foods, reduced physical activity and increased lipogenic metabolism. This indicates that people who inherit these genetic characteristics are prone to be obese regardless of their efforts to combat the condition. Therefore, a pharmacological agent is needed that can correct this problem of adiposity and allow the doctor to successfully treat obese patients despite their genetic inheritance. Physiologists have postulated for years that, when When a mammal is overfed, the resulting excess fat tells the brain that the body is obese, which, in turn, determines that the body eat less and burn more energy [G. R. Hervey, Nature, 222, 629-631 (1969)]. This "retroaction" model is supported by parabiotic experiments that involve a circulating hormone that controls adiposity.

The ob / ob mouse is a model of obesity and diabetes that is known to carry an autosomal recessive characteristic linked to a mutation in the sixth chromosome. Recently, Y. Zhang and colleagues published the positional cloning of mouse gene 5 linked to this condition [Y. Zhang et al. , Nature, 372, 425-432 (1994)]. This publication describes a gene encoding a 167 amino acid protein with a 21 amino acid signal peptide that is expressed exclusively in adipose tissue. Subsequently, the mouse obese gene was cloned and expressed [T. Murakami et al. , Biochem. Biophys. Res. Comm. , 209, 944-952 (1995)]. It is currently speculated that the protein, which is apparently encoded by the ob gene, is the hormone that regulates adiposity. The protein encoded by the ob gene is pharmacologically active, but the physical properties of the protein are lower than desirable. The human protein, for example, tends to precipitate and aggregate, both in a pharmaceutical formulation and in physiological conditions. Formulations of a protein that contain a precipitate, or that allow the precipitation of the protein after its injection, increase the risk of producing an immune response in the patient and can cause irritation at the site of the injection. Consequently, there remains a need to develop pharmacological agents that demonstrate pharmacological activity and that provide improved physical and chemical stability.

Applicants have discovered that the aggregation observed in the native human protein is due, in part, to hydrophobic interactions on the surface of the protein, particularly at residues 100 and 138. Applicants have further discovered that substituting these positions with charged amino acids the tendency of the ob protein to aggregate is surprisingly reduced, which provides a much improved pharmacological agent. Accordingly, the present invention provides biologically active antiobesity proteins. These agents allow patients to solve their obesity problem and lead a normal life with a more normalized risk of type 2 diabetes, cardiovascular diseases and cancer.

BRIEF DESCRIPTION OF THE INVENTION The present invention is directed to a protein of formula (I): SEQUENCE ID n ° 1 20 5 10 15 Val Pro lie Gln Lys Val Gln Asp Asp Thr Lys Thr Leu lie Lys 20 25 30 Thr lie Val Thr Arg lie Xaa Asp lie Ser His Thr Xaa Ser Val 25 35 40 45 Be Ser Lys Gln Lys Val Thr Gly Leu Asp Phe Lie Pro Gly Leu 50 55 60 His Pro lie Leu Thr Leu Ser Lys Met Asp Gln Thr Leu Ala Val 65 70 75 Tyr Gln Gln lie Leu Thr Ser Met Pro Ser Arg Xaa Xaa lie Gln 80 85 90 lie Ser Asn Asp Leu Glu Asn Leu Arg Asp Leu Leu His Val Leu 95 100 105 Wing Phe Ser Lys Ser Cys His Leu Pro Trp Wing Ser Gly Leu Glu 110 115 120 Thr Leu Asp Ser Leu Gly Gly Val Leu Glu Wing Ser Gly Tyr Ser 125 130 135 Thr Glu Val Val Ala Leu Ser Arg Leu Gln Gly Ser Leu Gln Asp 140 145 Met Leu Trp Gln Leu Asp Leu Ser Pro Gly Cys in which: Xaa from position 22 is Asn or Ser, Xaa from position 28 is Gln or is absent, Xaa from position 72 is Asn, Gln, Glu or Asp and Xaa from position 73 is Val or Met; said protein having at least one of the following substitutions: Trp of position 100 is substituted by Glu, Asp, His, Lys or Arg or Trp of position 138 is substituted by Glu, Asp, 'His, Lys or Arg; or a pharmaceutically acceptable salt thereof.

The invention further provides a method for treating obesity or conditions associated with obesity, comprising administering a protein of formula (I) to a mammal in need thereof. The invention further provides a pharmaceutical formulation comprising a protein of formula (I) together with one or more pharmaceutically acceptable diluents, carriers or excipients. The invention further provides proteins of formula (I) which additionally have a leader sequence attached to the amino terminus of the protein of formula I. Said proteins are useful for their anti-obesity activity and as intermediates in the preparation of proteins of formula (I). The invention further provides DNA encoding a protein of formula (I) or a protein of formula (I) having a leader sequence. A further embodiment of the present invention is a process for producing a protein of formula (I), which comprises: (a) transforming a host cell with DNA encoding the protein of formula (I) or a protein of formula (I) that has a leader sequence; (b) culturing the host cell under conditions that allow the expression of the protein; (c) recovering the expressed protein; and optionally (d) enzymatically cleaving the leader sequence to produce the protein of formula (I). The invention further provides a protein to be used to treat obesity or conditions associated with obesity and also a protein to be used in the manufacture of a medicament for treating obesity or conditions associated with obesity.

* Detailed description For the purposes of the present invention described and claimed, the following terms and abbreviations are defined as follows: Base pair (bp): refers to DNA or RNA. The abbreviations A, C, G and T correspond, respectively, to the 15 '5 -monophosphate forms of the nucleotides (deoxy) adenine, (deoxy) cytidine, (deoxy) guanine and (deoxy) thymine, when they appear in DNA molecules. The abbreviations U, C, G and T correspond, respectively, to the 5'-monophosphate forms of the nucleosides uracil, cytidine, guanine and thymine, when they occur in RNA molecules. In double-stranded DNA, base pair can refer to an association of A with T or of C with G. In a heteroduplex of DNA / RNA, the base pair can refer to an association of T or U with A or of C with G. 25 FMOC: abbreviation of 9-fluorenimetoxicarboni. Immunoreactive protein (s): a term used to collectively describe antibodies and antibody fragments capable of binding to antigens of a similar nature, such as the molecule of a matrix antibody from which they are derived, and single chain polypeptides they bind to molecules [E. R. Bird et al. , PCT Application No. PCT / US 87/02208, International Publication No. WO 88/01649, published March 10, 1988]. Plasmid: a self-replicating extrachromosomal genetic element. PAM: abbreviation of 4-hydroxymethylphenylacetamidomethyl. PMSF: abbreviation of phenylmethylsulfonyl fluroride. Reading frame: sequence of nucleotides from which the "read" translation takes place in triplets by the translational apparatus of the t-RNA, ribosomes and associated factors, each triplet corresponding to a particular amino acid. Since each triplet is different and of the same length, the coding sequence must be a multiple of three. An insertion or deletion of a pair of bases (termed mutation by change in the reading frame) can result in two different proteins that are encoded by the same segment of the DNA. To be sure against this, the triplet codons corresponding to the desired polypeptide must be aligned in multiples of three from the initiation codon, that is, the correct "reading frame" must be maintained. Recombinant DNA cloning vector: any * autonomously replicating agent including, but not limited to, plasmids and phages, comprising a DNA molecule to which one or more additional segments of DNA may be added or added. 5 Recombinant DNA expression vector: any recombinant DNA cloning vector in which a promoter has been incorporated. Replicon: DNA sequence that controls and allows the autonomous replication of a plasmid or another vector. 10 TFA: abbreviation of trifluoroacetic acid. Transcription: process by which information contained in a DNA nucleotide sequence is transferred to a complementary RNA sequence. Translation: process by which the genetic information of the messenger RNA is used to specify and direct the synthesis of the chain of a polypeptide. Tris: abbreviation for tris (hydroxymethyl) aminomethane. Treatment: describes the direction and care of a patient to combat a disease, condition or disorder, and includes the administration of a compound of the present invention to prevent the onset of symptoms or complications, to alleviate symptoms or complications or to eliminate the disease, condition or disorder. The treatment of obesity includes, therefore, the inhibition of food intake, the inhibition of weight gain and the induction of weight loss in patients who need it. Vector: replicon used for cell transformation by genetic manipulation that carries polynucleotide sequences corresponding to appropriate protein molecules that, when combined with appropriate control sequences, confer specific properties to the host cell to be transformed. Plasmids, viruses and bacteriophages are suitable vectors since they are replicons by themselves. Artificial vectors are constructed by cutting and joining DNA molecules from different sources using restriction enzymes and ligases. The vectors include recombinant DNA cloning vectors and recombinant DNA expression vectors. X-gal: abbreviation of 5-bromo-4-chloro-3-indolyl-jS-D-ga-lactoside. The abbreviations of amino acids are accepted by the United States Patent and Trademark Office, as indicated in 37 C.F.R. § 1,822 (b) (2) (1993). Unless indicated otherwise, the amino acids are in the L configuration. As indicated above, the present invention provides a protein of formula (I): SEQUENCE ID No. 1 10 15 Val Pro lie Gln Lys Val Gln Asp Asp Thr Lys Thr Leu lie Lys 20 25 30 Thr He Val Thr Arg He Xaa Asp He Ser His Thr Xaa Ser Val 35 40 45 Be Ser Lys Gln Lys Val Thr Gly Leu Asp Phe He Pro Gly Leu 50 55 60 His Pro He Leu Thr Leu Ser Lys Met Asp Gln Thr Leu Ala Val 65 70 75 Tyr Gln Gln He Leu Thr Ser Met Pro Ser Arg Xaa Xaa He Gln 80 85 90 He Be Asn Asp Leu Glu Asn Leu Arg Asp Leu Leu His Val Leu 95 100 105 Wing Phe Ser Lys Ser Cys His Leu Pro Trp Wing Ser Gly Leu Glu 110 115 120 Thr Leu Asp Ser Leu Gly Gly Val Leu Glu Wing Ser Gly Tyr Ser 125 130 135 Thr Glu Val Val Ala Leu Ser Arg Leu Gln Gly Ser Leu Gln Asp 140 145 Met Leu Trp Gln Leu Asp Leu Ser Pro Gly Cys in which: Xaa from position 22 is Asn or Ser, Xaa from position 28 is Gln or is absent, Xaa from position 72 is Asn, Gln, Glu or Asp and Xaa from position 73 is Val or Met; said protein having at least one of the following substitutions: Trp of position 100 is substituted by Glu, Asp, * His, Lys or Arg or Trp of position 138 is substituted by Glu, Asp, His, Lys or Arg; or a pharmaceutically acceptable salt thereof. The preferred proteins of the present invention are those of formula I wherein: Xaa from position 22 is Asn, Xaa from position 28 is Gln, > Xaa from position 72 is Asn or Asp and 10 Xaa from position 73 is Val. Other preferred proteins are those in which Trp of position 100 is substituted by Glu or Asp, or Trp of position 138 is substituted by Glu or Asp. Particularly preferred are proteins of formula I in which Xaa of position 72 is Asp. Additional preferred proteins are those in which Trp of position 100 is substituted by His, Lys or Arg. Other preferred proteins of formula I are those in which Trp of position 100 is substituted by Lys or Arg, or Trp of the position 138 is substituted by Lys or Arg. The most preferred species of the present invention include the species of sequences ID nos. 2-12: SEQUENCE ID n ° 2 25 5 10 15 Val Pro He Gln Lys Val Gln Asp Asp Thr Lys Thr Leu He Lys 20 25 30 Thr He Val Thr Arg He Asn Asp He Ser His Thr Gln Ser Val 35 40 45 Be Ser Lys Gln Lys Val Thr Gly Leu Asp Phe He Pro Gly Leu 5 50 55 60 His Pro He Leu Thr Leu Ser Lys Met Asp Gln Thr Leu Ala Val 65 70 75 Tyr Gln Gln He Leu Thr Ser Met Pro Ser Arg Asn Val He Gln 80 85 90 He As As Asp Leu Glu Asn Leu Arg Asp Leu Leu His Val Leu 95 100 105 Wing Phe Ser Lys Ser Cys His Leu Pro Asp Wing Ser Gly Leu Glu 110 115 120 Thr Leu Asp Ser Leu Gly Gly Val Leu Glu Wing Ser Gly Tyr Ser 125 130 135 Thr Glu Val Val Ala Leu Ser Arg Leu Gln Gly Ser Leu Gln Asp 140 145 Met Leu Trp Gln Leu Asp Leu Ser Pro Gly Cys SEQUENCE ID n ° 3 5 10 15 Val Pro He Gln Lys Val Gln Asp Asp Thr Lys Thr Leu He Lys 20 25 30 Thr He Val Thr Arg He Asn Asp He Ser His Thr Gln Ser Val 25 35 40 45 Be Ser Lys Gln Lys Val Thr Gly Leu Asp Phe He Pro Gly Leu 50 55 60 His Pro He Leu Thr Leu Ser Lys Met Asp Gln Thr Leu Ala Val 65 70 75 Tyr Gln Gln He Leu Thr Ser Met Pro Ser Arg Asn Val He Gln 5 80 85 90 He Be As Asp Leu Glu Asn Leu Arg Asp Leu Leu His Val Leu 95 100 105 Wing Phe Ser Lys Ser Cys His Leu Pro Glu Wing Ser Gly Leu Glu 110 115 120 Thr Leu Asp Ser Leu Gly Gly Val Leu Glu Wing Ser Gly Tyr Ser 125 130 135 Thr Glu Val Val Ala Leu Ser Arg Leu Gln Gly Ser Leu Gln Asp 140 145 Met Leu Trp Gln Leu Asp Leu Ser Pro Gly Cys 15 SEQUENCE ID n ° 4 5 10 15 Val Pro He Gln Lys Val Gln Asp Asp Thr Lys Thr Leu He Lys 20 25 30 Thr He Val Thr Arg He Asn Asp He Ser His Thr Gln Ser Val 35 40 45 Be Ser Lys Gln Lys Val Thr Gly Leu Asp Phe He Pro Gly Leu 50 55 60 His Pro He Leu Thr Leu Ser Lys Met Asp Gln Thr Leu Ala Val 25 65 70 75 Tyr Gln Gln He Leu Thr Ser Met Pro Ser Arg Asn Val He Gln 80 85 90 He Be As Asp Leu Glu Asn Leu Arg Asp Leu Leu His Val Leu 95 100 105 Wing Phe Ser Lys Ser Cys His Leu Pro Trp Wing Ser Gly Leu Glu 110 115 120 Thr Leu Asp Ser Leu Gly Gly Val Leu Glu Wing Ser Gly Tyr Ser 125 130 135 Thr Glu Val Val Ala Leu Ser Arg Leu Gln Gly Ser Leu Gln Asp 140 145 Met Leu Glu Gln Leu Asp Leu Ser Pro Gly Cys SEQUENCE ID n ° 5 5 10 15 Val Pro He Gln Lys Val Gln Asp Asp Thr, Lys Thr Leu He Lys 20 25 30 Thr He Val Thr Arg He Asn Asp He Ser His Thr Gln Ser Val 35 40 45 Be Ser Lys Gln Lys Val Thr Gly Leu Asp Phe He Pro Gly Leu 50 55 60 His Pro He Leu Thr Leu Ser Lys Met Asp Gln Thr Leu Ala Val 65 70 75 Tyr Gln Gln He Leu Thr Ser Met Pro Ser Arg Asn Val He Gln 80 85 90 He Be As Asp Leu Glu Asn Leu Arg Asp Leu Leu His Val Leu 95 100 105 Wing Phe Ser Lys Ser Cys His Leu Pro Glu Wing Ser Gly Leu Glu * 110 115 120 Thr Leu Asp Ser Leu Gly Gly Val Leu Glu Wing Ser Gly Tyr Ser 125 130 135 Thr Glu Val Val Ala Leu Ser Arg Leu Gln Gly Ser Leu Gln Asp 5 140 145 Met Leu Asp Gln Leu Asp Leu Ser Pro Gly Cys SEQUENCE ID n ° 6 5 10 15 Val Pro He Gln Lys Val Gln Asp Asp Thr Lys Thr Leu He Lys 20 25 30 Thr He Val Thr Arg He Asn Asp He Ser His Thr Gln Ser Val 15 35 40 45 Ser Ser Lys Gln Lys Val Thr Gly Leu Asp Phe He Pro Gly Leu 50 55 60 His Pro He Leu Thr Leu Ser Lys Met Asp Gln Thr Leu Wing Val 65 70 75 20 Tyr Gln Gln He Leu Thr Ser Met Pro Ser Arg Asp Val He Gln 80 85 90 He Ser Asn Asp Leu Glu Asn Leu Arg Asp Leu Leu His Val Leu 95 100 105 Wing Phe Ser Lys Ser Cys His Leu Pro Asp Wing Ser Gly Leu Glu 25 110 115 120 Thr Leu Asp Ser Leu Gly Val Leu Glu Wing Gly Tyr Ser 125 130 135 Thr Glu Val Val Ala Leu Ser Arg Leu Gln Gly Ser Leu Gln Asp 140 145 Met Leu Trp Gln Leu Asp Leu Ser Pro Gly Cys SEQUENCE ID n ° 7 5 10 15 Val Pro He Gln Lys Val Gln Asp Asp Thr Lys Thr Leu He Lys 20 25 30 Thr He Val Thr Arg He Asn Asp He Ser His Thr Gln Ser Val 35 40 45 Be Ser Lys Gln Lys Val Thr Gly Leu Asp. Phe He Pro Gly Leu 50 55 60 His Pro He Leu Thr Leu Ser Lys Met Asp Gln Thr Leu Ala Val 65 70 75 Tyr Gln Gln He Leu Thr Ser Met Pro Ser Arg Asn Val He Gln 80 85 90 He Be Asn Asp Leu Glu Asn Leu Arg Asp Leu Leu His Val Leu 95 100 105 Wing Phe Ser Lys Ser Cys Ser Leu Pro Lys Thr Ser Gly Leu Glu 110 115 120 Thr Leu Asp Ser Leu Gly Gly Val Leu Glu Wing Ser Gly Tyr Ser 125 130 135 Thr Glu Val Val Ala Leu Ser Arg Leu Gln Gly Ser Leu Gln Asp 140 145 Met Leu Trp Gln Leu Asp Leu Ser Pro Gly Cys SEQUENCE ID n ° 8 5 10 15 Val Pro He Gln Lys Val Gln Asp Asp Thr Lys Thr Leu He Lys 20 25 30 Thr He Val Thr Arg He Asn Asp He Ser His Thr Gln Ser Val 35 40 45 Be Ser Lys Gln Lys Val Thr Gly Leu Asp Phe He Pro Gly Leu 50 55 60 His Pro He Leu Thr Leu Ser Lys Met Asp Gln Thr Leu Ala Val 65 70 75 Tyr Gln Gln He Leu Thr Ser Met Pro Ser Arg Asn Val He Gln 80 85 90 He Be As Asp Leu Glu Asn Leu Arg Asp Leu Leu His Val Leu 95 100 105 Wing Phe Ser Lys Ser Cys Ser Leu Pro Trp Wing Ser Gly Leu Glu 110 115 120 Thr Leu Asp Ser Leu Gly Gly Val Leu Glu Wing Ser Gly Tyr Ser 125 130 135 Thr Glu Val Val Ala Leu Ser Arg Leu Gln Gly Ser Leu Gln Asp 140 145 Met Leu Lys Gln Leu Asp Leu Ser Pro Gly Cys SEQUENCE ID n ° 9 10 15 Met Arg Val Pro He Gln Lys Val Gln Asp Asp Thr Lys Thr Leu 20 25 30 He Lys Thr He Val Thr Arg He Asn Asp He Ser His Thr Gln 35 40 45 Be Val Be Ser Lys Gln Lys Val Thr Gly Leu Asp Phe He Pro 50 55 60 Gly Leu His Pro He Leu Thr Leu Ser Lys Met Asp Gln Thr Leu 65 70 75 Wing Val Tyr Gln Gln He Leu Thr Ser Met Pro Ser Arg Asp Val 80 85 90 He Gln He Be Asn Asp Leu Glu Asn Leu Arg Asp Leu Leu His 95 100 105 Val Leu Wing Phe Ser Lys Ser Cys His Leu Pro Glu Wing Ser Gly 110 115 120 Leu Glu Thr Leu Asp Ser Leu Gly Gly Val Leu Glu Wing Ser Gly 125 130 135 Tyr Ser Thr Glu Val Val Ala Leu Ser Arg Leu Gln Gly Ser Leu 140 145 Gln Asp Met Leu Trp Gln Leu Asp Leu Ser Pro Gly Cys SEQUENCE ID n ° 10 10 15 Met Arg Val Pro He Gln Lys Val Gln Asp Asp Thr Lys Thr Leu 20 25 30 He Lys Thr He Val Thr Arg He Asn Asp He Ser His Thr Gln 35 40 45 Be Val Be Ser Lys Gln Lys Val Thr Gly Leu Asp Phe He Pro 50 55 60 Gly Leu His Pro He Leu Thr Leu Ser Lys Met Asp Gln Thr Leu 65 70 75 Wing Val Tyr Gln Gln He Leu Thr Ser Met Pro Ser Arg Asp Val 80 85 90 He Gln He Be Asn Asp Leu Glu Asn Leu Arg Asp Leu Leu His 95 100 105 Val Leu Wing Phe Ser Lys Ser Cys His Leu Pro Asp Wing Ser Gly 110 115 120 Leu Glu Thr Leu Asp Ser Leu Gly Gly Val Leu Glu Wing Ser Gly 125 130 135 Tyr Ser Thr Glu Val Val Ala Leu Ser Arg Leu Gln Gly Ser Leu 140 145 Gln Asp Met Leu Trp Gln Leu Asp Leu Ser Pro Gly Cys SEQUENCE ID n ° 11 10 15 Met Arg Val Pro He Gln Lys Val Gln Asp Asp Thr Lys Thr Le 5 20 25 30 Be Val Be Ser Lys Gln Lys Val Thr Gly Leu Asp Phe He Pr 50 55 60 10 Gly Leu His Pro He Leu Thr Leu Ser Lys Met Asp Gln Thr Le 65 70 7 Wing Val Tyr Gln Gln He Leu- hr Ser Met Pro Ser Arg Asn V 80 85 9 He Gln He Ser Asn Asp Leu Glu Asn Leu Arg Asp Leu Leu H 15 95 100 1 Val Leu Wing Phe Ser Lys Ser Cys His Leu Pro Glu Wing Ser G 110 115 1 Leu Glu Thr Leu Asp Ser Leu Gly Gly Val Leu Glu Wing Ser G 125 130 1 20 Tyr Ser Thr Glu Val Val Ala Leu Ser Arg Leu Gln Gly Ser L 140 145 Gln Asp Met Leu Trp Gln Leu Asp Leu Ser Pro Gly Cys SEQUENCE ID n ° 12 10 15 Met Arg Val Pro He Gln Lys Val Gln Asp Asp Thr Lys Thr Leu 20 25 30 He Lys Thr He Val Thr Arg He Asn Asp He Ser His Thr Gln 35 40 45 Be Val Be Ser Lys Gln Lys Val Thr Gly Leu Asp Phe He Pro 50 55 60 Gly Leu His Pro He Leu Thr Leu Ser Lys Met Asp Gln Thr Leu 65 70 75 Wing Val Tyr Gln Gln He Leu Thr Ser Met Pro Ser Arg Asn Val 80 85 90 He Gln He Be Asn Asp Leu Glu Asn Leu Arg Asp Leu Leu His 95 100 105 Val Leu Wing Phe Ser Lys Ser Cys His Leu Pro Asp Wing Ser Gly 110 115 120 Leu Glu Thr Leu Asp Ser Leu Gly Gly Val Leu Glu Wing Ser Gly 125 130 135 Tyr Ser Thr Glu Val Val Ala Leu Ser Arg Leu Gln Gly Ser Leu 140 145 Gln Asp Met Leu Trp Gln Leu Asp Leu Ser Pro Gly Cys The present invention provides biologically active proteins that provide an effective treatment for obesity. Applicants have discovered that specific substitutions in the hydrophobic moieties on the surface of the protein result in improved properties. These remains can not be predicted from the primary sequence. Proteins that have these substitutions are pharmacologically active and have a reduced tendency to aggregate, compared to both mouse and human forms of the protein. The present invention makes it possible to more easily formulate anti-obesity proteins at higher concentrations and increase their pharmaceutical elegance because they are compatible with commonly used preservatives. The claimed proteins are usually prepared by recombinant techniques. The techniques for making substitutional mutations at predetermined sites in DNA having a known sequence are well known, for example, mutagenesis of the M13 primer. Mutations that can be made in the DNA encoding the present anti-obesity proteins should not place the sequence outside the reading frame and preferably will not create complementary regions that can produce secondary mRNA structure [H. A. DeBoer et al. , European Patent Publication No. 75,444 A2, published March 30, 1983]. The compounds of the present invention may be produced by recombinant DNA technology or by well-known chemical methods, such as synthesis of peptides in solution or in solid phase or by solution semi-synthesis starting with protein fragments coupled by conventional solution procedures.

A. Solid phase The synthesis of the claimed proteins can be produced by synthesis of solid phase peptides or by recombinant methods. The principles of chemical synthesis of solid phase polypeptides are well known in the art and can be found in general texts of the area, such as H. Dugas and C. Penney, Bioorganic Chemistry, 54-59, Springer-Verlag, New York ( 1981) . For example, peptides can be synthesized by solid-phase methodology using a peptide synthesizer PE-Applied Biosystems 433A (Perkin Elmer, Applied Biosystems Division, Foster City, California) and synthesis cycles provided by Applide Biosystems. BOC-amino acids and other reagents from PE-Applied Biosystems and other chemical suppliers can be commercially available. Sequential chemistry BOC, which uses double-coupling protocols, is applied to the starting p-methylbenzydrylamine resins for the production of C-terminal carboxamides. For the production of acids in the C terminal, the corresponding PAM resin is used. Arginine, asparagine, glutamine, histidine and methionine are coupled using preformed hydroxybenzotriazole esters. The following protection of the side chain may be used: Arg: tosyl Asp: cyclohexyl or benzyl Cys: 4-methylbenzyl Glu: cyclohexyl His: benzyloxymethyl 5 Lys: 2-chlorobenzyloxycarbonyl Met: sulfoxide Ser: benzyl Thr: benzyl Trp: formyl 10 Tyr : 4-bromocarbobenzoxy Deprotection of BOC can be achieved with trifluoroacetic acid (TFA) in methylene chloride. The formyl separation of Trp is achieved by treatment of the peptidyl resin with 20% piperidine in dimethylformamide for 15 minutes at 4 ° C. Met (O) can be reduced by treatment of the peptidyl resin with TFA / dimethyl sulfide / concentrated HCl (95/5/1) at 25 ° C for 60 minutes. After the aforementioned pretreatments, the peptides can be deprotected further and cleaved from the resin with anhydrous hydrogen fluoride containing a mixture of 10% m-cresol or m-cresol / 10% p-thiocresol or m-cresol. -cresol / p-thiocre-sol / dimethyl sulfide. Cleavage of the protecting group (s) from the side chain and the peptide from the resin is performed at 0o or less, preferably at -20 ° C for thirty minutes, followed by thirty minutes at 0 °. C. After separating the HF, the resin / peptide is washed with ether. The peptide is extracted with glacial acetic acid and lyophilized. Purification is achieved by reverse phase C18 chromatography in 0.1% TFA with a gradient of increasing concentration of acetonitrile, for example, on a 2.2 cm x 25 cm Vydac® column (The Separations Group Inc., Hesperia , CA). One skilled in the art should recognize that solid phase synthesis can also be achieved using the FMOC strategy and a TFA / captor excision mixture.

B. Recombinant synthesis The claimed proteins can also be produced by recombinant methods. Recombinant methods are preferred if a high yield is desired. The basic steps of the recombinant production of proteins include: a) construction of a synthetic or semi-synthetic DNA (or isolation from natural sources) that encodes the claimed protein, b) integrating the coding sequence into an expression vector in a suitable manner for the expression of the protein, alone or as a fusion protein c) transforming a eukaryotic or prokaryotic host cell with the expression vector and d) recovering and purifying the recombinantly produced protein. to. Gene building Synthetic genes can be constructed, whose in vitro or in vivo translation will lead to the production of the protein, by procedures well known in the art. Because of the natural degeneracy of the genetic code, the skilled artisan must recognize that a considerable but definite number of DNA sequences encoding the claimed proteins can be constructed. In the preferred practice of the invention, the synthesis is achieved by recombinant DNA technology. The methodology of synthetic gene construction is well known in the art [E. L. Brown et al. , Methods in Enzymology, 68, 109-151, Academic Press, New York, NY (1979)]. The DNA sequence corresponding to the claimed synthetic protein can be generated using a conventional DNA synthesis apparatus, such as the Applied Biosystems DNA Synthesizers models 380A or 380B (Perkin Elmer, Applied Biosystems Division, Foster City, California). In some applications it may be desirable to modify the coding sequence of the claimed protein to incorporate a convenient protease sensitive cleavage site, for example, between the signal peptide and the structural protein that facilitates controlled cleavage of the fusion protein signal peptide. built The gene encoding the claimed protein can also be created using a polymerase chain reaction (PCR) The template can be a cDNA library (commercially available from CLONETECH or STRATAGENE) or mRNA isolated from human adipose tissue. Said methodologies are well known in the art. See, for example, T. Maniatis et al. , Molecular Cloning: A Laboratory Manual, 2nd edition, Cold Spring Harbor Press, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York (1989). b. Direct Expression or Fusion Protein The claimed protein can be made by direct expression or as a fusion protein comprising the claimed protein, followed by enzymatic or chemical cleavage. A variety of peptidases (e.g., trypsin) that cleave a polypeptide at specific sites or that digest peptides from the amino or carboxy termini (e.g., diaminopeptidase) of the peptide chain are known. In addition, particular chemicals (eg, cyanogen bromide) can cleave the chain of a polypeptide at specific sites. The skilled artisan should appreciate the necessary modifications so that the amino acid sequence (and the synthetic or semisynthetic coding sequence, if recombinant means are employed) incorporates specific internal cleavage sites. See, for example, P. Cárter, chapter 13 of Protein Pur i f ication: From Molecular Mechanisms to Large Scale Processes, M. Ladisch et al. , (Ed.) American Chemical Soc., Washington, D.C. (1990). c. Construction of vectors The construction of suitable vectors containing the desired coding and control sequences employ standard ligation techniques. Isolated plasmids or DNA fragments are cleaved, adapted and religated in the desired form to form the required plasmids. To perform the translation of the desired protein, the projected synthetic DNA sequence is inserted into any one of a plethora of appropriate recombinant DNA expression vectors by the use of appropriate restriction endonucleases. A coding sequence is designed Synthetic to have cleavage sites for restriction endonucleases at either end of the transcript to facilitate isolation and integration into these expression and dextran plasmids. amplification and expression. The coding sequence of isolated cDNA can be modified easily using synthetic linkers to facilitate the incorporation of this sequence into the desired cloning vectors by methods well known in the art. The particular endonucleases employed will be governed by the model of excision of the expression vector matrix to be used. Restriction sites are chosen to appropriately orient the coding sequence with control sequences to achieve adequate reading and expression within the framework of the claimed protein. In general, vectors are used with these guests plasmids containing promoters and control sequences that are derived from species compatible with the host cell.

The vector ordinarily carries a replication site as well as marker sequences which are capable of providing a phenotypic selection in transformed cells. For example, -E. coli is typically transformed using pBR322, a plasmid 5 derived from an E. coli species [F. Bolivar et al. , Gene, 2, 95-113 (1977)]. Plasmid pBR322 contains genes for resistance to ampicillin and tetracycline and thus provides a means to identify transformed cells. Plasmid pBR322 or another microbial plasmid must also contain, or must be modified to contain, promoters or other control elements commonly used in recombinant DNA technology. The desired coding sequence is inserted into an expression vector in the appropriate orientation to be transcribed from a promoter and ribosomal binding site, both of which must be functional in the host cell in which the protein is to be expressed. An example of such an expression vector is a plasmid described in U.S. Patent No. 5,304,473, issued April 19, 1994. to R. M. Belagaje et al. , the description of which is incorporated herein by reference. The gene encoding proinsulin A-C-B, described in U.S. Patent No. 5,304,473, can be separated from plasmid pRB182 with the restriction enzymes Ndel and Ba Hl. The genes that code The protein of the present invention can be inserted into the plasmid scaffold in a cassette of Ndel / BamHl restriction fragments. d. Prokaryotic expression In general, prokaryotes are used to clone DNA sequences to construct the vectors useful in the invention. For example, E. coli K12 strain 294 (ATCC No. 31446) is particularly useful. Other microbial strains that can be used include E. coli B and E. coli X1776 (ATCC No. 31537). These examples are illustrative but not limiting. Prokaryotes are also used for expression. The aforementioned strains can be used, as well as E. coli W3110 (prototrophic, ATCC No. 27325), bacilli such as Bacillus subti-lis, and other enterobacteriaceae such as Salmonella typhimurium and Serratia marcescans, and various species of Pseudomonas. Promoters suitable for use with prokaryotic hosts include / 3-lactamase [vector pGX2907 (ATTC 39344) contains the replicon and the 3-lactamase gene] f and lactose promoter systems [A. C. Y. Chang et al. , Nature, 275, 617-624 (1978); and D. V. Goeddel et al. , Nature, 281, 544-548 (1979)], alkaline phosphatase, the tryptophan (trp) promoter system [the pATHl vector (ATCC 37695) is designed to facilitate the expression of an open reading frame, in the form of a trpE fusion protein, under control of the trp promoter] and hybrid promoters such as the tac promoter [isolable from plasmid pDR540 (ATCC 37282)]. However, other functional promoters in bacteria, whose nucleotide sequences are generally known, allow a person skilled in the art to bind them to the DNA encoding the protein using linkers or adapters to deliver any required restriction sites. The promoters to be used in bacterial systems must also contain a Shine-Dalgarno sequence operably linked to the DNA encoding the protein. and. Eukaryotic expression Protein can be produced recombinantly in eukaryotic expression systems. Preferred promoters that control transcription in mammalian host cells can be obtained from various sources, for example, from virus genomes [such as polyoma, simian virus 40 (SV40), adenovirus, retrovirus, hepatitis B virus and, most preferably, cytomegalovirus] and of heterologous mammalian promoters (e.g., / 3-actin promoter). The first and last promoters of the SV40 virus are conveniently obtained as an SV40 restriction fragment that also contains the SV40 viral origin of replication [W. Fiers et al. , Nature, 273, 113-120 (1978)]. The complete SV40 20 genome can be obtained from the pBRSV plasmid (ATCC 45019). The immediate first promoter of the human cytomegalovirus can be obtained from the plasmid pCMB / 3 (ATCC 77177). Of course, promoters derived from the host cell or related species are also useful. The transcription of a DNA encoding the protein claimed by higher eukaryotes is increased by inserting an enhancer sequence into the vector. Enhancers are cis-acting elements of DNA, usually about 10-300 base pairs, that act on a promoter to increase its transcription. The intensifiers are relatively independent of orientation and position, having been found 5 '[L. A. Laimins et al. , Proc. Nat 'l Acad. Sci. (USA), 78, 464-468 (1981)] and 3 '[M. Lusky et al. , Mol. Cell. Bio. , 3, 1108-1122 (1983)] to the transcription unit, inside an intron [J. Banerj i et al. , Cell, 33, 729-740 (1983)] as well as within the coding sequence itself [T. F. Osborne et al., Mol. Cell. Bio. , 4, 1293-1305 (1984)]. Many intensifier sequences from mammalian genes are currently known (globin, RSV, SV40, EMC, elastase, albumin, α-fetoprotein and insulin). However, an enhancer from a virus of a eukaryotic cell will typically be used. Examples include the late enhancer of SV40, the cytomegalovirus early promoter enhancer, the polyoma enhancer on the late region of the origin of replication and enhancing adenoviruses. Expression vectors in eukaryotic host cells (from yeasts, fungi, insects, plants, animals, humans or nucleated cells from other multicellular organisms) will also contain sequences necessary for the termination of transcription that can affect mRNA expression. These regions are transcribed as polyadenylated segments in the untranslated portion of the mRNA encoding the protein. Untranslated regions 31 also include transcription termination sites. The expression vectors may contain a selection gene, also called a selectable marker. Examples of selectable markers suitable for mammalian cells are dihydrofolate reductase [(DHFR), which can be derived from the BglII / HindIII restriction fragment from pJOD-10 (ATCC 68815)] and thymidine kinase [thymidine kinase from herpes simplex virus is contained in the BamH1 fragment of clone vP-5 (ATCC 2028)] or neomycin resistance genes (G418), which can be obtained from the yeast artificial chromosome vector pNN414 (ATCC 37682)]. When said selectable markers are successfully transferred to a mammalian host cell, the transfected mammalian host cell can survive if placed at a selective pressure. There are two distinct categories, widely used, of selective regimes. The first category is based on the metabolism of a cell and the use of a mutant cell line that lacks the capacity to develop without a supplemented medium. Two examples are: CHO DHFR 'cells (ATCC CRL-9096) and mouse LTKT cells [LM (TK ") ATCC CCL-2.3.] These cells lack the ability to develop without adding nutrients such as thymidine or hypoxanthine. lacking certain genes necessary for a complete nucleotide synthesis pathway, they can not survive unless the missing nucleotides are supplied in a supplemented medium.An alternative to supplement the medium is to introduce an intact DHFR or TK gene into cells lacking The individual genes that have not been transformed with the DHFR or TK gene will not be able to survive in an unsupplemented medium.The second category is the dominant selection, which refers to a selection scheme used in any type of cell and that does not require the use of a mutant cell line.These schemes typically use a drug to stop er the development of a host cell. These cells that have a new gene will express a protein that carries resistance to the drug and will survive selection. Examples of such dominant selection use the drugs neomycin [P. J. Southern et al. , J. Molec. Appl. Genet , 1, 327-341 (1982)], mycophenolic acid [R. C. Mulligan et al. , Sci ence, 209, 1,422-1,427 (1980)] or hygromycin [B. Sugden 20 et al. , Mol. Cell. Biol. , 5, 410-413 (1985)]. The three examples given above employ bacterial genes under eukaryotic control to bring resistance to the appropriate drug, such as G418, neomycin (geneticin), xgpt (mycophenolic acid) or hygromycin, respectively. A preferred vector for eukaryotic expression is pRc / CMV. The pRc / CMV vector is commercially available from Invitrogen Corporation, San Diego, CA. To confirm correct sequences in constructed plasmids, ligation mixtures are used to transform E. coli K12 strain DH5a (ATCC 31446) and the transformants achieved are selected for antibiotic resistance, when appropriate. Plasmids from the transformants are prepared, analyzed by restriction and / or sequenced by the method of J. Messing et al. , Nucleic Acids Res. , 9, 309-321 (1981). Host cells can be transformed with the expression vectors of this invention and can be cultured in conventional nutrient media modified as appropriate to induce promoters, select transformants or amplify genes. The culture conditions, co or temperature, pH and Similar conditions are those previously used with the host cell selected for expression, and will be apparent to the ordinary skilled artisan. The methods of transforming cells with the aforementioned vectors are well known in the art and can be found in general references, such as T. Maniatis et al. , Molecular Cloning: A Laboratory Manual, 2nd edition, Cold Spring Harbor Press, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York (1989), or Current Protocols in Molecular Biology (1989) and supplements. Suitable preferred host cells for expressing the vectors encoding the claimed proteins in higher eukaryotes include: African green monkey kidney cell line transformed by SV40 (COS-7, ATCCCRL-1651); line 293 of transformed human primary embryonic kidney cells [F. L. Graham et al. , J. Gen. Virol. , 36, 59-72 (1977); T. Harrison et al. , Virology, 77, 319-329 (1977); F. L. Graham et al. , Virology, 86, 10-21 (1978)]; small hamster kidney cells [BHK-21 (C-13), ATCCCCL-10; I. MacPherson et al. , Virology, 16, 147-151 (1962)]; Chinese hamster ovary cells [CHO-DHFR "(ATCCCRL-9096)], mouse Sertoli cells [TM4, ATCC CRL-1715, JP Mather, Biol. Reprod., 23, 243-252 (1980)]; African green monkey kidney (VERO 76, ATCC CRL-1587), human cervical epithelial carcinoma cells (HeLa, ATCC CCL-2), canine kidney cells (MDCK, ATCC CCL-34), buffalo rat liver cells (BRL 3A, ATCC CRL-1442), human lung diploid cells (WI-38, ATCC CCL-75), human hepatocellular carcinoma cells (Hep G2, ATCC HB-8065), and mouse breast tumor cells ( MMT 060562, ATCC CCL-51) f Expression in yeast In addition to mammalian and prokaryotic host cells, eukaryotic microorganisms, such as a yeast, can also be used as host cells Saccharomyces cerevisiae, the common bread yeast, is the microorganism eukaryotic most commonly used to express heterologous proteins, although a series is commonly available of other strains For example, for expression in Saccharomyces plasmid YRp7 is commonly used [ATCC-40053, D. T. Stinchcomb et al. , Nature, 282, 39-43 (1979); A. J. Kingsman et al. , Gene, 7, 141-152 (1979); G. Tschumper et al. , Gene, 10, 157-166 (1980)]. This plasmid already contains the trp gene which provides a selection marker for a strain of a mutant of a yeast lacking the ability to grow in tryptophan [eg, ATCC 44076 or PEP4-1; E. W. Jones, Genetics, 85, 23-33 (1977)]. Suitable promoter sequences for use with a host yeast include the promoters for 3-phosphoglycer-to-kinase, which is found in plasmid pAP12BD (ATCC 53231) (U.S. Patent No. 4,935,350, issued January 19, 1990 to AC Patel et al.) Or other glycolytic enzymes, such as enolase, which is found in plasmid pACl (ATCC 39532), glyceraldehyde-3-phosphate dehydrogenase, which is derived from the plasmid pHcGAPCl (ATCC 57090), zimomonas mobilis (patent of the United States No. 5,000,000, granted on March 19, 1991 to LO Ingram et al.), hexoquine-sa, pyruvate decarboxylase, phosphofructokinase, glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvate kinase, triose phosphate isomerase, phosphoglucose isomerase and glucoquina-sa. Other promoters of yeasts, which are inducible promoters that have the additional advantage of transcription controlled by the conditions of development, are the promoter regions for alcohol dehydrogenase 2, isocythochrome C, acid phosphatase, degrading enzymes associated with nitrogen metabolism, metallothionein, which is contained in the plasmid vector pCL28XhoLHBPV (ATCC 39475, U.S. Patent No. 4,840,896, issued June 20, 1989 to VB Reddy et al.), glyceraldehyde-3-phosphate dehydrogenase and enzymes responsible of the use of maltose and galactose, such as the GAL1 promoter, which can be found in plasmid pRY121 (ATCC 37658). Vectors and promoters suitable for use in expression in a Yeast are further described in European Patent Publication No. 73,657 Al, by R. A. Hitzeman et al. , published March 9, 1983. They are also used with promoters of yeast-enhancing yeasts, such as the UAS Gal of Saccharomyces cerevisiae, which is found together with the CYCl promoter in the plasmid YEpsec-hIIbeta (ATCC 67024).

The following examples are presented to further illustrate the preparation of the claimed proteins. The scope of the present invention should not be construed simply as the following examples.

Example 1 Construction of vector A sequence gene ID No. 13 is constructed from a segment of -220 base pairs and a segment of -240 base pairs, which are derived from chemically synthesized oligonucleotides. (SEQUENCE ID n ° 13) 1 CATATGAGGG TACCTATCCA AAAAGTACAA GATCACACCA AAACACTGAT 51 AAAGACAATA GTCACAAGGA TAAATGATAT CTCACACACA CAGTCAGTCT 101 CATCTAAACA GAAAGTCACA GGCTTGGACT TCATACCTGG GCTGCACCCC 151 ATACTGACAT TGTCTAAAAT GCACCAGACA CTGGCAGTCT ATCAACAGAT 201 CTTAACAAGT ATGCCTTCTA GAAACGTGAT ACAAATATCT AACGACCTGG 251 AGAACCTGCG GGATCTGCTG CACGTGCTGG CCTTCTCTAA AAGTTGCCAC 301 TTGCCATGGG CCAGTGGCCT GGAGACATTG GACAGTCTGG GGGGAGTCCT 351 GGAAGCCTCA GGCTATTCTA CAGAGGTGGT GGCCCTGAGC AGGCTGCAGG 401 GTCTCTGCA AGACATGCTG TGGCAGCTGG ACCTGAGCCC CGGGTGCTAA 451 TAGGATCC The 220 base pair segment extends from the Ndel site to the Xbal site at position 220 in the coding region and is constructed from 7 overlapping oligonucleotides, whose lengths vary between 34 and 83 bases. The 240-base pair segment extending from the Xbal site to the BamH1 site is also constructed from 7 overlapping oligonucleotides, whose lengths vary between 57 and 92 bases. To construct these fragments, the respective 7 nucleotides are mixed in equimolar amounts, usually at concentrations of about 1-2 picomoles per microliter. Prior to construction, almost all nucleotides at the 5 'ends of the segment are phosphorylated in standard kinase buffer with T4 DNA kinase using the conditions specified by the reagent supplier. The mixtures are heated to 95 ° C and allowed to cool slowly to room temperature over a period of 1-2 hours to ensure proper reassociation of the oligonucleotides. The oligonucleotides are then ligated together and into an appropriate cloning vector, such as pUC18 or pUC 19, using T4 DNA ligase. The buffers and conditions are those recommended by the supplier of the enzyme. Before use, the vector for the 220 base pair fragment is digested with Ndel and Xbal, while the vector for the 240 base pair fragment is digested with Xbal and BamHl. The ligation mixtures are used to transform E. coli DH10B cells (commercially available from Life Technologies, producer of GIBCO / BRL products, Grand Island, NY) and the transformed cells are plated on tryptone-yeast plates (TY, Difco, Detroit, MI) containing 100 μg / ml of ampicillin, X-gal and IPTG. Colonies that develop overnight are grown in TY liquid medium with 100 μg / ml ampicillin and are used for plasmid isolation and for DNA sequence analysis. The plasmids with the correct sequence are saved for the construction of the complete gene. This is done by gel purification of the 220 base pair and 240 base pair fragments, and ligation of these two fragments into an expression vector, such as pRB182, from which the sequence encoding proinsulin ACB and is digested with Ndel and BamHl, before using them. Alternatively, plasmid pET30 (Novagen, Madison, Wl) can be digested with Ndel and BamHI, and the desired DNA sequence encoding the proteins of the present invention can be inserted by art recognized procedures described herein. The source of DNA are synthetic oligonucleotides that are constructed by recognized methodology and described here.

Example 2 In a manner analogous to that of Example 1, plasmid pOJ722 (NRRL No. B-21,654), which contains the DNA sequence coding for the desired protein, was prepared. The plasmid was digested with Pmll and Bsu36I. The recognition sequences for these enzymes are in the region encoding the protein at positions 275 and 360 of the nucleotide, respectively. The cloning vector does not contain these recognition sequences. Consequently, only two fragments were seen after digestion of the restriction enzyme with Pmll and Bsu36I, one corresponding to the fragment of the vector and the other corresponding to the fragment of -85 base pairs released from the sequence encoding the protein. . This sequence was replaced by any DNA sequence encoding the amino acid substitutions of the present invention. These DNA sequences were synthesized chemically in the form of two oligonucleotides with complementary bases and ends that are compatible with the ends generated by digestion with P-mlJ and Bsr36I. The chemically synthesized oligonucleotides were mixed in equimolar amounts (1-10 picomoles / microliter), heated to 95 ° C and allowed to anneal, slowly lowering the temperature to 20-25 ° C. The reassociated oligonucleotides were used in a standard ligation reaction. The ligation products were transformed into E. coli DH10B cells (GIBCO BRL) and the transformed cells were plated on TY plates containing 10 μg / ml tetracycline (Sigma, St. Louis, MO). Colonies that developed overnight were grown in TY liquid medium with 10 μg / ml tetracycline and used for plasmid isolation and for DNA sequence analysis. The plasmids with the correct sequence were stored. Example 3 Expression Plasmid pHS787 (Sequence Protein ID No. 6) The vector pHS692, prepared in a manner analogous to that of Example 1, (40 μg) was digested with 20 units of P-mlJ (New England Biolab, Beverly, MA) in "buffer 1 of New England Biolab", at 37 ° C for two hours. The buffer salts were adjusted to "buffer 3 of New England Biolab" and 20 units of BstXI (New England Biolab) were added to initiate digestion, which lasted for 2 hours at 55 ° C. The digest was treated with 20 units of alkaline phosphatase (Boehringer Mannheim, Indianapolis, IN) at 37 ° C for 30 minutes. The resulting digest was purified on a 1% agarose gel and the 4,170 base pair fragment was isolated using the freeze-compression procedure. Oligonucleotide 1.3824 (5 '-TGA GGC TTC CAG GAC TCC CCC CAG ACT GTC CAA TGT CTC CAG GCC ACT GGC GTC TGG CAA GTG GCA ACT TTT AGA GAA GGC CAG CAC-3') (sequence ID No. 14) and the oligonucleotide 13825 (5 '-GTG CTG GCC TTC TCT AAA AGT TGC CAC TTG CCA GAC GCC AGT GGC CTG GAG ACA TTG GAC AGT CTG GGG GGA GTC CTG GAA GCC-3') (sequence ID No. 15) were kinased in the presence of buffer of kinase lx, 50mM Tris-HCl pH 8.0, MgCl2 lOmM, 0.5mM ATP, lmM DTT and 5 units of T4 polynucleotide kinase (GIBCO BRL) at 37 ° C for 30 minutes. Pmll / BstXI linearized the pHS692 vector, and oligonucleotide 13824 and linker 13825 were ligated in the presence of lx kinase buffer, 0.5mM ATP and 1 unit of T4 DNA ligase (GIBCO BRL) at 16 ° C overnight. The ligation products are transformed into E. coli BL21 (DE3) (NOVAGEN) and colonies that grow on TY plates supplemented with 10 μg / ml tetracycline are analyzed. The DNA is isolated from the plasmid and then subjected to DNA sequencing in a 370 DNA sequencer, from PE-Applied Biosystems. The plasmids containing the expected fragment Ndel to BamHI of -400 base pairs are saved and are designated pHS787.

Example 4 Expression Plasmid (Sequence Protein ID No. 3) Plasmid pOJ722 was digested with Pmll and Bsu36I. A synthetic DNA fragment sequence [5'-Sequence ID No. 16] (Sequence ID No. 16) TGAGGCTTCCAGGACTCCCCCCAGACTGTCCAATGTCTCCAGGCCACTGGCGTCTGGCAA GTGGCAACTTTTAGAGAAGGCCAGCAC annealed with the sequence [5'-Sequence ID No. 17] (sequence ID No 17) is GTGCTGGCCTTCTCTAAAAGTTGCCACTTGCCAGACGCCAGTGGCCTGGAGACATTGGAC AGTCTGGGGGGAGTCCTGGAAGCC inserted between the Pmll site and the Bsu36I site. After the ligation, transformation and isolation of the plasmid, the sequence of the synthetic fragment was verified by analysis of the DNA sequence. The methods of transforming cells with the aforementioned vectors are well known in the art [T. Maniatis et al. , Molecular Cloning: A Laboratory Manual, 2nd edition, Cold Spring Harbor Press, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York (1988); Id., Current Protocols in Molecular Biology (1989), and supplements thereof]. The methods involved in the transformation of E. coli cells into the preferred practice of the invention, exemplified herein, are well known in the art. The exact conditions in which the transformed E. coli cells are cultured depend on the nature of the host cell line of E. coli and on the expression or cloning vectors employed. For example, vectors incorporating thermoinducible promoter-operator regions, such as the thermo-inducible region cl857 of the promoter-operator of phage lambda, require a temperature range of about 30 ° C to about 40 ° C under culture conditions, to induce the synthesis of proteins. In the preferred embodiment of the invention, E. coli K12 RV308 cells are used as host cells, but numerous other cell lines are available, such as, but not limited to, E. coli K12 L201, L687, L693, L507, L640 , L641, L695, L814 (E. coli B). The transformed host cells are then plated with an appropriate medium at the selective pressure of the antibiotic corresponding to the resistance gene present in the expression plasmid. The cultures are then incubated for a time and at a temperature appropriate to the line of host cells used. Proteins that are expressed in expression systems rich in bacteria are typically added in granules or inclusion bodies containing high levels of the overexpressed protein [J. K. Kreuger et al. , Protein Folding, publication n ° 89-18S of the American Association for the Advancement of Science, Washington, D.C. 136-142 (1990)]. Said protein aggregates must be dissolved to provide additional purification and isolation of the desired protein (J. K. Kreuger et al., Supra). A variety of techniques are used to solubilize the proteins employing strongly denaturing solutions, such as guanidinium hydrochloride, and / or weakly denaturing solutions, such as urea. The gradual separation of the denaturing agents (often by dialysis) in a solution allows the denatured proteins to assume their natural conformation. The particular conditions of denaturation and folding are determined by the particular protein expression system and by the protein in question. Preferably, the present proteins are expressed with a leader secondary. One of ordinary skill in the art should recognize that one can work with numerous leader sequences; however, preferably the leader sequence is Met-Rt, where Rx is any amino acid except Pro or is absent, whereby the expressed proteins can be easily converted to the claimed protein with cathepsin C or with other suitable aminopeptidases. Preferably, Rx is Arg, Asp or Tyr and, most preferably, the proteins are expressed with a Met-Arg leader sequence. Interestingly, the leader sequence does not significantly affect the stability or activity of the protein. However, preferably the leader sequence is cleaved from the protein. Thus, proteins of formula [Met-Rj-sequence ID No. 1] are useful as biological agents and, preferably, as intermediates.

Example 5 The sequence protein ID No. 13, with a Met-Arg leader sequence, was expressed in E. coli, isolated and folded by techniques analogous to those of the previous examples. The pH of the protein solution was reduced to 2.8. The Met-Arg leader sequence was excised by the addition of 6-10 milliunits of dDAP per milligram of protein (dDAP is the abbreviation of a dipeptidylaminopeptidase isolated from the viscous mold Dicteostelium descoidium, described in U.S. Patent No. 5,565,330, granted on October 15, 1996 to PR Atkinson et al.). The conversion reaction was allowed to proceed for 2-8 hours at room temperature. The progress of the reaction was followed by high-resolution reverse phase chromatography. The reaction was terminated by adjusting the pH to 8 with NaOH. The des (Met-Arg) protein was further purified by cation exchange chromatography in the presence of 7-8M urea and by gel permeation chromatography in PBS. After the final purification of the proteins by gel permeation chromatography, the proteins were concentrated at 3-3.5 mg / ml in PBS. The purification of the claimed proteins is by methods known in the art and includes reverse phase chromatography, affinity chromatography, ion exchange chromatography and gel permeation chromatography.

The claimed proteins contain two cysteine residues. Therefore, a disulfide bond can be formed that stabilizes the protein. The present invention includes proteins of formula (I) in which Cys of position 96 is cross-linked to Cys of position 146 as well as proteins without said disulfide bonds. Preferably, Cys of position 96 is linked by a disulfide bond to Cys of position 146. In addition, the proteins of the present invention can exist, particularly when in a formulation, in the form of dimers, trimers, tetramers and other multimers. Said multimers are included in the scope of the present invention. The present invention provides a method for treating obesity. The method comprises administering to the organism an effective amount of an anti-obesity protein, in a dose of between about 1 and 1,000 μg / kg. A preferred dose is from about 10 to 100 μg of active compound / kg. A typical daily dose for an adult person is about 0.5 to 100 mg. In the practice of this method, the compounds of formula (I) can be administered in a single daily dose or in several doses per day. The treatment regimen may require administration for extended periods of time. The amount per dose administered or the total amount administered will be determined by the physician and depends on factors such as the nature and severity of the disease, age and general health of the patient and tolerance of the patient to the compound. The present invention further provides pharmaceutical formulations comprising compounds of the present invention. The proteins, preferably in the form of a pharmaceutically acceptable salt, can be formulated for parenteral administration for the therapeutic or prophylactic treatment of obesity. For example, compounds of formula (I) can be mixed with conventional pharmaceutical carriers and excipients. The compositions comprising the claimed proteins contain from about 0.1 to 95% by weight of the active protein, preferably in soluble form, and more generally from about 10 to 30%. In addition, the present proteins can be administered alone or in combination with other anti-obesity agents or with agents useful in the treatment of diabetes. For intravenous use, the protein is administered in commonly used fluid (s) and is administered by infusion. As such fluids, for example, physiological saline, Ringer's solution or 5% dextrose solution can be used. For intramuscular preparations, a sterile formulation, preferably a suitable soluble salt of a protein of formula (I), for example, the hydrochloride salt, can be dissolved and administered in a pharmaceutical diluent, such as pyrogen-free (distilled) water, physiological saline solution or of glucose at 5%. An insoluble form of the compound can be prepared and administered as a suspension in an aqueous base or in a pharmaceutically acceptable oily base, for example, an ester of a long-chain fatty acid, such as ethyl oleate. Preferably, pharmaceutically acceptable preservatives, such as an alkyl paraben (particularly methyl paraben, ethyl parabene, propyl paraben or butyl paraben) or chlorobutanol, are added to the formulation to allow various doses to be used. Significantly, the claimed proteins are also stable in the presence of a phenolic preservative, such as m-cresol or phenol. The stability of the proteins in the presence of a phenolic preservative offers advantages in the pharmaceutical contribution, including an improved preservation efficiency. Preferably the formulation is prepared in the absence of salts to minimize the ionic strength of the formulation. The ability of the present compounds to treat obesity is demonstrated in vivo, as follows.

Biological assays Parabiotic experiments suggest that a protein is released by peripheral adipose tissue and that the protein is able to control body weight gain in normal mice as well as in obese mice [D. L. Coleman, Diabeto-logia, 14, 141-148 (1978)]. Therefore the most intimately related biological assay is to inject the test article by any of the various routes of administration, for example, intravenous (iv), subcutaneous (se), intraperitoneal (ip) or by a minipump or cannula, and follow then the consumption of water and food, gain of body weight, chemistry or plasma hormones (glucose, insulin, ACTH, corticosterone, GH, T4) in various periods of time. Suitable test animals include normal mice (ICT, etc.) and obese mice (ob / ob, Acy / a, KK-Ay, tubby, fatty). The ob / ob mouse model of obesity and diabetes is generally accepted as indicative of the obesity condition. In the same animal controls are made of non-specific effects of these injections using a vehicle with or without the active agent of similar composition, following the same parameters or the active agent itself in animals that are believed to lack the receptor (db / db mice, fa / fa or rats cp / cp). Proteins that demonstrate activity in these models will demonstrate similar activity in other mammals, particularly in humans. Since it is assumed that the target tissue is the hypothalamus, where food intake and lipogenic status are regulated, a similar model is to inject the test article directly into the brain, for example, by injection into the lateral or third ventricle ( icv) or directly in specific hypothalamic nuclei, such as the arcuate, paraventricular or perifornical nucleus. The same parameters as mentioned above can be measured or the release of neurotransmitters which are known to regulate diet or metabolism can be monitored (for example, the release of NPY, galanin, norepinephrine, dopamine and / 3-endorphin). The representative proteins indicated in Examples 6 and 7 were prepared, according to the description and examples provided herein. The designation Met-Arg- indicates that the protein was prepared and tested with a Met-Arg sequence attached thereto. The amino acid sequences of the proteins of the examples were confirmed by mass spectroscopy and / or by amino acid analysis. When tested, the proteins were folded with the Cys residues joined transversely by a disulfide bond. Tables 1-3 show the ability of the present proteins to treat obesity in an ob / ob mouse. Similar studies were performed in vitro using isolated hypothalamic tissue in a tissue permeation or perfusion system. In In this situation, the release of neurotransmitters or electrophysiological changes is followed.

Example 6 Protein assay Ob of sequence ID nos. 2, 3 and 6 20 in male ob / ob mice. Male ob / ob mice [Harian Ltd., Blackthorn, England) were housed in groups of 5 animals each and were given Purina 5008 food and water ad libi tum. The mice were kept in a reverse lighting regime (the 25 lights went out at 9.00 in the morning and turned on at 9.00 at night). The mice were weighed daily at 8.30 in the morning. At the same time their consumption of water and food was determined. The treatment, indicated below, was made after weighing in the morning, just before turning off the lights. The mice were treated 5 times a day for 4 days.

Tables 1-3 illustrate the effects of these treatments on feed intake and cumulative body weight change for representative proteins of the present invention.

Table 1 Effect of sequence ID No. 2 on feed intake and cumulative body weight change in ob / ob mice Table 2 Effect of sequence ID No. 3 on feed intake and cumulative body weight change in ob / ob mice Table 3 Effect of sequence ID No. 6 on feed intake and cumulative change in body weight in mice or / ob Group 1: control group (PBS) Group 2: 30 μg / day of protein Group 3: 100 μg / day of protein Example 7 Analysis of aggregation by dynamic diffusion of light The physical properties of the present compounds are demonstrated as follows. Based on the availability of material, the solution to be analyzed by dynamic light diffusion (DLS) was prepared in one of two ways. The material was supplied in solution from the last purification step by gel permeation chromatography in PBS at approximately 1.5 mg / ml and concentrated to 3-5 mg / ml by diafiltration or dialyzed against water, lyophilized and reconstituted at 3-5 mg / ml. In the first procedure, the protein solution at about 1.5 mg / ml was concentrated to more than 3-5 mg / ml in a small volume shaking cell (10 ml) using a 25 mm Amicon YM10 membrane. This was done in cold environmental conditions (at approximately 5 ° C). The concentration of the protein was determined by absorption of ultraviolet radiation and the solution was diluted to 3.0 mg / ml with PBS (dilution to 10 times its volume with PBS lOx without Ca / Mg, GIBCO BRL). A second alternative procedure, used when lyophilization is feasible, consisted of dialyzing the protein solution against water, using three to five water changes at about 4 ° C. A typical dialysis membrane is the Spectra / Por dialysis membrane, a molecular weight cut-off membrane at 2,000 (Spectrum Medical Industries, Los Angeles, CA). The material was concentrated as above at a concentration of 3 to 4 mg / ml and typically a 2 mg amount was lyophilized in a 5 ml vial. For the analysis of DLS, this vial was reconstituted with water at a concentration higher than 3.3 mg / ml or higher than 5 mg / ml. Typically, several vials were collected. The concentration of the protein was determined by absorption of ultraviolet radiation at the maximum peak (typically 280 nm). The protein was diluted to a concentration of about 3 or 5 mg / ml with a combination of water and PBS lOx (without Ca / Mg, GIBCO BRL), giving a final concentration in PBS of lx. The solution of the protein at 3.0 mg / ml or at 5 mg / ml in PBS lx was adjusted to 7.4 with HCl / NaOH and passed through an Anotop® filter of 0.1 μm (Whatman International Ltd. , Maidstone, England). The average accumulated particle size was measured on a Brookhaven BI900 DLS instrument (Brookhaven Instruments, Holtsville, NY) with an Argon ion Lexel laser every 15 minutes using a duration of 30 seconds at a 90 ° angle. A 488 nm filter with a pore size of 400 μm was used. An incubation temperature of 37 ° C, a viscosity of 0.6915 centipoise (cp) and a refractive index of 1.333 were used. The average size of the light particles was calculated by a cumulative method using the measured autocorrelation reference. Table 4 shows the estimated time required for various anti-obesity proteins to reach an average particle size of 50 nm in a PBS solution at pH 7.4 and at a temperature of 37 ° C. The average size of the light particles was determined from a cumulative analysis of a binodal distribution composed of monomer populations and higher order aggregates. The time required to achieve an average size of 50 nm aggregates was estimated by plotting the size as a function of time. When assayed, the proteins were folded with the Cys residues cross-linked by a disulfide bond. For reference, the human Ob protein and the sequence ID protein No. 18 are presented. The notation "id" means impossible to determine and the notation "nd" means not determined. The sequence protein ID No. 18 has the sequence: Sequence ID n ° 18 10 15 Met Arg Val Pro He Gln Lys Val Gln Asp Asp Thr Lys Thr Leu 20 25 30 He Lys Thr He Val Thr Arg He Asn Asp He Ser His Thr Gln 35 40 45 Ser Val Ser Ser Lys Gln Lys Val Thr Gly Leu Asp Phe He Pro 50 55 60 Gly Leu His Pro He Leu Thr Leu Ser Lys Met Asp Gln Thr Leu 65 70 75 Wing Val Tyr Gln Gln He Leu Thr Ser Met Pro Ser Arg Asp Val 80 85 90 He Gln He Be Asn Asp Leu Glu Asn Leu Arg Asp Leu Leu His 95 100 105 Val Leu Wing Phe Ser Lys Ser Cys His Leu Pro Trp Wing Ser Gly 110 115 120 Leu Glu Thr Leu Asp Ser Leu Gly Gly Val Leu Glu Wing Ser Gly 125 130 135 Tyr Ser Thr Glu Val Val Ala Leu Ser Arg Leu Gln Gly Ser Leu 140 145 Gln Asp Met Leu Trp Gln Leu Asp Leu Ser Pro Gly Cys Table 4 Estimated time (minutes) required for various anti-obesity proteins to reach an average particle size of 50nm in a pBS solution at pH 7.4 at 37 ° C The compounds are active in at least one of the biological assays and are anti-obesity agents. As such, they are useful in the treatment of obesity and disorders involved in obesity. However, proteins are not only useful as therapeutic agents; a person skilled in the art should recognize that proteins are useful in the production of antibodies for diagnostic use and, as proteins, are useful as food additives for 5 animals. In addition, the compounds are useful for controlling weight for cosmetic purposes in mammals. A cosmetic purpose seeks to control the weight of a mammal to improve body appearance. The mammal is not necessarily obese. Said cosmetic use forms part of the present invention.

The principles, preferred embodiments and operating modes of the present invention have been described in the above specification. However, it should not be considered that the invention that is intended to be protected here is limited to the particular forms described, since these must be to consider as illustrative and not as restrictive. Those skilled in the art can make variations and changes without departing from the spirit of the invention.

Claims (12)

1. Having described the invention as above, the content of the following is claimed as property: CLAIMS 1. A protein of formula (I): Sequence ID No. 1 5 10 15 Val Pro He Gln Lys Val Gln Asp Asp Thr Lys Thr Leu He Lys 20 25 30 Thr He Val Thr Arg He Xaa Asp He Ser His Thr Xaa Ser Val 35 40 45 Be Ser Lys Gln Lys Val Thr Gly Leu Asp Phe He Pro Gly Leu 50 55 60 His Pro He Leu Thr Leu Ser Lys Met Asp Gln Thr Leu Wing Val 65 70 75 Tyr Gln Gln He Leu Thr Ser Met Pro Ser Arg Xaa He Gln 80 85 90 He Be As Asp Leu Glu Asn Leu Arg Asp Leu Leu His Val Leu 95 100 105 Wing Phe Ser Lys Ser Cys His Leu Pro Trp Wing Ser Gly Leu Glu 110 115 120 Thr Leu Asp Ser Leu Gly Gly Val Leu Glu Wing Ser Gly Tyr Ser 125 130 135 Thr Glu Val Val Ala Leu Ser Arg Leu Gln Gly Ser Leu Gln Asp 140 145 Met Leu Trp Gln Leu Asp Leu Ser Pro Gly Cys characterized oorque: Xaa from position 22 is Asn or Ser, Xaa from position 28 is Gln or is absent, Xaa from position 72 is Asn, Gln, Glu or Asp and Xaa from position 73 is Val or Met; said protein having at least one of the following substitutions: Trp of position 100 is substituted by Glu, Asp, His, Lys or Arg, or Trp of position 138 is substituted by Glu, Asp, His, Lys or Arg; or a pharmaceutically acceptable salt thereof.
2. 2. The protein according to claim 1, characterized by Cvs operon of position% is linked by a disulfide bond to Cys of position 146.
3. 3. The protein according to claim 2, characterized by Dorque: Xaa position. 22 is Asn, Xaa of position 28 is Gln, Xaa of position 72 is Asn or Asp, Xaa of position 73 is Val and Trp of position 100 is replaced by Glu or Asp. Four . The protein according to claim 1, characterized Dorque has a sequence selected from the compound qruoo; In the sequence TD not -? - the semen ID No. 3, the sequence ID No.
4. 4, the sequence ID No. 5, the sequence ID No. 6, the sequence ID No. 7 and the sequence ID No. 8 .
5. 5. A norcme characterized protein consists of the pro.pfna according to any one of the claims I to 4 and a leader sequence, in which the leader sequence is linked to the amino terminal qruoo of the nrotein, or a salt f ardé. iti rampnt.p acceptable of it.
6. 6. The protein according to claim 5, characterized noraue the leader sequence is Met-R,. in the? IIP R_! , is absent or is any amino acid except Pro.
7. 7. The protein according to claim 6, characterized in that the leader sequence is Met-Arq, Met-Aso or Met-Tyr.
8. 8. The protein according to claim 7, characterized in that it has a sequence selected from the qruoo -committee in nr ID sequence o.9. the sequence ID n ° 10, the sequence ID n ° II and sequence ID No. 12.
9. 9. A pharmaceutical formulation characterized ooraue-comorende, as an active agent, an orotein according to which one of claims 1 to 8, or a pharmaceutically acceptable salt thereof, together with one or more pharmaceutically acceptable diluents, vehicles or excipients.
10. 10. A DNA polynucleotide compound, characterized - Doraue comprises DNA encoding a protein according to any one of claims 1 to 8.
11. 11. A method of preparing a protein according to any one of claims 5 to 8, characterized because it comprises: (a) transforming a host cell with DNA encoding the protein, (b) culturing the transformed host cell so that the protein is expressed and (c) recovering the expressed protein.
12. 12. A method of preparing a protein according to any one of claims 1 to 4, characterized in that the process according to claim 10 is further understood. (d) enzymatically cleaving the expressed protein from the leader sequence to produce a protein according to any one of claims 1 to 4 and (e) recovering the protein.