Abstracts B1–Proteases as Molecular Targets of Drug Development B1-001 lin release from the beta cells. Furthermore, GLP-1 also stimu- DPP-IV structure and inhibitor design lates beta cell growth and insulin biosynthesis, inhibits glucagon H. B. Rasmussen1, S. Branner1, N. Wagtmann3, J. R. Bjelke1 and secretion, reduces free fatty acids and delays gastric emptying. A. B. Kanstrup2 GLP-1 has therefore been suggested as a potentially new treat- 1Protein Engineering, Novo Nordisk A/S, Bagsvaerd, Denmark, ment for type 2 diabetes. However, GLP-1 is very rapidly degra- 2Medicinal Chemistry, Novo Nordisk A/S, Maaloev, Denmark, ded in the bloodstream by the enzyme dipeptidyl peptidase IV 3Discovery Biology, Novo Nordisk A/S, Maaloev, DENMARK. (DPP-IV; EC 3.4.14.5). A very promising approach to harvest E-mail: [email protected] the beneficial effect of GLP-1 in the treatment of diabetes is to inhibit the DPP-IV enzyme, thereby enhancing the levels of The incretin hormones GLP-1 and GIP are released from the gut endogenously intact circulating GLP-1. The three dimensional during meals, and serve as enhancers of glucose stimulated insu- structure of human DPP-IV in complex with various inhibitors 138 Abstracts creates a better understanding of the specificity and selectivity of drug-like transition-state inhibitors but can be utilized for the this enzyme and allows for further exploration and design of new design of non-transition-state inhibitors that compete for sub- therapeutic inhibitors. The majority of the currently known DPP- strate binding. Besides carrying out proteolytic activity, the IV inhibitors consist of an alpha amino acid pyrrolidine core, to ectodomain of memapsin 2 also interacts with APP leading to which substituents have been added to optimize affinity, potency, the endocytosis of both proteins into the endosomes where APP enzyme selectivity, oral bioavailability, and duration of action. is hydrolyzed by memapsin 2 to produce Ab. A phosphorylated Various compound series and their SAR relative to alpha amino motif in the cytosolic domain of memapsin 2 is responsible acids will be presented. for the recognition of GGA proteins as part of the recycling mechanism that transports memapsin 2 from endosomes to trans-Golgi then back to cell surface. These interactions may B1-002 also be considered for the design of small-molecular compounds MEROPS: the peptidase database that interfere with memapsin 2 trafficking and thus reduce the N. D. Rawlings, F. R. Morton and A. J. Barrett production of Ab. Bioinformatics, Wellcome Trust Sanger Institute, Hinxton, Cambridgeshire, UK. E-mail: [email protected] Peptidases (also known proteases or proteinases) and their inhibi- B1-004 tors are of major importance to human health. The MEROPS Identification of human carnosinase – a brain- database is a comprehensive information resource on these pro- specific metalloprotease teins freely available to all. Central to the database is the hierarchi- M. Teufel cal classification system first introduced by Rawlings & Barrett Biochemistry, Exploratory Research, Sanofi Aventis, Strasbourg, (1993), which is now almost universally accepted. Peptidases are France. E-mail: michael.teufel@sanofi-synthelabo.com classified according to biochemical characterization, sequence homology and structural similarity. For each peptidase a region Metalloproteases form a large and diverse family of proteases known as the peptidase unit is defined, which encompasses the and are molecular targets that represent an opportunity for structural domains and residues important for activity. Each pepti- therapeutic intervention. In particular, the development of potent dase is given a unique MEROPS identifier, peptidases with homo- inhibitors has made progress for the family of matrix metallopro- logous peptidase unit sequences are grouped into a family, and teases (MMP). The sequencing of the human genome revealed peptidase units with similar structural folds are grouped into a clan that a significant percentage of the drugable genome is represen- (which can contain one or many families). A similar classification ted by proteases, many of them still with unknown function. In was developed for the protein inhibitors of peptidases in 2004. this presentation, data will be presented on the deorphanization Records can be accessed through the indexes, which list peptidases of two previously unknown genes by means of bioinformatics by MEROPS identifier, alphabetically by name (including numer- and classical biochemistry. This work led to the identification of ous synonyms), or by source organism name (both scientific and human carnosinase, a dipeptidase specifically expressed in the common). The database provides a gateway to the extensive litera- human brain and an ubiquitously expressed close homologue, ture on peptidases, and the current release of the database includes characterized to be a non-specific dipeptidase. over 20 000 references. There are annotated alignments at clan, family and peptidase levels showing peptidase units, active site resi- dues and other structural features, and evolutionary trees for each B1-005 family and peptidase. There are comprehensive searches of EST alignments showing alternatively spliced variants and SNPs that Stimulating serpins with synthetic tailor-made change the protein sequence. oligosaccharides: a new generation of antithrombotics B1-003 M. Petitou Thrombosis & Angiogenesis, Sanofi-Aventis, Toulouse, France. Structure and function relationship of E-mail: maurice.petitou@sanofi-aventis.com memapsin 2 (beta-secretase) J. Tang We will discuss our research on synthetic oligosaccharides able to Protein Studies Program, Oklahoma Medical Research selectively activate the inhibitory activity of antithrombin towards Foundation, Oklahoma City, OK, USA. various serine proteinases. We first synthesized pentasaccharides E-mail: [email protected] closely related to the antithrombin binding domain of heparin [1] (the active site), as well as analogues displaying different pharma- Memapsin 2 (b-secretase, BACE1) is the membrane-anchored cokinetic profiles. Selective inhibitors of coagulation factor Xa aspartic protease that initiates the cleavage of b-amyloid precur- were thus obtained that represent a new class of antithrombotic [2] sor protein (APP) leading to the production of amyloid-b (Ab), drugs currently being evaluated worldwide. We then designed lar- a major factor in the pathogenesis of Alzheimer’s disease (AD). ger oligosaccharides [3] that inhibit both factor Xa and thrombin Since memapsin 2 is a major target for the development of in the presence of antithrombin. They are devoid of undesired non- inhibitor drugs for the treatment of AD, its structure and phy- specific interactions with blood proteins, particularly with platelet siological functions are topics of intense research interest cur- factor 4. Clinical trials are ongoing to prove the therapeutic bene- rently. Here we discuss the structural features of memapsin 2 fits of this new type of coagulation inhibitors. and how do they contribute to the activity and inhibition of the References protease. Structural and kinetic evidence support the presence 1. van Boeckel CAA, Petitou M. Angew Chem Int Ed Engl 1993; of 11 subsites for substrate or inhibitor binding in the active- 32: 1671–1690. site cleft of memapsin 2. Subsites P3 to P2’ are most useful in 2. Turpie et al. New Engl J Med 2001; 344: 619–625; Eriksson the design of transition-state analogue inhibitors. Recent data et al. Ibid 2001; 345: 1298–1304; Bauer et al. Ibid 2001; 345: indicated that subsites P7, P6 and P5 have strong influence of 1305–1310. hydrolytic rate or inhibition potency. These subsites are, how- 3. Petitou et al. Nature 1999; 398: 417–422. Herbert et al. Thromb ever, too far from the transition-state isostere for the design of Haemost 2001; 85: 852–860. 139 Abstracts B1-006 bines experimental enzyme kinetics data and high level quantum Slow tight binding inhibitors in drug mechanical modeling of enzyme–inhibitor chemical interactions. discovery: in the case of DPPIV and elastase The method utilizes the principal catalytic reaction scheme of the target enzyme and does not require its 3D structure (a ligand inhibitors based approach). The method is valid for the prediction of the Z. Kapui, E. Boronkay, I. Bata, M. Varga, E. Mikus, trend in binding affinity of inhibitors not only for the specific K. Urban-Szabo, S. Ba´ tori and P. Ara´ nyi enzyme for which the QSAR model was optimized, but also for Discovery Research, CHINOIN member of Sanofi-Aventis Group, the whole enzyme family. The methodology would contribute sig- Budapest, Hungary. E-mail: zoltan.kapui@sanofi-aventis.com nificantly to overcoming the problem of fast mutational resist- Enzyme are extremely potent causing significant inhibition at very ance developed by pathogens in response to pharmaceutical low concentrations that may be comparable to the concentration treatment. It can be used as a computational tool for expert ana- of the target enzyme. When this inhibition is studied in vitro, com- lysis of various hypotheses about structure–activity relationships plexities arise because the concentration of the inhibitor is so low formulated for the design of new inhibitors. that it is altered significantly as a result of combination with the enzyme. This situation is referred to as tight-binding inhibition. B1-008P Partly as a result of their low concentrations, tight-binding inhibi- Dramatical role of ligand length in the tors often show slow-binding characteristics.