in 1997. She is currently completing her Ph.D. degree in the Centre for Drug Design and Development. Her Ph.D. studies are directed at designing and developing competitive reversible protease inhibitors, particularly for serine and cysteine proteases involved in viral and parasitic infections, Alzheimer's disease, and apoptosis. Giovanni (John) Abbenante received his B.Sc. (Hons) degree from the Department of Chemistry of the University of Adelaide and his Ph.D. degree on the synthesis of GABA b receptor antagonists from Flinders University in 1992. He undertook postdoctoral studies on the synthesis of ladderanes at Central Queensland University and of HIV-1 protease inhibitors at the University of Queensland. He is currently a Senior Research Officer in the Chemistry Group of the Centre for Drug Design and Development with interests in drug design and in the synthesis of protease inhibitors, macrocycles, receptor antagonists, and peptidomimetics. David P. Fairlie received a B.Sc. (Hons) degree from the University of Adelaide and a Ph.D. from the University of South Wales and did postdoctoral research at Stanford University and University of Toronto. He has held research/ teaching appointments in six Australian universities and has led the Chemistry Group in the Centre for Drug Design and Development since 1991. He is Scientific Director of Promics Pty Ltd. Research interests are in chemical synthesis (organic, inorganic), molecular recognition (DNA-, RNA-, protein-, metalbinding compounds), peptide and protein mimetics, enzyme inhibitors and receptor antagonists for viral infections, inflammatory disorders, cancers, and neurodegenerative diseases, and in mechanisms of chemical reactions, biological processes, disease development, and drug action.

Recombinant forms of the dengue 2 virus NS3 protease linked to a 40-residue co-factor, corresponding to part of NS2B, have been expressed in Escherichia coli and shown to be active against para-nitroanilide substrates comprising the P6-P1 residues of four substrate cleavage sequences. The enzyme is inactive alone or after the addition of a putative 13-residue co-factor peptide but is active when fused to the 40-residue co-factor, by either a cleavable or a noncleavable glycine linker. The NS4B/NS5 cleavage site was processed most readily, with optimal processing conditions being pH 9, I ‫؍‬ 10 mM, 1 mM CHAPS, 20% glycerol. A longer 10-residue peptide corresponding to the NS2B/NS3 cleavage site (P6-P4) was a poorer substrate than the hexapeptide (P6-P1) para-nitroanilide substrate under these conditions, suggesting that the prime side substrate residues did not contribute significantly to protease binding. We also report the first inhibitors of a co-factor-complexed, catalytically active flavivirus NS3 protease. Aprotinin was the only standard serine protease inhibitor to be active, whereas a number of peptide substrate analogues were found to be competitive inhibitors at micromolar concentrations.

Cyclic pentapeptides are not known to exist in alpha-helical conformations. CD and NMR spectra show that specific 20-membered cyclic pentapeptides, Ac-(cyclo-1,5) [KxxxD]-NH(2) and Ac-(cyclo-2,6)-R[KxxxD]-NH(2), are highly alpha-helical structures in water and independent of concentration, TFE, denaturants, and proteases. These are the smallest alpha-helical peptides in water.

This review describes the clinical status (based on available information) of experimental drugs that inhibit enzymes called proteases, or more precisely a sub-class of proteases called peptidases that catalyse the hydrolysis of polypeptide main chain amide bonds. These peptidases are classified by the key catalytic residue in the active site of the enzyme that effects hydrolysis, namely aspartic, serine, cysteine, metallo or threonine proteases. In this review we show structures for 108 inhibitors of these enzymes and update the clinical disposition of over 100 inhibitors that have been considered worthy enough by pharmaceutical, biotechnology or academic researchers and their financial backers to be trialed in humans as prospective medicines. We outline some of their chemical and pharmacological characteristics and compare the current status of protease inhibitors in the clinic with what was observed about 5 years ago (Leung et al, J. Med. Chem. 2000, 43, 305-341). We assess the progress of protease inhibitors into man, predict their futures, and outline some of the hurdles that have been overcome and that still remain for this promising class of new therapeutic agents.

The pathogenesis-related (PR) protein superfamily is widely distributed in the animal, plant, and fungal kingdoms and is implicated in human brain tumor growth and plant pathogenesis. The precise biological activity of PR proteins, however, has remained elusive. Here we report the characterization, cloning and structural homology modeling of Tex31 from the venom duct of Conus textile. Tex31 was isolated to >95% purity by activityguided fractionation using a para-nitroanilide substrate based on the putative cleavage site residues found in the propeptide precursor of conotoxin TxVIA. Tex31 requires four residues including a leucine N-terminal of the cleavage site for efficient substrate processing. The sequence of Tex31 was determined using two degenerate PCR primers designed from N-terminal and tryptic digest Edman sequences. A BLAST search revealed that Tex31 was a member of the PR protein superfamily and most closely related to the CRISP family of mammalian proteins that have a cysteine-rich C-terminal tail. A homology model constructed from two PR proteins revealed that the likely catalytic residues in Tex31 fall within a structurally conserved domain found in PR proteins. Thus, it is possible that other PR proteins may also be substrate-specific proteases.Of the genomes that have been completely sequenced, as many as 2% of the gene products encode known proteases, many of which regulate physiological processes such as blood coagulation, fibrinolysis, the complement system, and the processing of protein hormone precursors by specific convertases. Prohormones and neuropeptides (3-40 amino acids), important coordinators of cellular function in the endocrine and nervous systems, are often synthesized as propeptides. The propeptide is subsequently processed by substrate-specific proteases to yield the mature bioactive form (1). As many of the substratespecific proteases remain unidentified, predicting new bioactive peptides from cDNA sequences is presently difficult, if not impossible (2, 3). Linderström-Lang coined the term "limited proteolysis" to describe enzymes with restricted specificity (4). Enzyme activity can be restricted by substrate structure, enzyme processing state, endogenous protease inhibitor levels or a combination of these factors (5). Peptides derived from prohormones are typically flanked by a pair of dibasic residues (Lys-Arg, Arg-Arg, Lys-Lys, or Arg-Lys) and are cleaved by proteases found in secretory vesicles (1). However, many precursor peptides contain multiple sets of basic residues, suggesting that highly substrate specific or differentially expressed proteases can determine processing outcomes.The venom of cone snails (predatory marine molluscs of the genus Conus) has yielded a rich source of novel neuroactive peptides ("conotoxins") (6). However, the enzymes responsible for cleaving the conotoxins from their propeptide precursors have not been isolated. Here we report the purification, cloning, and initial characterization of Tex31, a protease from venom Conus textile. Interestingly,...