Studies with peptide-based and macromolecular inhibitors of the caspase family of cysteine proteases have helped to define a central role for these enzymes in inflammation and mammalian apoptosis. A clear interpretation of these studies has been compromised by an incomplete understanding of the selectivity of these molecules. Here we describe the selectivity of several peptide-based inhibitors and the coxpox serpin CrmA against 10 human caspases. The peptide aldehydes that were examined (Ac-WEHD-CHO, Ac-DEVD-CHO, Ac-YVAD-CHO, t-butoxycarbonyl-IETD-CHO, and t-butoxycarbonyl-AEVD-CHO) included several that contain the optimal tetrapeptide recognition motif for various caspases. These aldehydes display a wide range of selectivities and potencies against these enzymes, with dissociation constants ranging from 75 pM to >10 M. The halomethyl ketone benzyloxycarbonyl-VAD fluoromethyl ketone is a broad specificity irreversible caspase inhibitor, with second-order inactivation rates that range from 2.9 ؋ 10 2 M ؊1 s ؊1 for caspase-2 to 2.8 ؋ 10 M ؊1s ؊1 for caspase-1. The results obtained with peptidebased inhibitors are in accord with those predicted from the substrate specificity studies described earlier. The cowpox serpin CrmA is a potent (K i < 20 nM) and selective inhibitor of Group I caspases (caspase-1, -4, and -5) and most Group III caspases (caspase-8, -9, and -10), suggesting that this virus facilitates infection through inhibition of both apoptosis and the host inflammatory response.Members of the caspase family of cysteine proteases, which at present includes 11 homologues of human origin, are important mediators of both inflammation, where they are involved in the production of several inflammatory cytokines, and apoptosis, where they participate in signaling and effector pathways (for review, see Ref. 1). The evidence for the central role of these enzymes in both of these biological processes was initially obtained using potent peptide-based and macromolecular inhibitors. For example, the finding that Ac-YVAD-CHO, a potent inhibitor of caspase-1, prevented the release of interleukin-1␤ (IL-1␤) 1 from monocytes, suggested that this enzyme was, in fact, the pro-IL-1␤-processing enzyme (2). This was later confirmed with the description of caspase-1-deficient mice, which are defective in the production of this cytokine (3,4). Similarly, the observation that apoptosis could be attenuated by the cowpox serpin CrmA, also known to be a potent caspase-1 inhibitor, provided the first compelling evidence that caspases play an important role in mammalian cell death (5). This has recently been confirmed by several studies, including the description of caspase-3-deficient mice, which have a striking defect in the programmed cell deaths that occur during neuronal development (6).Studies using these and other caspase inhibitors continue to be an important component of the repertoire of scientists investigating these complex biological processes in whole cells and in vivo. Regarding the latter, there are numerous recent re...

A novel series of beta-amino amides incorporating fused heterocycles, i.e., triazolopiperazines, were synthesized and evaluated as inhibitors of dipeptidyl peptidase IV (DPP-IV) for the treatment of type 2 diabetes. (2R)-4-Oxo-4-[3-(trifluoromethyl)-5,6-dihydro[1,2,4]triazolo[4,3-a]pyrazin-7(8H)-yl]-1-(2,4,5-trifluorophenyl)butan-2-amine (1) is a potent, orally active DPP-IV inhibitor (IC(50) = 18 nM) with excellent selectivity over other proline-selective peptidases, oral bioavailability in preclinical species, and in vivo efficacy in animal models. MK-0431, the phosphate salt of compound 1, was selected for development as a potential new treatment for type 2 diabetes.

Dipeptidyl peptidase (DPP)-IV inhibitors are a new approach to the treatment of type 2 diabetes. DPP-IV is a member of a family of serine peptidases that includes quiescent cell proline dipeptidase (QPP), DPP8, and DPP9; DPP-IV is a key regulator of incretin hormones, but the functions of other family members are unknown. To determine the importance of selective DPP-IV inhibition for the treatment of diabetes, we tested selective inhibitors of DPP-IV, DPP8/DPP9, or QPP in 2-week rat toxicity studies and in acute dog tolerability studies. In rats, the DPP8/9 inhibitor produced alopecia, thrombocytopenia, reticulocytopenia, enlarged spleen, multiorgan histopathological changes, and mortality. In dogs, the DPP8/9 inhibitor produced gastrointestinal toxicity. The QPP inhibitor produced reticulocytopenia in rats only, and no toxicities were noted in either species for the selective DPP-IV inhibitor. The DPP8/9 inhibitor was also shown to attenuate T-cell activation in human in vitro models; a selective DPP-IV inhibitor was inactive in these assays. Moreover, we found DPP-IV inhibitors that were previously reported to be active in models of immune function to be more potent inhibitors of DPP8/9. These results suggest that assessment of selectivity of potential clinical candidates may be important to an optimal safety profile for this new class of antihyperglycemic agents. Diabetes

There is currently intense interest in the emerging group of proline-specific dipeptidases, and their roles in the regulation of biological processes. Dipeptidyl peptidase IV (DPP-IV) is involved in glucose metabolism by contributing to the regulation of glucagon family peptides and has emerged as a potential target for the treatment of metabolic diseases. Two other proline-specific dipeptidases, DPP-VII (also known as quiescent cell proline dipeptidase) and DPP-II, have unknown functions and have recently been suggested to be identical proteases based on a sequence comparison of human DPP-VII and rat DPP-II (78% identity) [Araki, Li, Yamamoto, Haneda, Nishi, Kikkawa and Ohkubo (2001) J. Biochem. 129, 279-288; Fukasawa, Fukasawa, Higaki, Shiina, Ohno, Ito, Otogoto and Ota (2001) Biochem. J. 353, 283-290]. To facilitate the identification of selective substrates and inhibitors for these enzymes, a complete biochemical profile of these enzymes was obtained. The pH profiles, substrate specificities as determined by positional scanning, Michaelis-Menten constants and inhibition profiles for DPP-VII and DPP-II were shown to be virtually identical, strongly supporting the hypothesis that they are the same protease. In addition, substrate specificities, catalytic constants and IC(50) values were shown to be markedly different from those of DPP-IV. Selective DPP-IV and DPP-VII substrates were identified and they can be used to design selective inhibitors and probe further into the biology of these enzymes.

Biosynthetically directed fractional incorporation of 13C into proteins results in nonrandom 13C-labeling patterns that can be investigated by analysis of the 13C-13C scalar coupling fine structures in heteronuclear 13C-1H or homonuclear 13C-13C correlation experiments. Previously this approach was used for obtaining stereospecific 1H and 13C assignments of the diastereotopic methyl groups of valine and leucine. In the present paper we investigate to what extent the labeling patterns are characteristic for other individual amino acids or groups of amino acids, and can thus be used to support the 1H spin-system identifications. Studies of the hydrolysates of fractionally 13C-labeled proteins showed that the 59 aliphatic carbon positions in the 20 proteinogenic amino acids exhibit 16 different types of 13C-13C coupling fine structures. These provide support for the assignment of the resonances of all methyl groups in a protein, which are otherwise often poorly resolved in homonuclear 1H NMR spectra. In particular, besides the individual methyl assignments in Val and Leu, unambiguous distinctions are obtained between the methyl groups of Ala and Thr, and between the gamma- and delta-methyl groups of Ile. In addition to the methyl resonances, the gamma CH2 groups of Glu and Gln can be uniquely assigned because of the large coupling constant with the delta-carbon, and the identification of most of the other spin systems can be supported on the basis of coupling patterns that are common to small groups of amino acid residues.