Peptidic inhibitors

The catalytic subunit of cAPK contains two domains connected by a peptide linker. ATP binds in a deep cleft between the two domains. Presently, crystal structures showed cAPK in three different conformations, (1) in a closed conformation in the ternary complex with ATP or other tight-binding ligands and a peptide inhibitor PKI(5-24), (2) in an intermediate conformation in the binary complex with adenosine, and (3) in an open conformation in the binary complex of mammalian cAPK with PKI(5-24). Fig.l shows a superposition of the three protein kinase configurations to visualize the type of conformational movement. 

Fig. 2. Conformational free energy of closed, intermediate and open protein kinase conformations. cAPK indicates the unbound form of cAMP-dependent protein kinase, cAPKiATP the binary complex of cAPK with ATP, cAPKiPKP the binary complex of cAPK with the peptide inhibitor PKI(5-24), and cAPK PKI ATP the ternary complex of cAPK with ATP and PKI(5-24). Shown are averaged values for the three crystal structures lATP.pdb, ICDKA.pdb, and ICDKB.pdb. All values have been normalized with respect to the free energy of the closed conformations.

DM Perguson, RJ Radmer, PA Kollman. Determination of the relative binding free energies of peptide inhibitors to the HIV-1 protease. J Med Chem 34 2654-2659, 1991. 

Inhibitors as well as substrates bind in this crevice between the domains. From the numerous studies of different inhibitors bound to serine pro-teinases we have chosen as an illustration the binding of a small peptide inhibitor, Ac-Pro-Ala-Pro-Tyr-COOH to a bacterial chymotrypsin (Figure 11.9). The enzyme-peptide complex was formed by adding a large excess of the substrate Ac-Pro-Ala-Pro-Tyr-CO-NHz to crystals of the enzyme. The enzyme molecules within the crystals catalyze cleavage of the terminal amide group to produce the products Ac-Pro-Ala-Pro-Tyr-COOH and NHs. The ammonium ions diffuse away, but the peptide product remains bound as an inhibitor to the active site of the enzyme. 

Figure 15.6 Chromatogram of a plasma standard of human leukocyte elastase inhibitors obtained by using LC-LC. Adapted from Journal of Liquid Chromatography and Related Technologies, 19, R. A. Earley and L. R Tini, Versatile multidimensional chromatographic system for di ug discovery as exemplified by the analysis of a non-peptidic inhibitor of human leukocyte elastase , pp. 2527-2540, 1996, by courtesy of Marcel DekkeiTnc.

Sticht J, Humbert M, Findlow S, Bodem J, Muller B, Dietrich U, Werner J, Krausslich HG (2005) A peptide inhibitor of HIV-1 assembly in vitro. Nat Struct Mol Biol 12 671-677 Stockman LJ, Bellamy R, Garner P (2006) SARS systematic review of treatment effects. PLoS... 

NMR structural characterization of peptide inhibitors bound to the Hepatitis C virus NS3 protease design of a new P2 substituent. J Med Chem 47 123-132... 

Sticht J, Humbert M, Eindlow S et al. (2005) A peptide inhibitor of HIV-1 assembly in vitro. Nat Struct Mol Biol 12 671-677... 

Many enveloped viruses share a common mechanism of fusion, mediated by a virus-encoded glycoprotein that contains heptad repeats in its extraceUnlar domain. Dnring the fnsion process, these domains rearrange to form highly structured and thermodynamically stable coiled-coils. Viruses encoding fusion proteins that have these domains inclnde members of the paramyxovirus family (e.g., respiratory syncytial virus, metapneumovirus, and measles virus), ebola virus, influenza, and members of the retroviridae (e.g., human T cell lenkemia virus type-1 and human immunodeficiency virus type-1, HlV-1). Peptide inhibitors of fusion that disrupt the... 

KUby JM, Hopkins S, Venetta TM, DiMassimo B, Cloud GA, Lee JY, Alldredge L, Hunter E, Lambert D, Bolognesi D, Matthews T, Johnson MR, Nowak MA, Shaw GM, Saag MS (1998) Potent suppression of HlV-1 replication in humans by T-20, a peptide inhibitor of gp41-mediated virus entry. Nat Med 4 1302-1307... 

KUby JM, Lalezari JP, Eron JJ, Carlson M, Cohen C, Arduino RC, Goodgame JC, Gallant JE, Volberding P, Murphy RL, Valentine F, Saag MS, Nelson EL, Sista PR, Dusek A (2002) The safety, plasma pharmacokinetics, and antiviral activity of subcutaneous enfuvirtide (T-20), a peptide inhibitor of gp41-mediated virus fusion, in HIV-infected adults. AIDS Res Hum Retroviruses 18 685-693... 

Wild C, Oas T, McDanal C, Bolognesi D, Matthews T (1992) A synthetic peptide inhibitor of human immunodeficiency virus replication correlation between solution structure and viral inhibition, Proc Natl Acad Sci USA 89 10537-10541... 

Haque TS, Skillman AG, Lee CE, Habashita H, Gluzman lY, Ewing TJA, Goldberg DE, Kuntz ID, Ellman JA. Potent, low-molecnlar-weight non-peptide inhibitors of malarial aspartyl protease plasmepsin II. J Med Chem 1999 42 1428-40. 

As an alternative to peptidic inhibitors, which display electrostatic interactions with the active site, covalent inhibitors have also been described recently. Such peptides bear a functional group that can react reversibly with the catalytic serine of the protease. These include aldehydes, a-ketoacid derivates, lactams and boronates. 

Fragment screening by NMR was applied recently in the search of non-peptidic small molecule inhibitors. Two scaffolds (13) and (14), which bind the enzyme at the S1-S3 and the S2 binding site respectively, as shown by chemical shift perturbation, were linked together to yield competitive inhibitors such as (15) with micromolar IC50 values [158]. There have been no reports of non-peptidic inhibitors with potency and pharmacokinetics similar to the peptidic or peptidomimetic inhibitors described above. 

A. Peptidic inhibitors Class Cpd. Ki (nMf MIC (pg/mL) Additional comments Reference... 

Maraganore J. M., Bourdon P Jablonskj J., Ramachandran K. L. Design and characterization of hirulogs A novel class of bivalent peptide inhibitors of thrombin. Biochemistry 1990 229, 7095-101. 

Luther M, Rusnak D, Sternbach DD, Mehrotra M, Peel M, Shampine L, Davis R, Robbins J, Patel IR, Kassel D, Burkhart W, Moyer M, Bradshaw T, Berman J. Peptide inhibitors of Src SH3-SH2-phosphoprotein interactions. J Biol Chem 1994 269 31711-31719. Also, for pTyr mimics see Ye B, Akamatsu M, Shoelson SE, Wolf G, Giorgetti-Peraldi S, Yan X, Roller PP, Burke TR Jr. L-0-(2-Malonyl)tyrosine a new phosphotyrosyl mimetic for the preparation of Src homology 2 domain inhibitory peptides. J Med Chem 1995 38 4270-4275. 

Poduslo JF, Curran GL, Kumar A, Frangione B, Soto C. Beta-sheet breaker peptide inhibitor of Alzheimer s amyloidogenesis with increased blood-brain barrier permeability and resistance to proteolytic degradation in plasma. J Neurobiol 1999 39 371-382. 

Figure 3.14 Idealized van t Hoff plot of the temperature dependence of the affinity of a peptide inhibitor for die enzyme hdm2.

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