Kaposi’s sarcoma-associated herpesvirus (KSHV) is the etiological agent of Kaposi’s sarcoma (KS), the most common cancer in AIDS patients. All herpesviruses express a conserved dimeric serine protease that is required for generating infectious virions and is therefore of pharmaceutical interest. Given the past challenges of developing drug-like active-site inhibitors to this class of proteases, small-molecules targeting allosteric sites are of great value. In light of evidence supporting a strong structural linkage between the dimer interface and the protease active site, we have focused our efforts on the dimer interface for identifying dimer disrupting inhibitors. Here, we describe a high throughput screening approach for identifying small molecule dimerization inhibitors of KSHV protease. The helical mimetic, small molecule library used, as well as general strategies for selecting compound libraries for this application will also be discussed. This methodology can be applicable to other systems where an alpha helical moiety plays a dominant role at the interaction site of interest, and in vitro assays to monitor function are in place.
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This work was supported by NIH grants T32 GMO7810, AIO67423 (C.S.C.), and by the American Lebanese and Syrian Associated Charities and St Jude Children’s Research Hospital (R.K.G.).
Borthwick AD et al (2002) Design and synthesis of pyrrolidine-5,5-trans-lactams (5-oxohexahydropyrrolo[3,2-b]pyrroles) as novel mechanism-based inhibitors of human cytomegalovirus protease. 2. Potency and chirality. J Med Chem 45:1–18PubMedCrossRefGoogle Scholar
Borthwick AD et al (2002) Pyrrolidine-5,5-trans-lactams as novel mechanism-based inhibitors of human cytomegalovirus protease. Part 3: potency and plasma stability. Bioorg Med Chem Lett 12:1719–22PubMedCrossRefGoogle Scholar
Borthwick AD et al (1998) Design and synthesis of monocyclic beta-lactams as mechanism-based inhibitors of human cytomegalovirus protease. Bioorg Med Chem Lett 8:365–70PubMedCrossRefGoogle Scholar
Gopalsamy A et al (2004) Design and syntheses of 1,6-naphthalene derivatives as selective HCMV protease inhibitors. J Med Chem 47:1893–9PubMedCrossRefGoogle Scholar
Waxman L, Darke PL (2000) The herpesvirus proteases as targets for antiviral chemotherapy. Antivir Chem Chemother 11:1–22PubMedGoogle Scholar
Marnett AB, Nomura AM, Shimba N, Ortiz de Montellano PR, Craik CS (2004) Communication between the active sites and dimer interface of a herpesvirus protease revealed by a transition-state inhibitor. Proc Natl Acad Sci USA 101:6870–5PubMedCrossRefGoogle Scholar
Nomura AM, Marnett AB, Shimba N, Dotsch V, Craik CS (2005) Induced structure of a helical switch as a mechanism to regulate enzymatic activity. Nat Struct Mol Biol 12:1019–20PubMedGoogle Scholar
Nomura AM, Marnett AB, Shimba N, Dotsch V, Craik CS (2006) One functional switch mediates reversible and irreversible inactivation of a herpesvirus protease. Biochemistry 45:3572–9PubMedCrossRefGoogle Scholar
Pray TR, Nomura AM, Pennington MW, Craik CS (1999) Auto-inactivation by cleavage within the dimer interface of Kaposi’s sarcoma-associated herpesvirus protease. J Mol Biol 289:197–203PubMedCrossRefGoogle Scholar
Pray TR, Reiling KK, Demirjian BG, Craik CS (2002) Conformational change coupling the dimerization and activation of KSHV protease. Biochemistry 41:1474–82PubMedCrossRefGoogle Scholar
Lazic A, Goetz DH, Nomura AM, Marnett AB, Craik CS (2007) Substrate modulation of enzyme activity in the herpesvirus protease family. J Mol Biol 373:913–23PubMedCrossRefGoogle Scholar
Reiling KK, Pray TR, Craik CS, Stroud RM (2000) Functional consequences of the Kaposi’s sarcoma-associated herpesvirus protease structure: regulation of activity and dimerization by conserved structural elements. Biochemistry 39:12796–803PubMedCrossRefGoogle Scholar
Shimba N, Nomura AM, Marnett AB, Craik CS (2004) Herpesvirus protease inhibition by dimer disruption. J Virol 78:6657–65PubMedCrossRefGoogle Scholar