Abstract
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.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
Similar content being viewed by others
References
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 45:1–18
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 12:1719–22
Borthwick AD et al (1998) Design and synthesis of monocyclic beta-lactams as mechanism-based inhibitors of human cytomegalovirus protease. Bioorg Med Chem Lett 8:365–70
Gopalsamy A et al (2004) Design and syntheses of 1,6-naphthalene derivatives as selective HCMV protease inhibitors. J Med Chem 47:1893–9
Ogilvie W et al (1997) Peptidomimetic inhibitors of the human cytomegalovirus protease. J Med Chem 40:4113–35
Waxman L, Darke PL (2000) The herpesvirus proteases as targets for antiviral chemotherapy. Antivir Chem Chemother 11:1–22
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 101:6870–5
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 12:1019–20
Nomura AM, Marnett AB, Shimba N, Dotsch V, Craik CS (2006) One functional switch mediates reversible and irreversible inactivation of a herpesvirus protease. Biochemistry 45:3572–9
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 289:197–203
Pray TR, Reiling KK, Demirjian BG, Craik CS (2002) Conformational change coupling the dimerization and activation of KSHV protease. Biochemistry 41:1474–82
Lazic A, Goetz DH, Nomura AM, Marnett AB, Craik CS (2007) Substrate modulation of enzyme activity in the herpesvirus protease family. J Mol Biol 373:913–23
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 39:12796–803
Shimba N, Nomura AM, Marnett AB, Craik CS (2004) Herpesvirus protease inhibition by dimer disruption. J Virol 78:6657–65
Lee GM, Craik CS (2009) Trapping moving targets with small molecules. Science 324:213–5
Shahian T et al (2009) Inhibition of a Viral Enzyme by a Small Molecule Dimer Disruptor. Nat Chem Biol 9:640–6
Lu F et al (2006) Proteomimetic libraries: design, synthesis, and evaluation of p53-MDM2 interaction inhibitors. J Comb Chem 8:315–25
Cummings CG, Hamilton AD (2010) Disrupting protein-protein interactions with non-peptidic, small molecule alpha-helix mimetics. Curr Opin Chem Biol 14:341–6
Davis JM, Tsou LK, Hamilton AD (2007) Synthetic non-peptide mimetics of alpha-helices. Chem Soc Rev 36:326–34
Gavathiotis E et al (2008) BAX activation is initiated at a novel interaction site. Nature 455:1076–81
Henchey LK, Jochim AL, Arora PS (2008) Contemporary strategies for the stabilization of peptides in the alpha-helical conformation. Curr Opin Chem Biol 12:692–7
Moellering RE et al (2009) Direct inhibition of the NOTCH transcription factor complex. Nature 462:182–8
Zhang JH, Chung TD, Oldenburg KR (1999) A Simple Statistical Parameter for Use in Evaluation and Validation of High Throughput Screening Assays. J Biomol Screen 4:67–73
Feng BY, Shelat A, Doman TN, Guy RK, Shoichet BK (2005) High-throughput assays for promiscuous inhibitors. Nat Chem Biol 1:146–8
Feng BY, Shoichet BK (2006) A detergent-based assay for the detection of promiscuous inhibitors. Nat Protoc 1:550–3
Acknowledgments
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.).
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2012 Springer Science+Business Media New York
About this protocol
Cite this protocol
Craik, C.S., Shahian, T. (2012). A Screening Strategy for Trapping the Inactive Conformer of a Dimeric Enzyme with a Small Molecule Inhibitor. In: Zheng, Y. (eds) Rational Drug Design. Methods in Molecular Biology, vol 928. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-008-3_9
Download citation
DOI: https://doi.org/10.1007/978-1-62703-008-3_9
Published:
Publisher Name: Humana Press, Totowa, NJ
Print ISBN: 978-1-62703-007-6
Online ISBN: 978-1-62703-008-3
eBook Packages: Springer Protocols