A Screening Strategy for Trapping the Inactive Conformer of a Dimeric Enzyme with a Small Molecule Inhibitor

Abstract

Summary 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.

Broad-Spectrum Allosteric Inhibition of Herpesvirus Proteases

“… 6 , 12 , 16 , 22 , 23 , 28 In doing so, we previously identified a small molecule inhibitor of KSHV Pr designated DD2 [compound 1 (Table 1 )]. 29 , 30 …”

“…As the dimer, the enzyme is active, and the disordered C-terminal residues of the monomer form two helices, one that functions as a major contact surface at the dimer interface and one that interacts with the catalytic site. This disorder-to-order transition links the dimer interface to the catalytic site. , Given the evidence supporting an allosteric link between Pr dimerization and activation, we have focused our efforts on identifying molecules that target the dimer interface. ,,,,, In doing so, we previously identified a small molecule inhibitor of KSHV Pr designated DD2 [compound 1 (Table )]. , …”

“…6,12,16,22,23,28 In doing so, we previously identified a small molecule inhibitor of KSHV Pr designated DD2 [compound 1 (Table 1)]. 29,30 DD2, a benzyl-substituted 4-(pyridine-2-amido)benzoic acid, is a helical peptide mimetic and allosteric inhibitor that prevents the disorder-to-order transition that activates KSHV Pr, thus trapping an inactive monomeric state. 27,30 The primary DD2 binding pocket, ∼15 Å from the active site, is formed by conformational changes that occur only in the partially disordered monomer.…”