Determination of in Vivo Enzyme Occupancy Utilizing Inhibitor Dissociation Kinetics

During drug discovery, assessment of in vivo target occupancy by therapeutic candidates is often required for predicting clinical efficacy. Current strategies for determining target occupancy include using radiolabeled or irreversible surrogates, which can be technically challenging, and the results are often not sufficiently quantitative. We developed a straightforward method by applying slow-dissociation kinetics to quantitatively determine enzyme occupancy without using specialized reagents. We applied this method to determine occupancy of Cathepsin K inhibitors in bone tissues harvested from rabbit femurs. Tissues from dosed animals were harvested, flash frozen, lysed, then analyzed by a jump-dilution assay with substrate. The rate of substrate turnover was monitored continuously until reaching steady state and progress curves were fit with the equation [product] = <i>v</i>_s<i>t</i> + ((<i>v</i>_i – <i>v</i>_s)/<i>k</i>_obs)­(1 – exp­(−<i>k</i>_obs<i>t</i>)). The initial rate <i>v</i>_<i>i</i> represents the residual activity of the enzyme before inhibitor dissociation; <i>v</i>_s is the reaction rate after dissociation of the inhibitor. Occupancy is derived from the ratio of <i>v</i>_<i>i</i>/<i>v</i>_s. A significant benefit of the method is that data from both the occupied and unoccupied states are obtained in the same assay under identical conditions, which provides greater consistency between studies. The Cat K inhibitor MK-0674 (in vitro IC₅₀ 1 nM) was tested in young rabbits (<6 month old) and showed a dose-dependent increase in occupancy, reaching essentially complete occupancy at 1.0 mg/kg. In addition the method enables measurement of the total Cat K in the target tissue. Results confirmed complete occupancy even as the osteoclasts responded to higher doses with increased enzyme production

Discovery of a Distinct Chemical and Mechanistic Class of Allosteric HIV‑1 Integrase Inhibitors with Antiretroviral Activity

Allosteric integrase inhibitors (ALLINIs) bind to the lens epithelial-derived growth factor (LEDGF) pocket on HIV-1 integrase (IN) and possess potent antiviral effects. Rather than blocking proviral integration, ALLINIs trigger IN conformational changes that have catastrophic effects on viral maturation, rendering the virions assembled in the presence of ALLINIs noninfectious. A high-throughput screen for compounds that disrupt the IN·LEDGF interaction was executed, and extensive triage led to the identification of a <i>t</i>-butylsulfonamide series, as exemplified by <b>1</b>. The chemical, biochemical, and virological characterization of this series revealed that <b>1</b> and its analogs produce an ALLINI-like phenotype through engagement of IN sites distinct from the LEDGF pocket. Key to demonstrating target engagement and differentiating this new series from the existing ALLINIs was the development of a fluorescence polarization probe of IN (FLIPPIN) based on the <i>t-</i>butylsulfonamide series. These findings further solidify the late antiviral mechanism of ALLINIs and point toward opportunities to develop structurally and mechanistically novel antiretroviral agents with unique resistance patterns