Enhanced potency of the metalloprotease inhibitor TAPI-2 by multivalent display.

Metalloproteases regulate a vast array of critical cellular processes such as proliferation, migration, repair, and invasion/metastasis. In so doing, metalloproteases have been shown to play key roles in the pathogenesis of multiple disorders including arteriosclerosis, arthritis, cancer metastasis, and ischemic brain injury. Therefore, much work has focused on developing metalloprotease inhibitors to provide a potential therapeutic benefit against the progression of these and other diseases. In order to produce a more potent inhibitor of metalloproteases, we synthesized multivalent displays of a metalloprotease inhibitor derived from the ring-opening metathesis polymerization (ROMP). Specifically, multivalent ligands of a broad-spectrum metalloprotease inhibitor, TAPI-2, were generated upon conjugation of the amine-bearing inhibitor with the ROMP-derived N-hydroxysuccinimide ester polymer. By monitoring the metalloprotease dependent cleavage of the transmembrane protein Semaphorin4D (Sema4D), we demonstrated an enhancement of inhibition by multivalent TAPI-2 compared to monovalent TAPI-2. To further optimize the potency of the multivalent inhibitor, we systematically varied the polymer length and inhibitor ligand density (mole fraction, χ). We observed that while ligand density plays a modest role in the potency of inhibition caused by the multivalent TAPI-2 display, the length of the polymer produces a much greater effect on inhibitor potency, with the shortest polymer achieving the greatest level of inhibition. These findings validate the use of multivalent display to enhance the potency of metalloprotease inhibitors and further, suggest this may be a useful approach to enhance potency of other small molecule towards their targets.

Inhibiting a dynamic viral protease by targeting a non-catalytic cysteine.

Viruses are responsible for some of the most deadly human diseases, yet available vaccines and antivirals address only a fraction of the potential viral human pathogens. Here, we provide a methodology for managing human herpesvirus (HHV) infection by covalently inactivating the HHV maturational protease via a conserved, non-catalytic cysteine (C161). Using human cytomegalovirus protease (HCMV Pr) as a model, we screened a library of disulfides to identify molecules that tether to C161 and inhibit proteolysis, then elaborated hits into irreversible HCMV Pr inhibitors that exhibit broad-spectrum inhibition of other HHV Pr homologs. We further developed an optimized tool compound targeted toward HCMV Pr and used an integrative structural biology and biochemical approach to demonstrate inhibitor stabilization of HCMV Pr homodimerization, exploiting a conformational equilibrium to block proteolysis. Irreversible HCMV Pr inhibition disrupts HCMV infectivity in cells, providing proof of principle for targeting proteolysis via a non-catalytic cysteine to manage viral infection.

Fragment-Based Protein-Protein Interaction Antagonists of a Viral Dimeric Protease.

Fragment-based drug discovery has shown promise as an approach for challenging targets such as protein-protein interfaces. We developed and applied an activity-based fragment screen against dimeric Kaposi's sarcoma-associated herpesvirus protease (KSHV Pr) using an optimized fluorogenic substrate. Dose-response determination was performed as a confirmation screen, and NMR spectroscopy was used to map fragment inhibitor binding to KSHV Pr. Kinetic assays demonstrated that several initial hits also inhibit human cytomegalovirus protease (HCMV Pr). Binding of these hits to HCMV Pr was also confirmed by NMR spectroscopy. Despite the use of a target-agnostic fragment library, more than 80 % of confirmed hits disrupted dimerization and bound to a previously reported pocket at the dimer interface of KSHV Pr, not to the active site. One class of fragments, an aminothiazole scaffold, was further explored using commercially available analogues. These compounds demonstrated greater than 100-fold improvement of inhibition. This study illustrates the power of fragment-based screening for these challenging enzymatic targets and provides an example of the potential druggability of pockets at protein-protein interfaces.

Determination of ubiquitin fitness landscapes under different chemical stresses in a classroom setting.

Ubiquitin is essential for eukaryotic life and varies in only 3 amino acid positions between yeast and humans. However, recent deep sequencing studies indicate that ubiquitin is highly tolerant to single mutations. We hypothesized that this tolerance would be reduced by chemically induced physiologic perturbations. To test this hypothesis, a class of first year UCSF graduate students employed deep mutational scanning to determine the fitness landscape of all possible single residue mutations in the presence of five different small molecule perturbations. These perturbations uncover 'shared sensitized positions' localized to areas around the hydrophobic patch and the C-terminus. In addition, we identified perturbation specific effects such as a sensitization of His68 in HU and a tolerance to mutation at Lys63 in DTT. Our data show how chemical stresses can reduce buffering effects in the ubiquitin proteasome system. Finally, this study demonstrates the potential of lab-based interdisciplinary graduate curriculum.