Research laboratory of Michelle Arkin, PhD
Chemical Biology and Biophysics Laboratory

Research & Projects

The Arkin Lab develops innovative approaches to screen for chemical tools and drug leads using biophysical approaches like fragment-based drug discovery and biological approaches including high-content imaging with primary cells and organisms. Our goal is to demonstrate “druggability” of new target classes and to use our compounds to discover new targets for drug discovery.

Small-molecule inhibitors for protein-protein interactions

Protein-protein interaction (PPI) networks are crucial to the signal transduction that underlies cellular processes as diverse as differentiation, proliferation, intercellular signaling, and cell death. In principle, small molecules and peptides would provide ideal tools to selectively inhibit a PPI with rapid and reversible kinetics. In practice, however, small-molecule inhibitors of PPI have been difficult to develop, and native peptides generally show poor cell permeability. Research in our laboratory is therefore aimed at developing the screening and fragment-based approaches that will allow the discovery of PPI inhibitors. Our ongoing PPI projects, some partnered with pharmaceutical companies, are focused on integrins, hormone/receptor complexes, and scaffolding sites on kinases. The long-term goals are two-fold:

  1. Develop molecular probes that selectively inhibit PPIs in order to advance the biological understanding of signal-transduction networks.
  2. Delineate the rules that underlie molecular-recognition events between small-molecule/protein and peptide/protein complexes.

Our earlier work with interleukin-2/IL2 receptor and ICAM/LFA1 led to PPI antagonists with nanomolar potency and underscored the role of protein dynamics in small-molecule binding.

Structure-function of proteases

In some ways, protease-substrate complexes are protein-protein complexes that carry out chemical reactions. We are thus extending our program in PPI inhibitor discovery to include dimeric proteases taspase1 (cancer) and caspases-2 and -6 (neurodegeneration). Caspases play key roles in inflammation, apoptosis, and neurodegeneration. However, it has been very difficult to identify reversible, cell-active, selective caspase inhibitors. We have used HTS, x-ray crystallography, and fragment-based discovery to address the structure and function of caspase/small-molecule interaction. In the case of caspase-2, we have solved the first crystal structures of apo caspase-2 and caspase-2 in complex with several peptide inhibitors. These complexes shed light on the recognition of pentapeptides by caspase-2 and the sequence of events leading to catalysis.

caspase-2

Snapshots along the catalytic pathway. Apo-caspase-2 is in an inactive form (1st structure) until binding of a peptide (green) breaks an inhibitory salt bridge (red and blue). Substrates bind to form the enzyme/substrate complex (2nd and 3rd structures), then react with the catalytic residue to form a covalent intermediate (3rd and 4th structures).  

For caspase-6 we have developed two series of compounds that selectively inhibit caspase-6 by different mechanisms. In collaboration with the Renslo Lab and Genentech, we identified potent caspase-6 inhibitors that exhibit exquisite selectivity over other caspases by binding uncompetitively to the enzyme/substrate complex. We have identified a second set of inhibitors through a fragment-based approach called disulfide trapping, described below. Our screening of wild-type caspase-6 selected compounds that bind to a non-conserved (non-active site) cysteine residue, which points toward the active site. Disulfide-bound fragments inhibit enzymatic activity in biochemical assays and in cell lysates and are highly selective for caspase-6 over other caspases. These two classes of compounds could be among the first inhibitors that can carefully probe caspase-6 activity in cellular assays of neurodegeneration.

caspase-6

Caspase-6 inhibitors found through high-throughput screening bind uncompetitively to caspase-6 + tetrapeptide substrate. (See Mechanistic and structural understanding of uncompetitive inhibitors of caspase-6.)

Cell-active probes

Cell-active compounds are crucial tools for dissecting biological pathways in disease. Disulfide trapping is a site-directed method in which a native or engineered cysteine residue on the protein captures thiol-containing fragments. We are developing this technology to rapidly identify such tools for protein-protein interactions. The potential of this concept is highlighted by the caspase-6 disulfide-trapped compounds and by cysteine-reactive compounds designed in the Cravatt Lab and the Taunton Lab. Our aim is to discover disulfide compounds that bind to specific sites on proteins, followed by chemical modification of the disulfide to make a reversible or irreversible electrophile. Tuning the chemical reactivity of this warhead will allow us to dial in potency and selectivity for the target in cells.

site-directed ligand discovery

Site-directed ligand discovery by disulfide-trapping (tethering)

Probe and lead discovery using high-content imaging

High-content analysis (HCA) is a powerful assay methodology that combines high-throughput microscopy and automated image analysis. One challenge with traditional cell-based screens is the use of engineered cell lines, which might not recapitulate all the relevant biological states. We hypothesize that cell-based screens designed with primary cells will yield small molecules that translate more directly to model organisms and humans. Toward this aim, we have developed HCA for human hematopoetic stem cells, primary immune cells from brain, and ciliated fibroblasts.

Another focus is screening and characterization of whole parasitic organisms (Giardia, Trypanosoma cruzi, Leishmania, Schistosoma mansoni), developed in partnership with the Center for Discovery and Innovation in Parasitic Diseases (CDIPD) at UCSF. One particularly interesting program is a collaboration with Conor Caffrey, PhD, (Pathology, UC San Francisco) and Rahul Singh, PhD, (Biocomputing, San Francisco State University) to develop high-throughput and quantitative methods to phenotype the helminth Schistosoma mansoni, the causative agent of schistosomiasis. Using live, time-resolved imaging, we define the shapes and motions of these worms in response to stimuli. Correlating phenotypes with known perturbations (drugs, siRNA, metabolites) is helping us understand these agents and is being used to find and characterize new hits from HTS. The current assay will dramatically increase our understanding of parasitic worms and will streamline the search for new anti-helminthics.

Schistosomula

Schistosomula, the youth-stage of the human parasite S. mansoni, swimming to the center of a well in a 96-well plate.