Disulfide-crosslink analysis of the ubiquitin ligase Hrd1 complex during endoplasmic reticulum-associated protein degradation.

Misfolded proteins in the lumen of the endoplasmic reticulum (ER) are retrotranslocated into the cytosol and degraded by the ubiquitin-proteasome system, a pathway termed luminal ER-associated protein degradation. Retrotranslocation is mediated by a conserved protein complex, consisting of the ubiquitin ligase Hrd1 and four associated proteins (Der1, Usa1, Hrd3, and Yos9). Photocrosslinking experiments provided preliminary evidence for the polypeptide path through the membrane but did not reveal specific interactions between amino acids in the substrate and Hrd1 complex.

A chemical genetics approach to examine the functions of AAA proteins.

The structural conservation across the AAA (ATPases associated with diverse cellular activities) protein family makes designing selective chemical inhibitors challenging. Here, we identify a triazolopyridine-based fragment that binds the AAA domain of human katanin, a microtubule-severing protein. We have developed a model for compound binding and designed ASPIR-1 (allele-specific, proximity-induced reactivity-based inhibitor-1), a cell-permeable compound that selectively inhibits katanin with an engineered cysteine mutation.

Chemical strategies to overcome resistance against targeted anticancer therapeutics.

Emergence of resistance is a major factor limiting the efficacy of molecularly targeted anticancer drugs. Understanding the specific mutations, or other genetic or cellular changes, that confer drug resistance can help in the development of therapeutic strategies with improved efficacies. Here, we outline recent progress in understanding chemotype-specific mechanisms of resistance and present chemical strategies, such as designing drugs with distinct binding modes or using proteolysis targeting chimeras, to overcome resistance.

Distinct Mechanisms of Resistance to a CENP-E Inhibitor Emerge in Near-Haploid and Diploid Cancer Cells.

Aberrant chromosome numbers in cancer cells may impose distinct constraints on the emergence of drug resistance-a major factor limiting the long-term efficacy of molecularly targeted therapeutics. However, for most anticancer drugs we lack analyses of drug-resistance mechanisms in cells with different karyotypes. Here, we focus on GSK923295, a mitotic kinesin CENP-E inhibitor that was evaluated in clinical trials as a cancer therapeutic.

Analyzing Resistance to Design Selective Chemical Inhibitors for AAA Proteins.

Drug-like inhibitors are often designed by mimicking cofactor or substrate interactions with enzymes. However, as active sites are comprised of conserved residues, it is difficult to identify the critical interactions needed to design selective inhibitors. We are developing an approach, named RADD (resistance analysis during design), which involves engineering point mutations in the target to generate active alleles and testing compounds against them.

Designing Allele-Specific Inhibitors of Spastin, a Microtubule-Severing AAA Protein.

The bump-hole approach is a powerful chemical biology strategy to specifically probe the functions of closely related proteins. However, for many protein families, such as the ATPases associated with diverse cellular activities (AAA), we lack structural data for inhibitor-protein complexes to design allele-specific chemical probes. Here we report the X-ray structure of a pyrazolylaminoquinazoline-based inhibitor bound to spastin, a microtubule-severing AAA protein, and characterize the residues involved in inhibitor binding.

Designing a chemical inhibitor for the AAA protein spastin using active site mutations.

Spastin is a microtubule-severing AAA (ATPases associated with diverse cellular activities) protein needed for cell division and intracellular vesicle transport. Currently, we lack chemical inhibitors to probe spastin function in such dynamic cellular processes. To design a chemical inhibitor of spastin, we tested selected heterocyclic scaffolds against wild-type protein and constructs with engineered mutations in the nucleotide-binding site that do not substantially disrupt ATPase activity.

Proteome labeling and protein identification in specific tissues and at specific developmental stages in an animal.

Identifying the proteins synthesized at specific times in cells of interest in an animal will facilitate the study of cellular functions and dynamic processes. Here we introduce stochastic orthogonal recoding of translation with chemoselective modification (SORT-M) to address this challenge. SORT-M involves modifying cells to express an orthogonal aminoacyl-tRNA synthetase/tRNA pair to enable the incorporation of chemically modifiable analogs of amino acids at diverse sense codons in cells in rich media.