The HTS by NMR approach consists of screening positional scanning combinatorial libraries of peptide mimetics using robust protein-NMR spectroscopy to identify initial PPIs ligands, that in turn can be iteratively optimized into low nanomolar possible leads. We found that a strategy to expedite the lengthy optimization process is the introduction of sulfonyl fluorides of fluorosulfates that when properly juxtaposed can covalently react with surface Lys or Tyr residues. The two strategies combined can be of general applicability to derive potent and selective PPIs ligands. I will present examples of the successful implementation of these strategies against several targets.

Stabilization of protein-protein interactions remains largely underexplored, compared to inhibition. Here, we apply a site-directed fragment-based screening for the systematic discovery of stabilizers, aiming to stabilize the interaction between the hub protein 14-3-3s and client peptides using covalent fragments. Orthogonal biophysical assays were applied for fragment validation and the mechanism of stabilization was confirmed by X-ray crystallography. Aspects of selectivity for representative 14-3-3 clients were also explored.

The presentation will highlight and exemplify a range of biophysical approaches that can be utilized during the identification as well as characterization of covalent inhibitors. Those techniques and strategies aim to inform about the mechanism of inhibition by simultaneously providing data on affinity and the rate of covalent bond formation. Particular focus will be on the use of regenerable, SPR-based Biosensors as novel means for covalent inhibitor characterization.

We describe here methods and equations to fit ligand affinity from irreversible protein denaturation. Irreversible denaturation occurs for most proteins, particularly in the space of human therapeutics, but equations to fit these data have eluded investigators for many years. These results suggest the kinetic energy barrier for unfolding is similar across proteins; application of these findings should allow investigators to calculate ligand affinity from a single thermal denaturation data point.

Most PPI inhibitors on the market are antibody-based because it’s easier to disrupt PPIs with larger molecules. I analyzed publicly available data to compare different binding spaces between PPIs and their antibody disrupters to discern general scaffold features amenable to smaller disruptors. Focusing on binding affinities, buried surface areas and epitope characteristics, differences and similarities will be highlighted with an attempt to draw general small molecule and non-immunoglobin selection principles.

Gamma-Secretase is an aspartyl protease that cleaves APP to generate Abeta species, including the neurotoxic Abeta-42 which is believed to play a causative role in Alzheimer’s disease. Gamma-Secretase modulators (GSMs) have emerged as a potential treatment for AD because they decrease the production of Abeta-42 without affecting the processing of other critical gamma-secretase substrates. We used clickable GSM photoaffinity probes to show that different classes of GSMs have distinct allosteric binding sites on the N-terminal fragment of presenilin 1 (PS1-NTF). In addition, we found that one photoprobe labeled IFITM3 and demonstrated the IFITM3 is part of the gamma-secretase complex.

g-Secretase (GS) is a key target for a potential Alzheimer’s disease treatment. Inhibiting GS led to serious side effects; modulating GS has greater safety potential. We report the discovery of a potent and selective gamma secretase modulator (GSM) (S)-3 (RO7185876), belonging to a novel chemical class, the triazolo-azepines demonstrating an excellent in-vitro and in-vivo DMPK profile. Furthermore, we propose a novel phenyl bioisostere with strongly improved drug-like properties.

Revolution Medicines is developing novel RAS(ON) Inhibitors based on our proprietary tri-complex technology platform, enabling a highly differentiated approach to inhibiting the active, GTP-bound form of RAS (RAS(ON)). We will discuss a portfolio of compounds that we believe are the first and only RAS(ON) inhibitors to use this mechanism of action. RMC-6291, our inhibitor targeting KRAS^G12C/NRAS^G12C(ON), and RMC-6236, our inhibitor of multiple RAS variants (RAS^MULTI(ON)), are in IND-enabling preclinical development.

KRAS, the most common oncogenic driver in human cancers, is controlled and signals through PPIs. Herein, we report the evolution of BI-3406, an orally available inhibitor of the SOS1-KRAS PPI. It decreases formation of GTP-KRAS, inhibits downstream signaling and limits proliferation of KRAS-driven cancers. BI-3406 served as probe for the study of SOS1 and KRAS biology and paved the way to clinical trials in combination with a MEK inhibitor.

The small GTPase, KRAS, is an oncogene and desirable drug target due to the prevalence of mutations with poor disease prognosis. To facilitate drug discovery targeting KRAS/MAPK pathway, we have produced the full spectrum of pathway proteins, including kinases, wild type, and mutated KRAS, and the upstream nucleotide exchange factor SOS1. In this talk, we will present our KRAS pathway assay portfolio, including biochemical and biophysical assays.