KRAS is the most frequently mutated driver of pancreatic, colorectal, and non-small cell lung cancers. Direct KRAS blockade has proven challenging and inhibition of a key downstream effector pathway, the RAF-MEK-ERK cascade, has shown limited success due to activation of feedback networks that keep the pathway in check. We hypothesized that inhibiting SOS1, a KRAS activator and important feedback node, represents an effective approach to treat KRAS-driven cancers. We report the discovery of a highly potent, selective and orally bioavailable small-molecule SOS1 inhibitor, BI-3406, that binds to the catalytic domain of SOS1 thereby preventing the interaction with KRAS. BI-3406 reduces formation of GTPloaded RAS and limits cellular proliferation of a broad range of KRAS-driven cancers.Importantly, BI-3406 attenuates feedback reactivation induced by MEK inhibitors and thereby enhances sensitivity of KRAS-dependent cancers to MEK inhibition. Combined SOS1 and MEK inhibition represents a novel and effective therapeutic concept to address KRAS-driven tumors. SignificanceTo date, there are no effective targeted pan-KRAS therapies. In-depth characterization of BI-3406 activity and identification of MEK inhibitors as effective combination partners provide an attractive therapeutic concept for the majority of KRAS mutant cancers, including those fueled by the most prevalent mutant KRAS oncoproteins G12D, G12V, G12C and G13D.Research.

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In contrast with the very well explored concept of structure-activity relationship, similar studies are missing for the dependency between binding kinetics and compound structure of a protein ligand complex, the structure-kinetic relationship. Here, we present a structure-kinetic relationship study of the cyclin-dependent kinase 8 (CDK8)/cyclin C (CycC) complex. The scaffold moiety of the compounds is anchored in the kinase deep pocket and extended with diverse functional groups toward the hinge region and the front pocket. These variations can cause the compounds to change from fast to slow binding kinetics, resulting in an improved residence time. The flip of the DFG motif ("DMG" in CDK8) to the inactive DFG-out conformation appears to have relatively little influence on the velocity of binding. Hydrogen bonding with the kinase hinge region contributes to the residence time but has less impact than hydrophobic complementarities within the kinase front pocket.kinetic profiling | structure-based drug design T he cyclin-dependent kinase 8/cyclin C (CDK8/CycC) complex is a potent oncogene (1-4), involved in transcription and regulation of transcriptional activity (5-10), linked to epigenetic processes (11), and regarded as an attractive drug target. The recent clinical success of the small molecule inhibitors sorafenib (BAY-43006, Bayer Pharma) and imatinib (STI-571, Novartis Pharma AG) has been attributed to their deep pocket binding mode (12). The "deep pocket" (13) is adjacent to the kinase ATP binding site and accessible in protein kinases by the rearrangement of the DFG motif (a short motif composed of the residues AspPhe-Gly near the N-terminal region of the activation loop) from the active (DFG-in) to the inactive state (DFG-out) (13,14). Binding of a type II compound to such a DFG-out conformation often includes slow binding kinetics (15) with a prolonged "residence time," which is defined as the period for which a target is occupied by a compound (16). Residence time is currently considered to be a key success factor for compound optimization during drug discovery and perhaps as important as the apparent affinity such as half-inhibitory concentration (IC 50 ) or dissociation constant (K d ) (16,17). Accordingly, residence time could be a key to providing enhanced potency in vivo (18) and a means to improve the correlation of in vitro and in vivo efficacy of drugs (19). Despite the increased acceptance of the need to understand binding kinetics, the interplay between compound-target interactions and binding kinetics is in general too complex and poorly understood to enable prediction of the essential dynamic properties. The impact of the combination of kinetic data of proteininhibitor interactions with crystallographic studies has been recognized with pioneer studies such as bovine trypsin in complex with the bovine pancreatic trypsin inhibitor (20). The concept of structure-activity relationships describing the interdependency between binding affinity and compound structure has been well explored in the l...

Here we report the fragment-based discovery of BI-9321 (17), a potent, selective, and cellular active antagonist of the NSD3-PWWP1 domain. The human NSD3 protein is encoded by the WHSC1L1 gene located in the 8p11-p12 amplicon, frequently amplified in breast and squamous lung cancer. Recently it was demonstrated that the PWWP1 domain of NSD3 is required for the viability of Acute Myeloid Leukemia cells. To further elucidate the relevance of NSD3 in cancer biology, we developed a chemical probe BI-9321 (17) targeting the methyl-lysine binding site of the PWWP1 domain with sub-micromolar in vitro activity and cellular target engagement at 1 µM. As a single agent BI-9321 (17) downregulates Myc mRNA expression and reduces proliferation in MOLM-13 cells. This first-in-class chemical probe BI-9321 (17), together with the negative control BI-9466 (12), will greatly facilitate the elucidation of the underexplored biological function of PWWP domains.

Potent, selective and broadly characterized small molecule modulators of protein function (chemical probes) are powerful research reagents. The pharmaceutical industry has generated many high-quality chemical probes and several of these have been made available to academia. However, probe-associated data and control compounds, such as inactive structurally related molecules and their associated data, are generally not accessible. The lack of data and guidance makes it difficult for researchers to decide which chemical tools to choose. Several pharmaceutical companies (AbbVie, Bayer, Boehringer Ingelheim, Janssen, MSD, Pfizer, and Takeda) have therefore entered into a pre-competitive collaboration to make available a large number of innovative high-quality probes, including all probe-associated data, control compounds and recommendations on use (https://openscienceprobes.sgc-frankfurt.de/). Here we describe the chemical tools and target-related knowledge that have been made available, and encourage others to join the project.