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.

Infections with the human immunodeficiency virus, which inevitably lead to the development of AIDS, are still among the most serious global health problems causing more than 2.5 million deaths per year. In the pathophysiological processes of this pandemic, HIV protease has proven to be an invaluable drug target because of its essential role in the virus' replication process. By use of pyrrolidine as core structure, symmetric 3,4-bis-N-alkylsulfonamides were designed and synthesized enantioselectively from D-(-)-tartaric acid as a new class of HIV protease inhibitors. Structure-guided design using the cocrystal structure of an initial lead as starting point resulted in a second series of inhibitors with improved affinity. The binding modes of four representatives were determined by X-ray crystallography to elucidate the underlying factors accounting for the SAR. With this information for further rational design, the combination of suitable side chains resulted in a final inhibitor showing a significantly improved affinity of K(i) = 74 nM.

KRAS, the most common oncogenic driver in human cancers, is controlled and signals primarily through protein−protein interactions (PPIs). The interaction between KRAS and SOS1, crucial for the activation of KRAS, is a typical, challenging PPI with a large contact surface area and high affinity. Here, we report that the addition of only one atom placed between Y884 SOS1 and A73 KRAS is sufficient to convert SOS1 activators into SOS1 inhibitors. We also disclose the discovery of BI-3406. Combination with the upstream EGFR inhibitor afatinib shows in vivo efficacy against KRAS G13D mutant colorectal tumor cells, demonstrating the utility of BI-3406 to probe SOS1 biology. These findings challenge the dogma that large molecules are required to disrupt challenging PPIs. Instead, a "foot in the door" approach, whereby single atoms or small functional groups placed between key PPI interactions, can lead to potent inhibitors even for challenging PPIs such as SOS1-KRAS.

◥Natural killer (NK) cells play a pivotal role in controlling cancer. Multiple extracellular receptors and internal signaling nodes tightly regulate NK activation. Cyclin-dependent kinases of the mediator complex (CDK8 and CDK19) were described as a signaling intermediates in NK cells. Here, we report for the first time the development and use of CDK8/19 inhibitors to suppress phosphorylation of STAT1 S727 in NK cells and to augment the production of the cytolytic molecules perforin and granzyme B (GZMB). Functionally, this resulted in enhanced NK-cell-mediated lysis of primary leukemia cells. Treatment with the CDK8/19 inhibitor BI-1347 increased the response rate and survival of mice bearing melanoma and breast cancer xenografts. In addition, CDK8/19 inhibition augmented the antitumoral activity of anti-PD-1 anti-body and SMAC mimetic therapy, both agents that promote T-cellmediated antitumor immunity. Treatment with the SMAC mimetic compound BI-8382 resulted in an increased number of NK cells infiltrating EMT6 tumors. Combination of the CDK8/19 inhibitor BI-1347, which augments the amount of degranulation enzymes, with the SMAC mimetic BI-8382 resulted in increased survival of mice carrying the EMT6 breast cancer model. The observed survival benefit was dependent on an intermittent treatment schedule of BI-1347, suggesting the importance of circumventing a hyporesponsive state of NK cells. These results suggest that CDK8/19 inhibitors can be combined with modulators of the adaptive immune system to inhibit the growth of solid tumors, independent of their activity on cancer cells, but rather through promoting NK-cell function.