BomTac:NMRI-Foxn1nu mice bearing human MIAPaCa-2 tumors were treated with vehicle, BI 847325 at 10 mg/kg daily p.o., AZD6244 at 25 mg/kg twice daily p.o., GSK 1120212 at 0.25 mg/kg twice daily p.o. Data is plotted as average tumor volume with standard error of the mean (SEM). Adjusted p values: BI 847325 vs AZD6244 p<0.0001(statistical significant difference); BI 847325 vs GSK 1120212 p<0.0001(statistical significant difference).

Chemical structure of BI 811283: 2,3-diamino-pyrimidine

Supplementary Materials and Methods, Tables S1 through S3 and Figures S1 and S2 - Supplementary Materials and Methods: The sources, application and dilution of the antibodies used for Western blotting (WB) and immunohistochemical (IHC) analyses in this study are listed. Supplementary Tables: The supplementary tables demonstrate the data and statistics from x-ray co-crystallization experiments with BI 885578 and the human INSR kinase domain (Tables S1 and S2) and the kinase selectivity of BI 885578 on a large panel of human kinases (Table S3). Supplementary Figures: The supplementary figures demonstrate the efficacy and tolerability of BI 885578 in the CL-14 colon cancer xenograft model (Figure S1) and the PD effects of BI 885578 on the phosphorylation of the IGF1R and INSR in GEO tumors (Figure S2).

Supplementary Table S1: Statistics of data collection and processing of AuroraB/INCENP in complex with BI 847325; Supplementary Table S2: Statistics of data collection and processing of MEK1 in complex with BI 847325; Supplementary Table S3: Statistics of data collection and processing of AuroraB/INCENP in complex with BI 811283; Supplementary Table S4: Pharmacokinetic parameters of BI 847325 in mice and humans. Human data from Schöffski et al (31).

Supplementary Data including Table S1-Table S5 and Figure S1 - Figure S9

(A), MEK1 and phospho-MEK1/2; (B) ERK1/2 and phospho-ERK1/2.

Supplementary Methods, Figure 1, Table 1 from BIBF 1120: Triple Angiokinase Inhibitor with Sustained Receptor Blockade and Good Antitumor Efficacy

Supplementary Data, Table S1, S3, S6, S8, S10-S13

Supplementary Figures S1-S5

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 Y884SOS1 and A73KRAS 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 KRASG13D 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.

PROTACs have shown promise as a new class of therapy, with a unique mechanism of action orthogonal to traditional small molecules that are used to regulate protein activity. Their novel MOA utilizing the body's natural protein degradation machinery degrades a protein of interest rather than inhibiting its function. This strategy has several advantages over conventional small-molecule inhibitors, e.g., higher sensitivity, less off-target effects, and greater target space. However, unlocking the potential of PROTACs necessitates drug discovery techniques that can support the complexity of the novel MOA. In this chapter, we describe the application of MicroScale Thermophoresis (MST) and Temperature-Related Intensity Change (TRIC) to characterize both the binary and ternary binding of PROTACs with target proteins and ubiquitin ligases along with an efficient determination of the cooperativity of the ternary complex formation. The assay development and experimental procedure to characterize the well-described BET PROTAC MZ1 show how MST and TRIC can be applied as a fast and highly sensitive method for PROTAC discovery.

Abstract KRAS is the most frequently mutated driver of pancreatic, colorectal, and non–small cell lung cancers. Direct KRAS blockade has proved challenging, and inhibition of a key downstream effector pathway, the RAF–MEK–ERK cascade, has shown limited success because of 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 GTP-loaded 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. Significance: To 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. See related commentary by Zhao et al., p. 17. This article is highlighted in the In This Issue feature, p. 1

AbstractNatural 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 STAT1S727 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 antibody and SMAC mimetic therapy, both agents that promote T-cell–mediated 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.

We thank Tran et al. (1) for their appreciation of our open innovation platform (https://opnme.com/) and interest in our manuscript (2) in which we demonstrate that the small-molecule KRAS inhibitor BI-2852 reduces pERK (EC50 = 6 µM) and inhibits proliferation (EC50 = 7 µM) in NCI-H358 cells. Tran et al. (1) propose that BI-2852 exerts its cellular effects on the MAPK pathway at least in part through stabilization of a nonfunctional KRAS dimer. No cellular data for KRAS dimer induction are provided and the hypothesis is primarily based on KRAS dimer formation observed with size exclusion chromatography using concentrations 1,000 times higher (3 mM) than those in which the cellular effects of BI-2852 are observed. Hence, inhibiting the protein–protein interactions between KRAS and its GEFs, GAPs, and downstream effectors is a more plausible explanation than … [↵][1]1To whom correspondence should be addressed. Email: darryl.mcconnell{at}boehringer-ingelheim.com. [1]: #xref-corresp-1-1

Focal adhesion tyrosine kinase (PTK2) is often overexpressed in human hepatocellular carcinoma (HCC), and several reports have linked PTK2 depletion and/or pharmacological inhibition to reduced tumorigenicity. However, the clinical relevance of targeting PTK2 still remains to be proven. Here, we present two highly selective and functional PTK2 proteolysis-targeting chimeras utilizing von Hippel-Lindau and cereblon ligands to hijack E3 ligases for PTK2 degradation. BI-3663 (cereblon-based) degrades PTK2 with a median DC50 of 30 nM to >80% across a panel of 11 HCC cell lines. Despite effective PTK2 degradation, these compounds did not phenocopy the reported antiproliferative effects of PTK2 depletion in any of the cell lines tested. By disclosing these compounds, we hope to provide valuable tools for the study of PTK2 degradation across different biological systems.

Here, we report the fragment-based discovery of BI-9321, 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, 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 downregulates Myc messenger RNA expression and reduces proliferation in MOLM-13 cells. This first-in-class chemical probe BI-9321, together with the negative control BI-9466, will greatly facilitate the elucidation of the underexplored biological function of PWWP domains. A chemical probe BI-9321 for the PWWP1 domain of NSD3 and its inactive analog were identified. BI-9321 binds to the methyl-lysine binding site, reduces the association of NSD3 with chromatin and inhibits proliferation of acute myeloid leukemia cells.

Phosphoglycerate dehydrogenase (PHGDH) is known to be the rate-limiting enzyme in the serine synthesis pathway in humans. It converts glycolysis-derived 3-phosphoglycerate to 3-phosphopyruvate in a co-factor-dependent oxidation reaction. Herein, we report the discovery of BI-4916, a prodrug of the co-factor nicotinamide adenine dinucleotide (NADH/NAD(+))-competitive PHGDH inhibitor BI-4924, which has shown high selectivity against the majority of other dehydrogenase targets. Starting with a fragment-based screening, a subsequent hit optimization using structure-based drug design was conducted to deliver a single-digit nanomolar lead series and to improve potency by 6 orders of magnitude. To this end, an intracellular ester cleavage mechanism of the ester prodrug was utilized to achieve intracellular enrichment of the actual carboxylic acid based drug and thus overcome high cytosolic levels of the competitive cofactors NADH/NAD(+).

Targeting subunits of BAF/PBAF chromatin remodeling complexes has been proposed as an approach to exploit cancer vulnerabilities. Here, we develop proteolysis targeting chimera (PROTAC) degraders of the BAF ATPase subunits SMARCA2 and SMARCA4 using a bromodomain ligand and recruitment of the E3 ubiquitin ligase VHL. High-resolution ternary complex crystal structures and biophysical investigation guided rational and efficient optimization toward ACBI1, a potent and cooperative degrader of SMARCA2, SMARCA4 and PBRM1. ACBI1 induced anti-proliferative effects and cell death caused by SMARCA2 depletion in SMARCA4 mutant cancer cells, and in acute myeloid leukemia cells dependent on SMARCA4 ATPase activity. These findings exemplify a successful biophysics- and structure-based PROTAC design approach to degrade high profile drug targets, and pave the way toward new therapeutics for the treatment of tumors sensitive to the loss of BAF complex ATPases.

The 3 human RAS genes, KRAS, NRAS, and HRAS, encode 4 different RAS proteins which belong to the protein family of small GTPases that function as binary molecular switches involved in cell signaling. Activating mutations in RAS are among the most common oncogenic drivers in human cancers, with KRAS being the most frequently mutated oncogene. Although KRAS is an excellent drug discovery target for many cancers, and despite decades of research, no therapeutic agent directly targeting RAS has been clinically approved. Using structure-based drug design, we have discovered BI-2852 (1), a KRAS inhibitor that binds with nanomolar affinity to a pocket, thus far perceived to be "undruggable," between switch I and II on RAS; 1 is mechanistically distinct from covalent KRASG12C inhibitors because it binds to a different pocket present in both the active and inactive forms of KRAS. In doing so, it blocks all GEF, GAP, and effector interactions with KRAS, leading to inhibition of downstream signaling and an antiproliferative effect in the low micromolar range in KRAS mutant cells. These findings clearly demonstrate that this so-called switch I/II pocket is indeed druggable and provide the scientific community with a chemical probe that simultaneously targets the active and inactive forms of KRAS.