The discovery of druggable pockets within proteins that lack traditional active sites remains a significant challenge in the development of therapeutics. To address this, we developed Cysteine Mapping of Accessible Pockets (CysMAP), a method for identifying druggable pockets in proteins. CysMAP employs systematic pooled cysteine (Cys)-variant libraries screened against diverse covalent compound libraries by intact LC-MS. We applied CysMAP to 189 KRAS(G12D) variants, purifying KRAS Cys-variants and screening them against 47 covalent compounds, quantifying accessibility, and reactivity across KRAS(G12D). We discovered previously unidentified ligand-bound states of Cys-variants surrounding the KRAS switch-II pocket. Structural studies of the D92C variant in complex with the compound BI-1830 uncovered a distinct novel binding pocket, highlighting the inherent plasticity of the region between switch-II and α3, that can accommodate diverse chemical entities in various conformations. This method holds significant potential for advancing drug discovery efforts against elusive targets such as oncogenic RAS mutants.

The Son of Sevenless (SOS) protein family includes two highly homologous proteins, SOS1 and SOS2, that act as guanine nucleotide exchange factors (GEFs) for RAS proteins. They catalyze the GDP-to-GTP exchange, resulting in an increase of the active GTP-bound form of RAS. Despite highly similar structures and expression patterns, SOS1 is generally accepted as the dominant RAS GEF for downstream signaling in pathological states. Nonetheless, SOS2 has been reported to critically impact the RAS-PI3K/AKT signaling axis, especially in KRAS-driven cancer cell lines and in the absence of SOS1. Hence, therapeutic targeting of SOS2 may be an attractive strategy to target RAS-driven malignancies. Herein, we report the discovery and initial optimization of a selective quinazoline-based compound series that binds with micromolar affinity to the catalytic site of SOS2. We also disclose an additional, previously unreported binding site on SOS2 occupied by a different small molecule class.

Targeted protein degradation with compounds like proteolysis targeting chimeras (PROTACs) directs disease-associated proteins to the E3 ligase ubiquitin-proteasome system for removal. However, commonly employed E3 ligases such as cereblon (CRBN) are broadly expressed. To metabolically gate PROTAC activity, we developed an enzymatic activation strategy by integrating an O-GlcNAc modification to the cyclimids, ligands derived from the natural motifs recognized by CRBN. These sugar-coated PROTACs (SCPs) were designed using structural analyses of representative cyclimid degraders complexed with CRBN and target protein BRD4. We found that glycosylation of the cyclimid reduced CRBN binding and complex formation with BRD4 until enzymatic removal of the O-GlcNAc moiety by O-GlcNAcase (OGA). The requirement for enzymatic activation is demonstrated by in vitro biochemical binding, cellular degradation, and cell viability assays in engineered and native cell lines. O-GlcNAc is thus an effective mechanism to gate targeted protein degradation modalities that motivates the development of similar strategies to enhance selectivity with other protein modifications.

Transcription factors are generally considered challenging, if not "undruggable", targets but they promise new therapeutic options due to their fundamental involvement in many diseases. In this study, we aim to assess the ligandability of the C-terminal Rel-homology domain of nuclear factor of activated T cells 1 (NFAT1), a TF implicated in T-cell regulation. Using a combination of experimental and computational approaches, we demonstrate that small molecule fragments can indeed bind to this protein domain. The newly identified binder is the first small molecule binder to NFAT1 validated with biophysical methods and an elucidated binding mode by X-ray crystallography. The reported eutomer/distomer pair provides a strong basis for potential exploration of higher potency binders on the path toward degrader or glue modalities.

The WWE domain is a relatively under-researched domain found in twelve human proteins and characterized by a conserved tryptophan-tryptophan-glutamate (WWE) sequence motif. Six of these WWE domain-containing proteins also contain domains with E3 ubiquitin ligase activity. The general recognition of poly-ADP-ribosylated substrates by WWE domains suggests a potential avenue for development of Proteolysis-Targeting Chimeras (PROTACs). Here, we present novel crystal structures of the HUWE1, TRIP12, and DTX1 WWE domains in complex with PAR building blocks and their analogs, thus enabling a comprehensive analysis of the PAR binding site structural diversity. Furthermore, we introduce a versatile toolbox of biophysical and biochemical assays for the discovery and characterization of novel WWE domain binders, including fluorescence polarization-based PAR binding and displacement assays, 15N-NMR-based binding affinity assays and 19F-NMR-based competition assays. Through these assays, we have characterized the binding of monomeric iso-ADP-ribose (iso-ADPr) and its nucleotide analogs with the aforementioned WWE proteins. Finally, we have utilized the assay toolbox to screen a small molecule fragment library leading to the successful discovery of novel ligands targeting the HUWE1 WWE domain. Six WWE domain-containing proteins also contain domains with E3 ubiquitin ligase activity. We report structures, biophysical and biochemical assays for the discovery of novel WWE domain binders and their respective application.

Abstract Pancreatic ductal adenocarcinoma (PDAC) is predicted to become the second most common cause of cancer related death within the next decade. Late detection and limited therapeutic options drive the dismal prognosis for PDAC patients. Activating mutations within the KRAS gene occur within the vast majority (>85%) of PDAC tumors. KRAS mutations occur at an early stage within PDAC development but remain necessary for continued tumor growth. Clinical responses observed in patients with KRASG12C mutant PDAC treated with selective KRASG12C inhibitors have driven drug development programmes that can address other, more frequently occurring KRAS alleles. Here we report the characterization of a KRASmulti inhibitor, BI-2493, that potently inhibits the proliferation of mutant KRAS PDAC cancer cell lines in vitro. The DMPK properties of the compound have been optimized so that oral administration resulted in regression of KRAS mutant PDAC xenograft models. These data support the continued development of compounds capable of addressing the most prevalent KRAS mutant alleles in PDAC with the potential to provide a therapeutic option to patients in an indication with a high unmet medical need. Citation Format: Antonio Tedeschi, Otmar Schaaf, Michael Gmachl, Lorenz Herdeis, Francesca Rocchetti, Johannes Popow, Fiorella Schischlik, Daniel Gerlach, Jesse Lipp, Joachim Bröker, Andreas Mantoulidis, Chris Smethurst, Dirk Kessler, Jark Boettcher, Tobias Wunberg, Valeria Santoro, Alex Waterson, Jason Phan, Andrew Little, Jason Abbott, Qi Sun, Stephen Fesik, Darryl Mcconnell, Mark A. Pearson, Norbert Kraut, Dorothea Rudolph. Development and characterisation of KRASmulti inhibitors for the treatment of KRAS mutant pancreatic ductal adenocarcinoma [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr B103.

Identifying promising chemical starting points for small molecule inhibitors of active, GTP-loaded KRAS “on” remains of great importance to clinical oncology and represents a significant challenge in medicinal chemistry. Here, we describe broadly applicable learnings from a KRAS hit finding campaign: While we initially identified KRAS inhibitors in a biochemical high-throughput screen, we later discovered that compound potencies were all but assay artifacts linked to metal salts interfering with KRAS AlphaScreen assay technology. The source of the apparent biochemical KRAS inhibition was ultimately traced to unavoidable palladium impurities from chemical synthesis. This discovery led to the development of a Metal Ion Interference Set (MIIS) for up-front assay development and testing. Profiling of the MIIS across 74 assays revealed a reduced interference liability of label-free biophysical assays and, as a result, provided general estimates for luminescence- and fluorescence-based assay susceptibility to metal salt interference.

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).

Large-scale cancer sequencing efforts coupled with deep functional genomic interrogation of cancer models provides us an increasingly detailed view of the critical cancer drivers required for persistent growth and survival of cancer cells. Targeting such drivers has provided significant therapeutic benefit to patients across a wide range of cancers, yet progress has been, in large part, confined to the targeting of catalytic domains in kinases. To expand the reach of therapeutics into new classes of therapeutic targets, we need to identify and validate specific mechanisms and pockets to enable drug discovery. To enable this, we are developing a method called Hit-finding by Cysteine-Scanning (HCS) for identifying druggable pockets in proteins of interest. Here, we seek to couple the ability to create systematic variant libraries across proteins of interest with increasingly diverse covalent small molecule/fragment libraries. Thus, HCS involves screening pooled cysteine (cys)-variants against libraries of covalent compounds. To demonstrate the utility of this approach, we applied HCS to systematically screen 189 KRAS-G12D variants. We successfully purified KRAS cys-variant recombinant proteins in pools of seven or eight achieving coverage of 184/189 variants. KRAS variant pools were then screened against a pilot library of 47 covalent compounds with diverse reactivity detecting reactive variant-ligand pairs by intact LC-MS. This allowed us to quantify the accessibility and reactivity across the entire KRAS-G12D protein. The data confirmed the known SII-pocket in KRAS and revealed potentially novel ligand-bound conformations of oncogenic KRAS mutant proteins. In parallel, the same KRAS cys-variant library was generated as a barcoded library in a mammalian expression system. Upon screening this library in intact cells we observed that Sotorasib functionally inhibit the relevant KRAS SII-pocket cys-variants, enabling enrichment of these variants in a negative selection screen. Importantly, binding of a panel of SII-pocket compounds to variant H95C was found to be independent of the KRAS mutant context. Our results indicate that HCS could be a useful tool for the development of inhibitors for currently undruggable oncogenic KRAS mutants as well as potentially other drug targets. Citation Format: Laurens Moore van Tienen, Shadwa Bayoumi, Khaja Muneeruddin, Nancy Leymarie, David Kornfilt, Yuemin Bian, Devishi Kesar, Ruitong Li, Thomas Atack, Alexandra-Mariela Popa, Zuzana Jandova, Philipp Trollmann, Andreas Bergner, Klaus Rumpel, Dirk Kessler, William R. Sellers. Hit-finding by Cysteine-Scanning (HCS): A method for finding druggable pockets [abstract]. In: Proceedings of the AACR Special Conference: Targeting RAS; 2023 Mar 5-8; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Res 2023;21(5_Suppl):Abstract nr B037.

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

Supplementary Figures S1-S5

Activating mutations in KRAS are the most frequent oncogenic alterations in cancer. The oncogenic hotspot position 12, located at the lip of the switch II pocket, offers a covalent attachment point for KRASG12C inhibitors. To date, KRASG12C inhibitors have been discovered by first covalently binding to the cysteine at position 12 and then optimizing pocket binding. We report on the discovery of the in vivo active KRASG12C inhibitor BI-0474 using a different approach, in which small molecules that bind reversibly to the switch II pocket were identified and then optimized for non-covalent binding using structure-based design. Finally, the Michael acceptor containing warhead was attached. Our approach offers not only an alternative approach to discovering KRASG12C inhibitors but also provides a starting point for the discovery of inhibitors against other oncogenic KRAS mutants.

Aberrant WNT pathway activation, leading to nuclear accumulation of β-catenin, is a key oncogenic driver event. Mutations in the tumor suppressor gene APC lead to impaired proteasomal degradation of β-catenin and subsequent nuclear translocation. Restoring cellular degradation of β-catenin represents a potential therapeutic strategy. Here, we report the fragment-based discovery of a small molecule binder to β-catenin, including the structural elucidation of the binding mode by X-ray crystallography. The difficulty in drugging β-catenin was confirmed as the primary screening campaigns identified only few and very weak hits. Iterative virtual and NMR screening techniques were required to discover a compound with sufficient potency to be able to obtain an X-ray co-crystal structure. The binding site is located between armadillo repeats two and three, adjacent to the BCL9 and TCF4 binding sites. Genetic studies show that it is unlikely to be useful for the development of protein-protein interaction inhibitors but structural information and established assays provide a solid basis for a prospective optimization towards β-catenin proteolysis targeting chimeras (PROTACs) as alternative modality.

It has taken four decades of research to see the first major breakthrough for KRAS-driven cancers. In particular, the last decade has seen a paradigm shift with the discovery of drug-gable pockets on KRAS and clinical efficacy with covalent KRAS(G12C) inhibitors, culminating in the first approval of sotorasib monotherapy as second-line treatment in KRAS(G12C)-driven non-small-cell lung cancer. Nevertheless, 85% of all KRAS-mutated cancers still lack novel agents. In this review, we will outline the structure, function, and post-translational modifications of KRAS and highlight the various approaches being adopted to drug KRAS, ranging from selective to pan concepts. The range of molecular modalities being explored, including PROTACs and glues, will also be described. Finally, an outlook toward the next wave of KRAS drugs and the challenges of resistance will be given.

Son of Sevenless (SOS) is a guanine nucleotide exchange factor that activates the important cell signaling switch KRAS. SOS acts as a pacemaker for KRAS, the beating heart of cancer, by catalyzing the “beating” from the KRAS(off) to the KRAS(on) conformation. Activating mutations in SOS1 are common in Noonan syndrome and oncogenic alterations in KRAS drive 1 in seven human cancers. Promising clinical efficacy has been observed for selective KRASG12C inhibitors, but the vast majority of oncogenic KRAS alterations remain undrugged. The discovery of a druggable pocket on SOS1 has led to potent SOS1 inhibitors such as BI-3406. SOS1 inhibition leads to antiproliferative effects against all major KRAS mutants. The first SOS1 inhibitor has entered clinical trials for KRAS-mutated cancers. In this review, we provide an overview of SOS1 function, its association with cancer and RASopathies, known SOS1 activators and inhibitors, and a future perspective is provided.

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.

The Front Cover shows the discovered fragment in the process of binding to β-catenin bound to the transcription factor TCF4 and co-activator BCL9 (PDB ID: 2GL7). The electron density surrounding the ligand and the chemical shift perturbations observed in the fragment binding site represent the strong biophysical validation. The outreaching arm represents the prospective utility of this discovery to be used as a handle for proteolysis targeting chimeras (PROTACs) upon further optimization. More information can be found in the Communication by Dirk Kessler, Jark Böttcher et al. Cover art created by Mila Böttcher, Jark Böttcher, and Darryl B. McConnell.

HUWE1 is a universal quality-control E3 ligase that marks diverse client proteins for proteasomal degradation. Although the giant HECT enzyme is an essential component of the ubiquitin-proteasome system closely linked with severe human diseases, its molecular mechanism is little understood. Here, we present the crystal structure of Nematocida HUWE1, revealing how a single E3 enzyme has specificity for a multitude of unrelated substrates. The protein adopts a remarkable snake-like structure, where the C-terminal HECT domain heads an extended alpha-solenoid body that coils in on itself and houses various protein-protein interaction modules. Our integrative structural analysis shows that this ring structure is highly dynamic, enabling the flexible HECT domain to reach protein targets presented by the various acceptor sites. Together, our data demonstrate how HUWE1 is regulated by its unique structure, adapting a promiscuous E3 ligase to selectively target unassembled orphan proteins.