Tilting the Scales toward EGFR Mutant Selectivity: Expanding the Scope of Bivalent "Type V" Kinase Inhibitors.

Binding multiple sites within proteins with bivalent compounds is a strategy for developing uniquely active agents. A new class of dual-site inhibitors has emerged targeting the epidermal growth factor receptor (EGFR) anchored to both the orthosteric (ATP) and allosteric sites. Despite proof-of-concept successes, enabling selectivity against oncogenic activating mutations has not been achieved and classifying these inhibitors among kinase inhibitors remains underexplored.

Biochemical analysis of EGFR exon20 insertion variants insASV and insSVD and their inhibitor sensitivity.

Somatic mutations in the epidermal growth factor receptor (EGFR) are a major cause of non-small cell lung cancer. Among these structurally diverse alterations, exon 20 insertions represent a unique subset that rarely respond to EGFR tyrosine kinase inhibitors (TKIs). Therefore, there is a significant need to develop inhibitors that are active against this class of activating mutations.

Molecular Bidents with Two Electrophilic Warheads as a New Pharmacological Modality.

A systematic strategy to develop dual-warhead inhibitors is introduced to circumvent the limitations of conventional covalent inhibitors such as vulnerability to mutations of the corresponding nucleophilic residue. Currently, all FDA-approved covalent small molecules feature one electrophile, leaving open a facile route to acquired resistance. We conducted a systematic analysis of human proteins in the protein data bank to reveal ∼400 unique targets amendable to dual covalent inhibitors, which we term "molecular bidents".

Structural Analysis of the Macrocyclic Inhibitor BI-4020 Binding to EGFR Kinase.

A novel macrocyclic inhibitor of mutant EGFR (BI-4020) has shown promise in pre-clinical studies of T790M and C797S drug-resistant non-small cell lung cancer. To better understand the molecular basis for BI-4020 selectivity and potency, we have carried out biochemical activity assays and structural analysis with X-ray crystallography. Biochemical potencies agree with previous studies indicating that BI-4020 is uniquely potent against drug-resistant L858R/T790M and L858R/T790M/C797S variants.

Linking ATP and allosteric sites to achieve superadditive binding with bivalent EGFR kinase inhibitors.

Bivalent molecules consisting of groups connected through bridging linkers often exhibit strong target binding and unique biological effects. However, developing bivalent inhibitors with the desired activity is challenging due to the dual motif architecture of these molecules and the variability that can be introduced through differing linker structures and geometries. We report a set of alternatively linked bivalent EGFR inhibitors that simultaneously occupy the ATP substrate and allosteric pockets.

Signaling from RAS to RAF: The Molecules and Their Mechanisms.

RAF family protein kinases are a key node in the RAS/RAF/MAP kinase pathway, the signaling cascade that controls cellular proliferation, differentiation, and survival in response to engagement of growth factor receptors on the cell surface. Over the past few years, structural and biochemical studies have provided new understanding of RAF autoregulation, RAF activation by RAS and the SHOC2 phosphatase complex, and RAF engagement with HSP90-CDC37 chaperone complexes. These studies have important implications for pharmacologic targeting of the pathway.

ZNL0325, a Pyrazolopyrimidine-Based Covalent Probe, Demonstrates an Alternative Binding Mode for Kinases.

The pyrazolopyrimidine (PP) heterocycle is a versatile and widely deployed core scaffold for the development of kinase inhibitors. Typically, a 4-amino-substituted pyrazolopyrimidine binds in the ATP-binding pocket in a conformation analogous to the 6-aminopurine of ATP. Here, we report the discovery of ZNL0325 which exhibits a flipped binding mode where the C3 position is oriented toward the ribose binding pocket.

A Constitutive EGFR Kinase Dimer to Study Inhibitor Pharmacology.

Lung cancer is commonly caused by activating mutations in the epidermal growth factor receptor (EGFR). Allosteric kinase inhibitors are unaffected by common ATP-site resistance mutations and represent a promising therapeutic strategy for targeting drug-resistant EGFR variants. However, allosteric inhibitors are antagonized by kinase dimerization, and understanding this phenomenon has been limited to cellular experiments.

Linking ATP and allosteric sites to achieve superadditive binding with bivalent EGFR kinase inhibitors.

Bivalent molecules consisting of groups connected through bridging linkers often exhibit strong target binding and unique biological effects. However, developing bivalent inhibitors with the desired activity is challenging due to the dual motif architecture of these molecules and the variability that can be introduced through differing linker structures and geometries. We report a set of alternatively linked bivalent EGFR inhibitors that simultaneously occupy the ATP substrate and allosteric pockets.

Cryo-EM structure of a RAS/RAF recruitment complex.

RAF-family kinases are activated by recruitment to the plasma membrane by GTP-bound RAS, whereupon they initiate signaling through the MAP kinase cascade. Prior structural studies of KRAS with RAF have focused on the isolated RAS-binding and cysteine-rich domains of RAF (RBD and CRD, respectively), which interact directly with RAS. Here we describe cryo-EM structures of a KRAS bound to intact BRAF in an autoinhibited state with MEK1 and a 14-3-3 dimer.

Oncogenic K-Ras suppresses global miRNA function.

K-Ras frequently acquires gain-of-function mutations (K-Ras being the most common) that trigger significant transcriptomic and proteomic changes to drive tumorigenesis. Nevertheless, oncogenic K-Ras-induced dysregulation of post-transcriptional regulators such as microRNAs (miRNAs) during oncogenesis is poorly understood. Here, we report that K-Ras promotes global suppression of miRNA activity, resulting in the upregulation of hundreds of targets.

Structural elements that enable specificity for mutant EGFR kinase domains with next-generation small-molecule inhibitors.

Specificity for a desired enzyme target is an essential property of small-molecule inhibitors. Molecules targeting oncogenic driver mutations in the epidermal growth factor receptor (EGFR) kinase domain have had a considerable clinical impact due to their selective binding to cancer-causing mutants compared to wild type. Despite the availability of clinically approved drugs for cancers driven by EGFR mutants, persistent challenges in drug resistance in the past decades have led to newer generations of drugs with divergent chemical structures.

Macrocyclization of Quinazoline-Based EGFR Inhibitors Leads to Exclusive Mutant Selectivity for EGFR L858R and Del19.

Activating mutations in the epidermal growth factor receptor (EGFR) are frequent oncogenic drivers of non-small-cell lung cancer (NSCLC). The most frequent alterations in EGFR are short in-frame deletions in exon 19 (Del19) and the missense mutation L858R, which both lead to increased activity and sensitization of NSCLC to EGFR inhibition. The first approved EGFR inhibitors used for first-line treatment of NSCLC, gefitinib and erlotinib, are quinazoline-based.

PH domain-mediated autoinhibition and oncogenic activation of Akt.

Akt is a Ser/Thr protein kinase that plays a central role in metabolism and cancer. Regulation of Akt's activity involves an autoinhibitory intramolecular interaction between its pleckstrin homology (PH) domain and its kinase domain that can be relieved by C-tail phosphorylation. PH domain mutant E17K Akt is a well-established oncogene.

Structure of the MRAS-SHOC2-PP1C phosphatase complex.

RAS-MAPK signalling is fundamental for cell proliferation and is altered in most human cancers. However, our mechanistic understanding of how RAS signals through RAF is still incomplete. Although studies revealed snapshots for autoinhibited and active RAF-MEK1-14-3-3 complexes, the intermediate steps that lead to RAF activation remain unclear.

Molecular basis for cooperative binding and synergy of ATP-site and allosteric EGFR inhibitors.

Lung cancer is frequently caused by activating mutations in the epidermal growth factor receptor (EGFR). Allosteric EGFR inhibitors offer promise as the next generation of therapeutics, as they are unaffected by common ATP-site resistance mutations and synergize with the drug osimertinib. Here, we examine combinations of ATP-competitive and allosteric inhibitors to better understand the molecular basis for synergy.

An allosteric inhibitor against the therapy-resistant mutant forms of EGFR in non-small cell lung cancer.

Epidermal growth factor receptor (EGFR) therapy using small-molecule tyrosine kinase inhibitors (TKIs) is initially efficacious in patients with EGFR-mutant lung cancer, although drug resistance eventually develops. Allosteric EGFR inhibitors, which bind to a different EGFR site than existing ATP-competitive EGFR TKIs, have been developed as a strategy to overcome therapy-resistant EGFR mutations. Here we identify and characterize JBJ-09-063, a mutant-selective allosteric EGFR inhibitor that is effective across EGFR TKI-sensitive and resistant models, including those with EGFR T790M and C797S mutations.

Quinazolinones as allosteric fourth-generation EGFR inhibitors for the treatment of NSCLC.

The C797S mutation confers resistance to covalent EGFR inhibitors used in the treatment of lung tumors with the activating L858R mutation. Isoindolinones such as JBJ-4-125-02 bind in an allosteric pocket and are active against this mutation, with high selectivity over wild-type EGFR. The most potent examples we developed from that series have a potential chemical instability risk from the combination of the amide and phenol groups.

A Novel HER2-Selective Kinase Inhibitor Is Effective in HER2 Mutant and Amplified Non-Small Cell Lung Cancer.

Unlabelled: In-frame insertions in exon 20 of HER2 are the most common HER2 mutations in patients with non-small cell lung cancer (NSCLC), a disease in which approved EGFR/HER2 tyrosine kinase inhibitors (TKI) display poor efficiency and undesirable side effects due to their strong inhibition of wild-type (WT) EGFR. Here, we report a HER2-selective covalent TKI, JBJ-08-178-01, that targets multiple HER2 activating mutations, including exon 20 insertions as well as amplification. JBJ-08-178-01 displayed strong selectivity toward HER2 mutants over WT EGFR compared with other EGFR/HER2 TKIs.

The structural basis of PTEN regulation by multi-site phosphorylation.

Phosphatase and tensin homolog (PTEN) is a phosphatidylinositol-3,4,5-triphosphate (PIP) phospholipid phosphatase that is commonly mutated or silenced in cancer. PTEN's catalytic activity, cellular membrane localization and stability are orchestrated by a cluster of C-terminal phosphorylation (phospho-C-tail) events on Ser380, Thr382, Thr383 and Ser385, but the molecular details of this multi-faceted regulation have remained uncertain. Here we use a combination of protein semisynthesis, biochemical analysis, NMR, X-ray crystallography and computational simulations on human PTEN and its sea squirt homolog, VSP, to obtain a detailed picture of how the phospho-C-tail forms a belt around the C2 and phosphatase domains of PTEN.

Allosteric MEK inhibitors act on BRAF/MEK complexes to block MEK activation.

The RAF/MEK/ERK pathway is central to the control of cell physiology, and its dysregulation is associated with many cancers. Accordingly, the proteins constituting this pathway, including MEK1/2 (MEK), have been subject to intense drug discovery and development efforts. Allosteric MEK inhibitors (MEKi) exert complex effects on RAF/MEK/ERK pathway signaling and are employed clinically in combination with BRAF inhibitors in malignant melanoma.