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. X-ray structures with wild-type (2.4 Å) and T790M/V948R (3.1 Å) EGFR kinase domains show that BI-4020 is likely rendered selective due to interactions with the kinase domain hinge region as well as T790M, akin to Osimertinib. Additionally, BI-4020 is also rendered more potent due to its constrained macrocycle geometry as well as additional H-bonds to conserved K745 and T845 residues in both active and inactive conformations. These findings taken together show how this novel macrocyclic inhibitor is both highly potent and selective for mutant EGFR in a reversible mechanism and motivate structure-inspired approaches to developing targeted therapies in medicinal oncology.

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. Crystal structures show that initial and redesigned linkers bridging a trisubstituted imidazole ATP-site inhibitor and dibenzodiazepinone allosteric-site inhibitor proved successful in spanning these sites. The re-engineered linker yielded a compound that exhibited significantly higher potency (~60 pM) against the drug-resistant EGFR L858R/T790M and L858R/T790M/C797S, which was superadditive as compared with the parent molecules. The enhanced potency is attributed to factors stemming from the linker connection to the allosteric-site group and informs strategies to engineer linkers in bivalent agent design.

TFE3-Splicing Factor Fusions Represent Functional Drivers and Druggable Targets in Translocation Renal Cell Carcinoma.

TFE3 is a member of the basic helix-loop-helix leucine zipper MiT transcription factor family, and its chimeric proteins are associated with translocation renal cell carcinoma (tRCC). Despite the variety of gene fusions, most TFE3 fusion partner genes are related to spliceosome machinery. Dissecting the function of TFE3 fused to spliceosome machinery factors (TFE3-SF) could direct the development of effective therapies for this lethal disease, which is refractory to standard treatments for kidney cancer. Here, by using a combination of in silico structure prediction, transcriptome profiling, molecular characterization, and high-throughput high-content screening (HTHCS), we interrogated a number of oncogenic mechanisms of TFE3-SF fusions. TFE3-SF fusions drove the transformation of kidney cells and promoted distinct oncogenic phenotypes in a fusion partner-dependent manner, differentially altering the transcriptome and RNA splicing landscape and activating different oncogenic pathways. Inhibiting TFE3-SF dimerization reversed its oncogenic activity and represented a potential target for therapeutic intervention. Screening the FDA-approved drugs library LOPAC and a small-molecule library (Microsource) using HTHCS combined with FRET technology identified compounds that inhibit TFE3-SF dimerization. Hit compounds were validated in 2D and 3D patient-derived xenograft models expressing TFE3-SF. The antihistamine terfenadine decreased cell proliferation and reduced in vivo tumor growth of tRCC. Overall, these results unmask therapeutic strategies to target TFE3-SF dimerization for treating patients with tRCC. SIGNIFICANCE: TFE3-splicing factor fusions possess both transcription and splicing factor functions that remodel the transcriptome and spliceosome and can be targeted with dimerization inhibitors to suppress the growth of translocation renal cell carcinoma.

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. To facilitate the study of allosteric inhibitor pharmacology, we designed and purified a constitutive EGFR kinase dimer harboring the clinically relevant L858R/T790M mutations. Kinetic characterization revealed that the EGFR kinase dimer is more active than monomeric EGFR(L858R/T790M) kinase and has the same Km,ATP Biochemical profiling of a large panel of ATP-competitive and allosteric EGFR inhibitors showed that allosteric inhibitor potency decreased by >500-fold in the kinase dimer compared with monomer, yielding IC50 values that correlate well with Ba/F3 cellular potencies. Thus, this readily purifiable constitutive asymmetric EGFR kinase dimer represents an attractive tool for biochemical evaluation of EGFR inhibitor pharmacology, in particular for allosteric inhibitors. SIGNIFICANCE STATEMENT: Drugs targeting epidermal growth factor receptor (EGFR) kinase are commonly used to treat lung cancers but are affected by receptor dimerization. Here, we describe a locked kinase dimer that can be used to study EGFR inhibitor pharmacology.

Pitfalls and Considerations in Determining the Potency and Mutant Selectivity of Covalent Epidermal Growth Factor Receptor Inhibitors.

Enzyme inhibitors that form covalent bonds with their targets are being increasingly pursued in drug development. Assessing their biochemical activity relies on time-dependent assays, which are distinct and more complex compared with methods commonly employed for reversible-binding inhibitors. To provide general guidance to the covalent inhibitor development community, we explored methods and reported kinetic values and experimental factors in determining the biochemical activity of various covalent epidermal growth factor receptor (EGFR) inhibitors. We showcase how liquid handling and assay reagents impact kinetic parameters and potency interpretations, which are critical for structure-kinetic relationships and covalent drug design. Additionally, we include benchmark kinetic values with reference inhibitors, which are imperative, as covalent EGFR inhibitor kinetic values are infrequently consistent in the literature. This overview seeks to inform best practices for developing new covalent inhibitors and highlight appropriate steps to address gaps in knowledge presently limiting assay reliability and reproducibility.

Inhibition of a lower potency target drives the anticancer activity of a clinical p38 inhibitor.

The small-molecule drug ralimetinib was developed as an inhibitor of the p38α mitogen-activated protein kinase, and it has advanced to phase 2 clinical trials in oncology. Here, we demonstrate that ralimetinib resembles EGFR-targeting drugs in pharmacogenomic profiling experiments and that ralimetinib inhibits EGFR kinase activity in vitro and in cellulo. While ralimetinib sensitivity is unaffected by deletion of the genes encoding p38α and p38ß, its effects are blocked by expression of the EGFR-T790M gatekeeper mutation. Finally, we solved the cocrystal structure of ralimetinib bound to EGFR, providing further evidence that this drug functions as an ATP-competitive EGFR inhibitor. We conclude that, though ralimetinib is >30-fold less potent against EGFR compared to p38α, its ability to inhibit EGFR drives its primary anticancer effects. Our results call into question the value of p38α as an anticancer target, and we describe a multi-modal approach that can be used to uncover a drug's mechanism-of-action.

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. The current clinical challenges are mainly due to acquired resistance to third-generation inhibitors, including by the acquisition of the C797S mutation. Several diverse fourth-generation candidates and tool compounds that inhibit the C797S mutant have emerged, and their structural characterization has revealed molecular factors that allow for EGFR mutant selective binding. Here, we have reviewed all known structurally-characterized EGFR TKIs targeting clinically-relevant mutations to identify specific features that enable C797S inhibition. Newer generation EGFR inhibitors exhibit consistent and previously underutilized hydrogen bonding interactions with the conserved K745 and D855 residue side chains. We also consider binding modes and hydrogen bonding interactions of inhibitors targeting the classical ATP and the more unique allosteric sites.

Structural Basis for Inhibition of Mutant EGFR with Lazertinib (YH25448).

Lazertinib (YH25448) is a novel third-generation tyrosine kinase inhibitor (TKI) developed as a treatment for EGFR mutant non-small cell lung cancer. To better understand the nature of lazertinib inhibition, we determined crystal structures of lazertinib in complex with both WT and mutant EGFR and compared its binding mode to that of structurally related EGFR TKIs. We observe that lazertinib binds EGFR with a distinctive pyrazole moiety enabling hydrogen bonds and van der Waals interactions facilitated through hydrophilic amine and hydrophobic phenyl groups, respectively. Biochemical assays and cell studies confirm that lazertinib effectively targets EGFR(L858R/T790M) and to a lesser extent HER2. The molecular basis for lazertinib inhibition of EGFR reported here highlights previously unexplored binding interactions leading to improved medicinal chemistry properties compared to clinically approved osimertinib (AZD9291) and offers novel strategies for structure-guided design of tyrosine kinase inhibitors.

Genomic and biological study of fusion genes as resistance mechanisms to EGFR inhibitors.

The clinical significance of gene fusions detected by DNA-based next generation sequencing remains unclear as resistance mechanisms to EGFR tyrosine kinase inhibitors in EGFR mutant non-small cell lung cancer. By studying EGFR inhibitor-resistant patients treated with a combination of an EGFR inhibitor and a drug targeting the putative resistance-causing fusion oncogene, we identify patients who benefit and those who do not from this treatment approach. Through evaluation including RNA-seq of potential drug resistance-imparting fusion oncogenes in 504 patients with EGFR mutant lung cancer, we identify only a minority of them as functional, potentially capable of imparting EGFR inhibitor resistance. We further functionally validate fusion oncogenes in vitro using CRISPR-based editing of EGFR mutant cell lines and use these models to identify known and unknown drug resistance mechanisms to combination therapies. Collectively, our results partially reveal the complex nature of fusion oncogenes as potential drug resistance mechanisms and highlight approaches that can be undertaken to determine their functional significance.

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. We identify a subset of irreversible EGFR inhibitors that display positive binding cooperativity and synergy with the allosteric inhibitor JBJ-04-125-02 in several EGFR variants. Structural analysis of these complexes reveals conformational changes occur mainly in the phosphate-binding loop (P-loop). Mutation of F723 in the P-loop reduces cooperative binding and synergy, supporting a mechanism in which F723-mediated contacts between the P-loop and the allosteric inhibitor are critical for synergy. These structural and mechanistic insights will aid in the identification and development of additional inhibitor combinations with potential clinical value.

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. We further uncover that EGFR homo- or heterodimerization with other ERBB family members, as well as the EGFR L747S mutation, confers resistance to JBJ-09-063, but not to ATP-competitive EGFR TKIs. Overall, our studies highlight the potential clinical utility of JBJ-09-063 as a single agent or in combination with EGFR TKIs to define more effective strategies to treat EGFR-mutant lung cancer.

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. We explored a scaffold hopping approach to identify new series of allosteric EGFR inhibitors that retained good potency in the absence of the phenol group. The 5-F quinazolinone 34 demonstrated tumor regression in an H1975 efficacy model upon once daily oral dosing at 25 mg/kg.

Architecture of the NADPH oxidase family of enzymes.

The NADPH Oxidases (NOX) catalyze the deliberate production of reactive oxygen species (ROS) and are established regulators of redox-dependent processes across diverse biological settings. Proper management of their activity is controlled through a conserved electron transfer (ET) cascade from cytosolic NADPH substrate through the plasma membrane to extracellular O2. After decades-long investigations of their biological functions, including potential as drug targets, only very recently has atomic-resolution information of NOX enzymes been made available. In this graphical review, we summarize the present structural biology understanding of the NOX enzymes afforded by X-ray crystallography and cryo-electron microscopy. Combined molecular-level insights predominantly informed by DUOX1 full-length Cryo-EM structures suggest a general structural basis for the control of their catalytic activity by intracellular domain-domain stabilization.

Design of a "Two-in-One" Mutant-Selective Epidermal Growth Factor Receptor Inhibitor That Spans the Orthosteric and Allosteric Sites.

Inhibitors targeting the epidermal growth factor receptor (EGFR) are an effective therapy for patients with non-small cell lung cancer harboring drug-sensitive activating mutations in the EGFR kinase domain. Drug resistance due to treatment-acquired mutations has motivated the development of successive generations of inhibitors that bind in the ATP site. The third-generation agent osimertinib is now a first-line treatment for this disease. Recently, allosteric inhibitors have been developed to overcome drug-resistant mutations that confer a resistance to osimertinib. Here, we present the structure-guided design and synthesis of a mutant-selective lead compound, which consists of a pyridinyl imidazole-fused benzylisoindolinedione scaffold that simultaneously occupies the orthosteric and allosteric sites. The compound potently inhibits enzymatic activity in L858R/T790M/C797S mutant EGFR (4.9 nM), with a significantly lower activity for wild-type EGFR (47 nM). Additionally, this compound achieves modest cetuximab-independent and mutant-selective cellular efficacies on the L858R (1.2 µM) and L858R/T790M (4.4 µM) variants.

Oxidation-Dependent Activation of Src Kinase Mediates Epithelial IL-33 Production and Signaling during Acute Airway Allergen Challenge.

The respiratory epithelium forms the first line of defense against inhaled pathogens and acts as an important source of innate cytokine responses to environmental insults. One critical mediator of these responses is the IL-1 family cytokine IL-33, which is rapidly secreted upon acute epithelial injury as an alarmin and induces type 2 immune responses. Our recent work highlighted the importance of the NADPH oxidase dual oxidase 1 (DUOX1) in acute airway epithelial IL-33 secretion by various airborne allergens associated with H2O2 production and reduction-oxidation-dependent activation of Src kinases and epidermal growth factor receptor (EGFR) signaling. In this study, we show that IL-33 secretion in response to acute airway challenge with house dust mite (HDM) allergen critically depends on the activation of Src by a DUOX1-dependent oxidative mechanism. Intriguingly, HDM-induced epithelial IL-33 secretion was dramatically attenuated by small interfering RNA- or Ab-based approaches to block IL-33 signaling through its receptor IL1RL1 (ST2), indicating that HDM-induced IL-33 secretion includes a positive feed-forward mechanism involving ST2-dependent IL-33 signaling. Moreover, activation of type 2 cytokine responses by direct airway IL-33 administration was associated with ST2-dependent activation of DUOX1-mediated H2O2 production and reduction-oxidation-based activation of Src and EGFR and was attenuated in Duox1 -/- and Src +/- mice, indicating that IL-33-induced epithelial signaling and subsequent airway responses involve DUOX1/Src-dependent pathways. Collectively, our findings suggest an intricate relationship between DUOX1, Src, and IL-33 signaling in the activation of innate type 2 immune responses to allergens, involving DUOX1-dependent epithelial Src/EGFR activation in initial IL-33 secretion and in subsequent IL-33 signaling through ST2 activation.

A structural perspective on targeting the RTK/Ras/MAP kinase pathway in cancer.

Precision oncology is premised on identifying and drugging proteins and pathways that drive tumorigenesis or are required for survival of tumor cells. Across diverse cancer types, the signaling pathway emanating from receptor tyrosine kinases on the cell surface to RAS and the MAP kinase pathway is the most frequent target of oncogenic mutations, and key proteins in this signaling axis including EGFR, SHP2, RAS, BRAF, and MEK have long been a focus in cancer drug discovery. In this review, we provide an overview of historical and recent efforts to develop inhibitors targeting these nodes with an emphasis on the role that an understanding of protein structure and regulation has played in inhibitor discovery and characterization. Beyond its well-established role in structure-based drug design, structural biology has revealed mechanisms of allosteric regulation, distinct effects of activating oncogenic mutations, and other vulnerabilities that have opened new avenues in precision cancer drug discovery.

Structural insights into redox-active cysteine residues of the Src family kinases.

The Src Family Kinases (SFKs) are pivotal regulators of cellular signal transduction and highly sought-after targets in drug discovery. Their actions within cells are controlled by alterations in protein phosphorylation that switch the SFKs from autoinhibited to active states. The SFKs are also well recognized to contain redox-active cysteine residues where oxidation of certain residues directly contribute to kinase function. To more completely understand the factors that influence cysteine oxidation within the SFKs, a review is presented of the local structural environments surrounding SFK cysteine residues compared to their quantified oxidation in vivo from the Oximouse database. Generally, cysteine local structure and degree of redox sensitivity vary with respect to sequence conservation. Cysteine residues found in conserved positions are more mildly redox-active as they are found in hydrophobic environments and not fully exposed to solvent. Non-conserved redox-active cysteines are generally the most reactive with direct solvent access and/or in hydrophilic environments. Results from this analysis motivate future efforts to conduct comprehensive proteome-wide analysis of redox-sensitivity, conservation, and local structural environments of proteins containing reactive cysteine residues.

Rapid Decay of the Native Intermediate in the Metallooxidase Fet3p Enables Controlled FeII Oxidation for Efficient Metabolism.

The multicopper oxidases (MCOs) couple four 1e- oxidations of substrate to the 4e- reduction of O2 to H2O. These divide into two groups: those that oxidize organic substrates with high turnover frequencies (TOFs) up to 560 s-1 and those that oxidize metal ions with low TOFs, ∼1 s-1 or less. The catalytic mechanism of the organic oxidases has been elucidated, and the high TOF is achieved through rapid intramolecular electron transfer (IET) to the native intermediate (NI), which only slowly decays to the resting form. Here, we uncover the factors that govern the low TOF in Fet3p, a prototypical metallooxidase, in the context of the MCO mechanism. We determine that the NI decays rapidly under optimal turnover conditions, and the mechanism thereby becomes rate-limited by slow IET to the resting enzyme. Development of a catalytic model leads to the important conclusions that proton delivery to the NI controls the mechanism and enables the slow turnover in Fet3p that is functionally significant in Fe metabolism enabling efficient ferroxidase activity while avoiding ROS generation.

Structural Basis for EGFR Mutant Inhibition by Trisubstituted Imidazole Inhibitors.

Acquired drug resistance in epidermal growth factor receptor (EGFR) mutant non-small-cell lung cancer is a persistent challenge in cancer therapy. Previous studies of trisubstituted imidazole inhibitors led to the serendipitous discovery of inhibitors that target the drug resistant EGFR(L858R/T790M/C797S) mutant with nanomolar potencies in a reversible binding mechanism. To dissect the molecular basis for their activity, we determined the binding modes of several trisubstituted imidazole inhibitors in complex with the EGFR kinase domain with X-ray crystallography. These structures reveal that the imidazole core acts as an H-bond acceptor for the catalytic lysine (K745) in the "αC-helix out" inactive state. Selective N-methylation of the H-bond accepting nitrogen ablates inhibitor potency, confirming the role of the K745 H-bond in potent, noncovalent inhibition of the C797S variant. Insights from these studies offer new strategies for developing next generation inhibitors targeting EGFR in non-small-cell lung cancer.