Direct and selective pharmacological disruption of the YAP-TEAD interface by IAG933 inhibits Hippo-dependent and RAS-MAPK-altered cancers.

The YAP-TEAD protein-protein interaction mediates YAP oncogenic functions downstream of the Hippo pathway. To date, available YAP-TEAD pharmacologic agents bind into the lipid pocket of TEAD, targeting the interaction indirectly via allosteric changes. However, the consequences of a direct pharmacological disruption of the interface between YAP and TEADs remain largely unexplored. Here, we present IAG933 and its analogs as potent first-in-class and selective disruptors of the YAP-TEAD protein-protein interaction with suitable properties to enter clinical trials. Pharmacologic abrogation of the interaction with all four TEAD paralogs resulted in YAP eviction from chromatin and reduced Hippo-mediated transcription and induction of cell death. In vivo, deep tumor regression was observed in Hippo-driven mesothelioma xenografts at tolerated doses in animal models as well as in Hippo-altered cancer models outside mesothelioma. Importantly this also extended to larger tumor indications, such as lung, pancreatic and colorectal cancer, in combination with RTK, KRAS-mutant selective and MAPK inhibitors, leading to more efficacious and durable responses. Clinical evaluation of IAG933 is underway.

Dissecting the Interaction Fingerprints and Binding Affinity of BYL719 Analogs Targeting PI3Kα.

Phosphatidylinositol-3-kinase Alpha (PI3Kα) is a lipid kinase which regulates signaling pathways involved in cell proliferation. Dysregulation of these pathways promotes several human cancers, pushing for the development of anticancer drugs to target PI3Kα. One such medicinal chemistry campaign at Novartis led to the discovery of BYL719 (Piqray, Alpelicib), a PI3Kα inhibitor approved by the FDA in 2019 for treatment of HR+/HER2-advanced breast cancer with a PIK3CA mutation. Structure-based drug design played a key role in compound design and optimization throughout the discovery process. However, further characterization of potency drivers via structural dynamics and energetic analyses can be advantageous for ensuing PI3Kα programs. Here, our goal is to employ various in-silico techniques, including molecular simulations and machine learning, to characterize 14 ligands from the BYL719 analogs and predict their binding affinities. The structural insights from molecular simulations suggest that although the ligand-hinge interaction is the primary driver of ligand stability at the pocket, the R group positioning at C2 or C6 of pyridine/pyrimidine also plays a major role. Binding affinities predicted via thermodynamic integration (TI) are in good agreement with previously reported IC50s. Yet, computationally demanding techniques such as TI might not always be the most efficient approach for affinity prediction, as in our case study, fast high-throughput techniques were capable of classifying compounds as active or inactive, and one docking approach showed accuracy comparable to TI.

Identification of Brain-Penetrant ATP-Competitive mTOR Inhibitors for CNS Syndromes.

The allosteric inhibitor of the mechanistic target of rapamycin (mTOR) everolimus reduces seizures in tuberous sclerosis complex (TSC) patients through partial inhibition of mTOR functions. Due to its limited brain permeability, we sought to develop a catalytic mTOR inhibitor optimized for central nervous system (CNS) indications. We recently reported an mTOR inhibitor (1) that is able to block mTOR functions in the mouse brain and extend the survival of mice with neuronal-specific ablation of the Tsc1 gene. However, 1 showed the risk of genotoxicity in vitro. Through structure-activity relationship (SAR) optimization, we identified compounds 9 and 11 without genotoxicity risk. In neuronal cell-based models of mTOR hyperactivity, both corrected aberrant mTOR activity and significantly improved the survival rate of mice in the Tsc1 gene knockout model. Unfortunately, 9 and 11 showed limited oral exposures in higher species and dose-limiting toxicities in cynomolgus macaque, respectively. However, they remain optimal tools to explore mTOR hyperactivity in CNS disease models.

Leveraging Advanced In Silico Techniques in Early Drug Discovery: A Study of Potent Small-Molecule YAP-TEAD PPI Disruptors.

Disruption of the YAP-TEAD protein-protein interaction is an attractive therapeutic strategy in oncology to suppress tumor progression and cancer metastasis. YAP binds to TEAD at a large flat binding interface (∼3500 Å2) devoid of a well-defined druggable pocket, so it has been difficult to design low-molecular-weight compounds to abrogate this protein-protein interaction directly. Recently, work by Furet and coworkers (ChemMedChem 2022, DOI: 10.1002/cmdc.202200303) reported the discovery of the first class of small molecules able to efficiently disrupt the transcriptional activity of TEAD by binding to a specific interaction site of the YAP-TEAD binding interface. Using high-throughput in silico docking, they identified a virtual screening hit from a hot spot derived from their previously rationally designed peptidic inhibitor. Structure-based drug design efforts led to the optimization of the hit compound into a potent lead candidate. Given advances in rapid high-throughput screening and rational approaches to peptidic ligand discovery for challenging targets, we analyzed the pharmacophore features involved in transferring from the peptidic to small-molecule inhibitor that could enable small-molecule discovery for such targets. Here, we show retrospectively that pharmacophore analysis augmented by solvation analysis of molecular dynamics trajectories can guide the designs, while binding free energy calculations provide greater insight into the binding conformation and energetics accompanying the association event. The computed binding free energy estimates agree well with experimental findings and offer useful insight into structural determinants that influence ligand binding to the TEAD interaction surface, even for such a shallow binding site. Taken together, our results demonstrates the utility of advanced in silico methods in structure-based design efforts for difficult-to-drug targets such as the YAP-TEAD transcription factor complex.

Optimization of a Class of Dihydrobenzofurane Analogs toward Orally Efficacious YAP-TEAD Protein-Protein Interaction Inhibitors.

The inhibition of the YAP-TEAD protein-protein interaction constitutes a promising therapeutic approach for the treatment of cancers linked to the dysregulation of the Hippo signaling pathway. The identification of a class of small molecules which potently inhibit the YAP-TEAD interaction by binding tightly to the Ω-loop pocket of TEAD has previously been communicated. This report details the further multi-parameter optimization of this class of compounds resulting in advanced analogs combining nanomolar cellular potency with a balanced ADME and off-target profile, and efficacy of these compounds in tumor bearing mice is demonstrated for the first time.

The First Class of Small Molecules Potently Disrupting the YAP-TEAD Interaction by Direct Competition.

Inhibition of the YAP-TEAD protein-protein interaction is an attractive therapeutic concept under intense investigation with the objective to treat cancers associated with a dysregulation of the Hippo pathway. However, owing to the very extended surface of interaction of the two proteins, the identification of small drug-like molecules able to efficiently prevent YAP from binding to TEAD by direct competition has been elusive so far. We disclose here the discovery of the first class of small molecules potently inhibiting the YAP-TEAD interaction by binding at one of the main interaction sites of YAP at the surface of TEAD. These inhibitors, providing a path forward to pharmacological intervention in the Hippo pathway, evolved from a weakly active virtual screening hit advanced to high potency by structure-based design.

Identification of NVP-CLR457 as an Orally Bioavailable Non-CNS-Penetrant pan-Class IA Phosphoinositol-3-Kinase Inhibitor.

Balanced pan-class I phosphoinositide 3-kinase inhibition as an approach to cancer treatment offers the prospect of treating a broad range of tumor types and/or a way to achieve greater efficacy with a single inhibitor. Taking buparlisib as the starting point, the balanced pan-class I PI3K inhibitor 40 (NVP-CLR457) was identified with what was considered to be a best-in-class profile. Key to the optimization to achieve this profile was eliminating a microtubule stabilizing off-target activity, balancing the pan-class I PI3K inhibition profile, minimizing CNS penetration, and developing an amorphous solid dispersion formulation. A rationale for the poor tolerability profile of 40 in a clinical study is discussed.

p53 dynamics vary between tissues and are linked with radiation sensitivity.

Radiation sensitivity varies greatly between tissues. The transcription factor p53 mediates the response to radiation; however, the abundance of p53 protein does not correlate well with the extent of radiosensitivity across tissues. Given recent studies showing that the temporal dynamics of p53 influence the fate of cultured cells in response to irradiation, we set out to determine the dynamic behavior of p53 and its impact on radiation sensitivity in vivo. We find that radiosensitive tissues show prolonged p53 signaling after radiation, while more resistant tissues show transient p53 activation. Sustaining p53 using a small molecule (NMI801) that inhibits Mdm2, a negative regulator of p53, reduced viability in cell culture and suppressed tumor growth. Our work proposes a mechanism for the control of radiation sensitivity and suggests tools to alter the dynamics of p53 to enhance tumor clearance. Similar approaches can be used to enhance killing of cancer cells or reduce toxicity in normal tissues following genotoxic therapies.

Correction to "2-Formylpyridyl Ureas as Highly Selective Reversible-Covalent Inhibitors of Fibroblast Growth Factor Receptor 4".

[This corrects the article DOI: 10.1021/acsmedchemlett.7b00485.].

A new perspective on the interaction between the Vg/VGLL1-3 proteins and the TEAD transcription factors.

The most downstream elements of the Hippo pathway, the TEAD transcription factors, are regulated by several cofactors, such as Vg/VGLL1-3. Earlier findings on human VGLL1 and here on human VGLL3 show that these proteins interact with TEAD via a conserved amino acid motif called the TONDU domain. Surprisingly, our studies reveal that the TEAD-binding domain of Drosophila Vg and of human VGLL2 is more complex and contains an additional structural element, an Ω-loop, that contributes to TEAD binding. To explain this unexpected structural difference between proteins from the same family, we propose that, after the genome-wide duplications at the origin of vertebrates, the Ω-loop present in an ancestral VGLL gene has been lost in some VGLL variants. These findings illustrate how structural and functional constraints can guide the evolution of transcriptional cofactors to preserve their ability to compete with other cofactors for binding to transcription factors.

Discovery of Roblitinib (FGF401) as a Reversible-Covalent Inhibitor of the Kinase Activity of Fibroblast Growth Factor Receptor 4.

FGF19 signaling through the FGFR4/ß-klotho receptor complex has been shown to be a key driver of growth and survival in a subset of hepatocellular carcinomas, making selective FGFR4 inhibition an attractive treatment opportunity. A kinome-wide sequence alignment highlighted a poorly conserved cysteine residue within the FGFR4 ATP-binding site at position 552, two positions beyond the gate-keeper residue. Several strategies for targeting this cysteine to identify FGFR4 selective inhibitor starting points are summarized which made use of both rational and unbiased screening approaches. The optimization of a 2-formylquinoline amide hit series is described in which the aldehyde makes a hemithioacetal reversible-covalent interaction with cysteine 552. Key challenges addressed during the optimization are improving the FGFR4 potency, metabolic stability, and solubility leading ultimately to the highly selective first-in-class clinical candidate roblitinib.

Identification of FAM181A and FAM181B as new interactors with the TEAD transcription factors.

The Hippo pathway is a key signaling pathway in the control of organ size and development. The most distal elements of this pathway, the TEAD transcription factors, are regulated by several proteins, such as YAP (Yes-associated protein), TAZ (transcriptional co-activator with PDZ-binding motif) and VGLL1-4 (Vestigial-like members 1-4). In this article, combining structural data and motif searches in protein databases, we identify two new TEAD interactors: FAM181A and FAM181B. Our structural data show that they bind to TEAD via an Ω-loop as YAP/TAZ do, but only FAM181B possesses the LxxLF motif (x any amino acid) found in YAP/TAZ. The affinity of different FAM181A/B fragments for TEAD is in the low micromolar range and full-length FAM181A/B proteins interact with TEAD in cells. These findings, together with a recent report showing that FAM181A/B proteins have a role in nervous system development, suggest a potential new involvement of the TEAD transcription factors in the development of this tissue.

FGF401, A First-In-Class Highly Selective and Potent FGFR4 Inhibitor for the Treatment of FGF19-Driven Hepatocellular Cancer.

Hepatocellular carcinoma (HCC) is the most common primary malignancy of the liver and it is the third leading cause of cancer-related deaths worldwide. Recently, aberrant signaling through the FGF19/FGFR4 axis has been implicated in HCC. Here, we describe the development of FGF401, a highly potent and selective, first in class, reversible-covalent small-molecule inhibitor of the kinase activity of FGFR4. FGF401 is exquisitely selective for FGFR4 versus the other FGFR paralogues FGFR1, FGFR2, FGFR3, and all other kinases in the kinome. FGF401 has excellent drug-like properties showing a robust pharmacokinetic/pharmacodynamics/efficacy relationship, driven by a fraction of time above the phospho-FGFR4 IC90 value. FGF401 has remarkable antitumor activity in mice bearing HCC tumor xenografts and patient-derived xenograft models that are positive for FGF19, FGFR4, and KLB. FGF401 is the first FGFR4 inhibitor to enter clinical trials, and a phase I/II study is currently ongoing in HCC and other solid malignancies.

Structure-based design of potent linear peptide inhibitors of the YAP-TEAD protein-protein interaction derived from the YAP omega-loop sequence.

The YAP-TEAD protein-protein interaction is a potential therapeutic target to treat cancers in which the Hippo signaling pathway is deregulated. However, the extremely large surface of interaction between the two proteins presents a formidable challenge for a small molecule interaction disrupter approach. We have accomplished progress towards showing the feasibility of this approach by the identification of a 15-mer peptide able to potently (nanomolar range) disrupt the YAP-TEAD interaction by targeting only one of the two important sites of interaction. This peptide, incorporating non-natural amino acids selected by structure-based design, is derived from the Ω-loop sequence 85-99 of YAP.

Structural States of Hdm2 and HdmX: X-ray Elucidation of Adaptations and Binding Interactions for Different Chemical Compound Classes.

Hdm2 (human MDM2, human double minute 2 homologue) counteracts p53 function by direct binding to p53 and by ubiquitin-dependent p53 protein degradation. Activation of p53 by inhibitors of the p53-Hdm2 interaction is being pursued as a therapeutic strategy in p53 wild-type cancers. In addition, HdmX (human MDMX, human MDM4) was also identified as an important therapeutic target to efficiently reactivate p53, and it is likely that dual inhibition of Hdm2 and HdmX is beneficial. Herein we report four new X-ray structures for Hdm2 and five new X-ray structures for HdmX complexes, involving different classes of synthetic compounds (including the worldwide highest resolutions for Hdm2 and HdmX, at 1.13 and 1.20 Å, respectively). We also reveal the key additive 18-crown-ether, which we discovered to enable HdmX crystallization and show its stabilization of various Lys residues. In addition, we report the previously unpublished details of X-ray structure determinations for eight further Hdm2 complexes, including the clinical trial compounds NVP-CGM097 and NVP-HDM201. An analysis of all compound binding modes reveals new and deepened insight into the possible adaptations and structural states of Hdm2 (e.g., flip of F55, flip of Y67, reorientation of H96) and HdmX (e.g., flip of H55, dimer induction), enabling key binding interactions for different compound classes. To facilitate comparisons, we used the same numbering for Hdm2 (as in Q00987) and HdmX (as in O15151, but minus 1). Taken together, these structural insights should prove useful for the design and optimization of further selective and/or dual Hdm2/HdmX inhibitors.

Molecular and structural characterization of a TEAD mutation at the origin of Sveinsson's chorioretinal atrophy.

Four TEAD transcription factors (TEAD1-4) mediate the signalling output of the Hippo pathway that controls organ size in humans. TEAD transcriptional activity is regulated via interactions with the YAP, TAZ and VGLL proteins. A mutation in the TEAD1 gene, Tyr421His, has been identified in patients suffering from Sveinsson's chorioretinal atrophy (SCA), an autosomal dominant eye disease. This mutation prevents the YAP/TAZ:TEAD1 interaction. In this study, we measure the affinity of YAP, TAZ and VGLL1 for the four human TEADs and find that they have a similar affinity for all TEADs. We quantitate the effect of the mutation found in SCA patients and show that it destabilizes the YAP/TAZ:TEAD interaction by about 3 kcal·mol-1 . We determine the structure of YAP in complex with this mutant form of TEAD and show that the histidine residue adopts different conformations at the binding interface. The presence of this flexible residue induces the destabilization of several H-bonds and the loss of van der Waals contacts, which explains why the Tyr421HisTEAD1 mutation has such a large destabilizing effect on the formation of the YAP:TEAD complex. DATABASE: The crystallographic data have been deposited at the RSCB Protein Data Bank (PDB, www.pdb.org) with the access codes: 6HIL (wtYAP :Tyr421HisTEAD1 ), 6HIK (wtYAP :Tyr429HisTEAD4 ).

Capmatinib (INC280) Is Active Against Models of Non-Small Cell Lung Cancer and Other Cancer Types with Defined Mechanisms of MET Activation.

PURPOSE: The selective MET inhibitor capmatinib is being investigated in multiple clinical trials, both as a single agent and in combination. Here, we describe the preclinical data of capmatinib, which supported the clinical biomarker strategy for rational patient selection. EXPERIMENTAL DESIGN: The selectivity and cellular activity of capmatinib were assessed in large cellular screening panels. Antitumor efficacy was quantified in a large set of cell line- or patient-derived xenograft models, testing single-agent or combination treatment depending on the genomic profile of the respective models. RESULTS: Capmatinib was found to be highly selective for MET over other kinases. It was active against cancer models that are characterized by MET amplification, marked MET overexpression, MET exon 14 skipping mutations, or MET activation via expression of the ligand hepatocyte growth factor (HGF). In cancer models where MET is the dominant oncogenic driver, anticancer activity could be further enhanced by combination treatments, for example, by the addition of apoptosis-inducing BH3 mimetics. The combinations of capmatinib and other kinase inhibitors resulted in enhanced anticancer activity against models where MET activation co-occurred with other oncogenic drivers, for example EGFR activating mutations. CONCLUSIONS: Activity of capmatinib in preclinical models is associated with a small number of plausible genomic features. The low fraction of cancer models that respond to capmatinib as a single agent suggests that the implementation of patient selection strategies based on these biomarkers is critical for clinical development. Capmatinib is also a rational combination partner for other kinase inhibitors to combat MET-driven resistance.

In vitro and in vivo characterization of a novel, highly potent p53-MDM2 inhibitor.

Small molecule inhibitors of the p53-MDM2 protein complex are under intense investigation in clinical trials as anti-cancer agents, including our first generation inhibitor NVP-CGM097. We recently described the rational design of a novel pyrazolopyrrolidinone core as a new lead structure and now we report on the synthesis and optimization of this to provide a highly potent lead compound. This new compound displayed excellent oral efficacy in our preclinical mechanistic in vivo model and marked a significant milestone towards the identification of our second generation clinical candidate NVP-HDM201.

Comparison of the Kinase Profile of Midostaurin (Rydapt) with That of Its Predominant Metabolites and the Potential Relevance of Some Newly Identified Targets to Leukemia Therapy.

The multitargeted protein kinase inhibitor midostaurin is approved for the treatment of both newly diagnosed FLT3-mutated acute myeloid leukemia (AML) and KIT-driven advanced systemic mastocytosis. AML is a heterogeneous malignancy, and investigational drugs targeting FLT3 have shown disparate effects in patients with FLT3-mutated AML, probably as a result of their inhibiting different targets and pathways at the administered doses. However, the efficacy and side effects of drugs do not just reflect the biochemical and pharmacodynamic properties of the parent compound but are often comprised of complex cooperative effects between the properties of the parent and active metabolites. Following chronic dosing, two midostaurin metabolites attain steady-state plasma trough levels greater than that of the parent drug. In this study, we characterized these metabolites and determined their profiles as kinase inhibitors using radiometric transphosphorylation assays. Like midostaurin, the metabolites potently inhibit mutant forms of FLT3 and KIT and several additional kinases that either are directly involved in the deregulated signaling pathways or have been implicated as playing a role in AML via stromal support, such as IGF1R, LYN, PDPK1, RET, SYK, TRKA, and VEGFR2. Consequently, a complex interplay between the kinase activities of midostaurin and its metabolites is likely to contribute to the efficacy of midostaurin in AML and helps to engender the distinctive effects of the drug compared to those of other FLT3 inhibitors in this malignancy.

Discovery of Asciminib (ABL001), an Allosteric Inhibitor of the Tyrosine Kinase Activity of BCR-ABL1.

Chronic myelogenous leukemia (CML) arises from the constitutive activity of the BCR-ABL1 oncoprotein. Tyrosine kinase inhibitors (TKIs) that target the ATP-binding site have transformed CML into a chronic manageable disease. However, some patients develop drug resistance due to ATP-site mutations impeding drug binding. We describe the discovery of asciminib (ABL001), the first allosteric BCR-ABL1 inhibitor to reach the clinic. Asciminib binds to the myristate pocket of BCR-ABL1 and maintains activity against TKI-resistant ATP-site mutations. Although resistance can emerge due to myristate-site mutations, these are sensitive to ATP-competitive inhibitors so that combinations of asciminib with ATP-competitive TKIs suppress the emergence of resistance. Fragment-based screening using NMR and X-ray yielded ligands for the myristate pocket. An NMR-based conformational assay guided the transformation of these inactive ligands into ABL1 inhibitors. Further structure-based optimization for potency, physicochemical, pharmacokinetic, and drug-like properties, culminated in asciminib, which is currently undergoing clinical studies in CML patients.