Discovery of reversible and covalent TEAD 1 selective inhibitors MSC-1254 and MSC-5046 based on one scaffold.

The Transcriptional Enhanced Associated Domain (TEAD) family of transcription factors are key components of the Hippo signalling family which play a crucial role in the regulation of cell proliferation, differentiation and apoptosis. The identification of inhibitors of the TEAD transcription factors are an attractive strategy for the development of novel anticancer therapies. A HTS campaign identified hit 1, which was optimised using structure-based drug design, to deliver potent TEAD1 selective inhibitors with both a reversible and covalent mode of inhibition. The preference for TEAD1 could be rationalised by steric differences observed in the lower pocket of the palmitoylation-site between subtypes, with TEAD1 having the largest available volume to accommodate substitution in this region.

Novel reversible methionine aminopeptidase-2 (MetAP-2) inhibitors based on purine and related bicyclic templates.

The natural product fumagillin 1 and derivatives like TNP-470 2 or beloranib 3 bind to methionine aminopeptidase 2 (MetAP-2) irreversibly. This enzyme is critical for protein maturation and plays a key role in angiogenesis. In this paper we describe the synthesis, MetAP-2 binding affinity and structural analysis of reversible MetAP-2 inhibitors. Optimization of enzymatic activity of screening hit 10 (IC50: 1µM) led to the most potent compound 27 (IC50: 0.038µM), with a concomitant improvement in LLE from 2.1 to 4.2. Structural analysis of these MetAP-2 inhibitors revealed an unprecedented conformation of the His339 side-chain imidazole ring being co-planar sandwiched between the imidazole of His331 and the aryl-ether moiety, which is bound to the purine scaffold. Systematic alteration and reduction of H-bonding capability of this metal binding moiety induced an unexpected 180° flip for the triazolo[1,5-a]pyrimdine bicyclic template.

Discovery of novel 7-azaindoles as PDK1 inhibitors.

A combined screening strategy using HTS together with focused kinase library and virtual screening led to the identification of diverse chemical series as PDK1 inhibitors. We focused our medicinal chemistry efforts on 7-azaindoles with low micromolar IC50s (e.g., 16: IC50=1.1µM) in the biochemical PDK1 assay. Our structure-guided optimization efforts considered also PDK1 X-ray structures with weaker binding fragments and resulted in 7-azaindoles with significantly improved biochemical PDK1 potency in the two-digit nanomolar range. However, the most potent analogues only showed moderate activities in a cellular mechanistic assay (42: IC50=2.3µM) together with either low microsomal stability or low permeability. The described structure-activity relationship together with PDK1 X-ray structures and early ADME data provided the basis for our subsequent hit-to-lead program.

Novel Methionine Aminopeptidase 2 Inhibitor M8891 Synergizes with VEGF Receptor Inhibitors to Inhibit Tumor Growth of Renal Cell Carcinoma Models.

N-terminal processing by methionine aminopeptidases (MetAP) is a crucial step in the maturation of proteins during protein biosynthesis. Small-molecule inhibitors of MetAP2 have antiangiogenic and antitumoral activity. Herein, we characterize the structurally novel MetAP2 inhibitor M8891. M8891 is a potent, selective, reversible small-molecule inhibitor blocking the growth of human endothelial cells and differentially inhibiting cancer cell growth. A CRISPR genome-wide screen identified the tumor suppressor p53 and MetAP1/MetAP2 as determinants of resistance and sensitivity to pharmacologic MetAP2 inhibition. A newly identified substrate of MetAP2, translation elongation factor 1-alpha-1 (EF1a-1), served as a pharmacodynamic biomarker to follow target inhibition in cell and mouse studies. Robust angiogenesis and tumor growth inhibition was observed with M8891 monotherapy. In combination with VEGF receptor inhibitors, tumor stasis and regression occurred in patient-derived xenograft renal cell carcinoma models, particularly those that were p53 wild-type, had Von Hippel-Landau gene (VHL) loss-of-function mutations, and a mid/high MetAP1/2 expression score.

Fragment-based discovery of focal adhesion kinase inhibitors.

Chemically diverse fragment hits of focal adhesion kinase (FAK) were discovered by surface plasmon resonance (SPR) screening of our in-house fragment library. Site specific binding of the primary hits was confirmed in a competition setup using a high-affinity ATP-site inhibitor of FAK. Protein crystallography revealed the binding mode of 41 out of 48 selected fragment hits within the ATP-site. Structural comparison of the fragment binding modes with a DFG-out inhibitor of FAK initiated first synthetic follow-up optimization leading to improved binding affinity.

Comparative analysis of YAP/TEAD inhibitors in 2D and 3D cultures of primary human hepatocytes reveals a novel non-canonical mechanism of CYP induction.

Induction of cytochrome P450 (CYP) genes constitutes an important cause of drug-drug interactions and preclinical evaluation of induction liability is mandatory for novel drug candidates. YAP/TEAD signaling has emerged as an attractive target for various oncological indications and multiple chemically distinct YAP/TEAD inhibitors are rapidly progressing towards clinical stages. Here, we tested the liability for CYP induction of a diverse set of YAP/TEAD inhibitors with different modes of action and TEAD isoform selectivity profiles in monolayers and 3D spheroids of primary human hepatocytes (PHH). We found that YAP/TEAD inhibition resulted in broad induction of CYPs in 2D monolayers, whereas, if at all, only marginal induction was seen in spheroid culture. Comprehensive RNA-Seq indicated that YAP/TEAD signaling was increased in 2D culture compared to spheroids, which was paralleled by elevated activities of the interacting transcription factors LXR and ESRRA, likely at least in part due to altered mechanosensing. Inhibition of this YAP/TEAD hyperactivation resulted in an overall reduction of hepatocyte dedifferentiation marked by increased hepatic functionality, including CYPs. These results thus demonstrate that the observed induction is due to on-target effects of the compounds rather than direct activation of xenobiotic sensing nuclear receptors. Combined, the presented data link hepatocyte dedifferentiation to YAP/TEAD dysregulation, reveal a novel non-canonical pathway of CYP induction and highlight the advantage of organotypic 3D cultures to predict clinically relevant pharmacokinetic properties, particularly for atypical induction mechanisms.

MSC-1186, a Highly Selective Pan-SRPK Inhibitor Based on an Exceptionally Decorated Benzimidazole-Pyrimidine Core.

The highly conserved catalytic sites in protein kinases make it difficult to identify ATP competitive inhibitors with kinome-wide selectivity. Serendipitously, during a dedicated fragment campaign for the focal adhesion kinase (FAK), a scaffold that had lost its initial FAK affinity showed remarkable potency and selectivity for serine-arginine-protein kinases 1-3 (SRPK1-3). Non-conserved interactions with the uniquely structured hinge region of the SRPK family were the key drivers of the exclusive selectivity of the discovered fragment hit. Structure-guided medicinal chemistry efforts led to the SRPK inhibitor MSC-1186, which fulfills all hallmarks of a reversible chemical probe, including nanomolar cellular potency and excellent kinome-wide selectivity. The combination of MSC-1186 with CDC2-like kinase (CLK) inhibitors showed additive attenuation of SR-protein phosphorylation compared to the single agents. MSC-1186 and negative control (MSC-5360) are chemical probes available via the Structural Genomics Consortium chemical probe program (https://www.sgc-ffm.uni-frankfurt.de/).

Extract2Chip-Bypassing Protein Purification in Drug Discovery Using Surface Plasmon Resonance.

Modern drug discovery relies on combinatorial screening campaigns to find drug molecules targeting specific disease-associated proteins. The success of such campaigns often relies on functional and structural information of the selected therapeutic target, only achievable once its purification is mastered. With the aim of bypassing the protein purification process to gain insights on the druggability, ligand binding, and/or characterization of protein-protein interactions, herein, we describe the Extract2Chip method. This approach builds on the immobilization of site-specific biotinylated proteins of interest, directly from cellular extracts, on avidin-coated sensor chips to allow for the characterization of molecular interactions via surface plasmon resonance (SPR). The developed method was initially validated using Cyclophilin D (CypD) and subsequently applied to other drug discovery projects in which the targets of interest were difficult to express, purify, and crystallize. Extract2Chip was successfully applied to the characterization of Yes-associated protein (YAP): Transcriptional enhancer factor TEF (TEAD1) protein-protein interaction inhibitors, in the validation of a ternary complex assembly composed of Dyskerin pseudouridine synthase 1 (DKC1) and RuvBL1/RuvBL2, and in the establishment of a fast-screening platform to select the most suitable NUAK family SNF1-like kinase 2 (NUAK2) surrogate for binding and structural studies. The described method paves the way for a potential revival of the many drug discovery campaigns that have failed to deliver due to the lack of suitable and sufficient protein supply.

MCT4 blockade increases the efficacy of immune checkpoint blockade.

BACKGROUND & AIMS: Intratumoral lactate accumulation and acidosis impair T-cell function and antitumor immunity. Interestingly, expression of the lactate transporter monocarboxylate transporter (MCT) 4, but not MCT1, turned out to be prognostic for the survival of patients with rectal cancer, indicating that single MCT4 blockade might be a promising strategy to overcome glycolysis-related therapy resistance. METHODS: To determine whether blockade of MCT4 alone is sufficient to improve the efficacy of immune checkpoint blockade (ICB) therapy, we examined the effects of the selective MCT1 inhibitor AZD3965 and a novel MCT4 inhibitor in a colorectal carcinoma (CRC) tumor spheroid model co-cultured with blood leukocytes in vitro and the MC38 murine CRC model in vivo in combination with an antibody against programmed cell death ligand-1(PD-L1). RESULTS: Inhibition of MCT4 was sufficient to reduce lactate efflux in three-dimensional (3D) CRC spheroids but not in two-dimensional cell-cultures. Co-administration of the MCT4 inhibitor and ICB augmented immune cell infiltration, T-cell function and decreased CRC spheroid viability in a 3D co-culture model of human CRC spheroids with blood leukocytes. Accordingly, combination of MCT4 and ICB increased intratumoral pH, improved leukocyte infiltration and T-cell activation, delayed tumor growth, and prolonged survival in vivo. MCT1 inhibition exerted no further beneficial impact. CONCLUSIONS: These findings demonstrate that single MCT4 inhibition represents a novel therapeutic approach to reverse lactic-acid driven immunosuppression and might be suitable to improve ICB efficacy.

Synthesis and crystal structure of rac-2-(1,3-dioxo-isoindolin-2-yl)ethyl 4-methyl-N-phenyl-N'-(tri-iso-propyl-sil-yl)benzene-sulfondiimidoate: the first member of a new substance class.

The title compound {systematic name: rac-2-[7-methyl-4-(4-methylphenyl)-4-(phenylimino)-6,6-bis(propan-2-yl)-3-oxa-4λ6-thia-5-aza-6-silaoct-4-en-1-yl]-2,3-dihydro-1H-isoindole-1,3-dione}, C32H41N3O3SSi, was synthesized by desoxychlorination of 4-methyl-N-phenyl-N'-(triisopropyl-sil-yl)benzene-sul-fon-imid-am-ide and subsequent reaction with 2-(2-hy-droxy-eth-yl)isoindoline-1,3-dione. The racemic compound was crystallized from isopropanol. The structural characterization by single-crystal X-ray diffraction revealed two double-bonded nitro-gen atoms to the central sulfur atom and an overall crystal packing driven by its aromatic inter-actions.

Optimization of TEAD P-Site Binding Fragment Hit into In Vivo Active Lead MSC-4106.

The dysregulated Hippo pathway and, consequently, hyperactivity of the transcriptional YAP/TAZ-TEAD complexes is associated with diseases such as cancer. Prevention of YAP/TAZ-TEAD triggered gene transcription is an attractive strategy for therapeutic intervention. The deeply buried and conserved lipidation pocket (P-site) of the TEAD transcription factors is druggable. The discovery and optimization of a P-site binding fragment (1) are described. Utilizing structure-based design, enhancement in target potency was engineered into the hit, capitalizing on the established X-ray structure of TEAD1. The efforts culminated in the optimized in vivo tool MSC-4106, which exhibited desirable potency, mouse pharmacokinetic properties, and in vivo efficacy. In close correlation to compound exposure, the time- and dose-dependent downregulation of a proximal biomarker could be shown.

Structure-kinetic relationship reveals the mechanism of selectivity of FAK inhibitors over PYK2.

There is increasing evidence of a significant correlation between prolonged drug-target residence time and increased drug efficacy. Here, we report a structural rationale for kinetic selectivity between two closely related kinases: focal adhesion kinase (FAK) and proline-rich tyrosine kinase 2 (PYK2). We found that slowly dissociating FAK inhibitors induce helical structure at the DFG motif of FAK but not PYK2. Binding kinetic data, high-resolution structures and mutagenesis data support the role of hydrophobic interactions of inhibitors with the DFG-helical region, providing a structural rationale for slow dissociation rates from FAK and kinetic selectivity over PYK2. Our experimental data correlate well with computed relative residence times from molecular simulations, supporting a feasible strategy for rationally optimizing ligand residence times. We suggest that the interplay between the protein structural mobility and ligand-induced effects is a key regulator of the kinetic selectivity of inhibitors of FAK versus PYK2.

Discovery of 5-{2-[5-Chloro-2-(5-ethoxyquinoline-8-sulfonamido)phenyl]ethynyl}-4-methoxypyridine-2-carboxylic Acid, a Highly Selective in Vivo Useable Chemical Probe to Dissect MCT4 Biology.

Due to increased lactate production during glucose metabolism, tumor cells heavily rely on efficient lactate transport to avoid intracellular lactate accumulation and acidification. Monocarboxylate transporter 4 (MCT4/SLC16A3) is a lactate transporter that plays a central role in tumor pH modulation. The discovery and optimization of a novel class of MCT4 inhibitors (hit 9a), identified by a cellular screening in MDA-MB-231, is described. Direct target interaction of the optimized compound 18n with the cytosolic domain of MCT4 was shown after solubilization of the GFP-tagged transporter by fluorescence cross-correlation spectroscopy and microscopic studies. In vitro treatment with 18n resulted in lactate efflux inhibition and reduction of cellular viability in MCT4 high expressing cells. Moreover, pharmacokinetic properties of 18n allowed assessment of lactate modulation and antitumor activity in a mouse tumor model. Thus, 18n represents a valuable tool for investigating selective MCT4 inhibition and its effect on tumor biology.

Syntheses of novel 2,3-diaryl-substituted 5-cyano-4-azaindoles exhibiting c-Met inhibition activity.

Herein we report the syntheses of 2,3-diaryl-substituted pyrrolo[3,2-b]pyridine-5-carbonitriles via a one-pot 5-endo-dig-cyclization/protection reaction followed by palladium catalyzed arylation. In addition, a complementary synthesis route employing Larock methodology is applied to efficiently explore further aryl moieties in the 2-position. The novel compounds' expedient c-Met receptor tyrosine kinase inhibition activity is discussed.

Discovery and Structure-Based Optimization of Next-Generation Reversible Methionine Aminopeptidase-2 (MetAP-2) Inhibitors.

Co- and post-translational processing are crucial maturation steps to generate functional proteins. MetAP-2 plays an important role in this process, and inhibition of its proteolytic activity has been shown to be important for angiogenesis and tumor growth, suggesting that small-molecule inhibitors of MetAP-2 may be promising options for the treatment of cancer. This work describes the discovery and structure-based hit optimization of a novel MetAP-2 inhibitory scaffold. Of critical importance, a cyclic tartronic diamide coordinates the MetAP-2 metal ion in the active site while additional side chains of the molecule were designed to occupy the lipophilic methionine side chain recognition pocket as well as the shallow cavity at the opening of the active site. The racemic screening hit from HTS campaign 11a was discovered with an enzymatic IC50 of 150 nM. The resynthesized eutomer confirmed this activity and inhibited HUVEC proliferation with an IC50 of 1.9 µM. Its structural analysis revealed a sophisticated interaction pattern of polar and lipophilic contacts that were used to improve cellular potency to an IC50 of 15 nM. In parallel, the molecular properties were optimized on plasma exposure and antitumor efficacy which led to the identification of advanced lead 21.

Identification of Methionine Aminopeptidase-2 (MetAP-2) Inhibitor M8891: A Clinical Compound for the Treatment of Cancer.

The recently disclosed next generation of reversible, selective, and potent MetAP-2 inhibitors introduced a cyclic tartronic diamide scaffold. However, the lead compound 1a suffered from enterohepatic circulation, preventing further development. Nevertheless, 1a served as a starting point for further optimization. Maintaining potent antiproliferation activity, while improving other compound properties, enabled the generation of an attractive array of new MetAP-2 inhibitors. The most promising derivatives were identified by a multiparameter analysis of the compound properties. Essential for the efficient selection of candidates with in vivo activity was the identification of molecules with a long residence time on the target protein, high permeability, and low efflux ratio not only in Caco-2 but also in the MDR-MDCK cell line. Orally bioavailable, potent, and reversible MetAP-2 inhibitors impede the growth of primary endothelial cells and demonstrated antitumoral activity in mouse models. This assessment led to the nomination of the clinical development compound M8891, which is currently in phase I clinical testing in oncology patients.

Higher-order risk preferences in social settings.

We study prudence and temperance (next to risk aversion) in social settings. Previous experimental studies have shown that these higher-order risk preferences affect the choices of individuals deciding privately on lotteries that only affect their own payoff. Yet, many risky and financially relevant decisions are made in the social settings of households or organizations. We elicit higher-order risk preferences of individuals and systematically vary how an individual's decision is made (alone or while communicating with a partner) and who is affected by the decision (only the individual or the partner as well). In doing so, we can isolate the effects of other-regarding concerns and communication on choices. Our results reveal that the majority of choices are risk averse, prudent, and temperate across social settings. We also observe that individuals are influenced significantly by the preferences of a partner when they are able to communicate and choices are payoff-relevant for both of them.

Indolebutylamines as selective 5-HT(1A) agonists.

A series of new 1-[4-(indol-3-yl)butyl]-4-arylpiperazines was prepared to identify highly selective and potent 5-HT(1A) agonists as potential pharmacological tools in studies of mood disorders. The combination of structural elements (indole-alkyl-amine and aryl-piperazine) known to introduce 5-HT(1A) receptor affinity and the proper selection of substituents (R on the indole moiety and R' on the aryl moiety) led to compounds with high receptor specificity and affinity. In particular, the introduction of the methyl ether or the unsubstituted carboxamide as substituents in position 5 of the indole (R) guaranteed serotonergic 5-HT(1A) affinity compared to the unsubstituted analogue. Para-substituted arylpiperazines (R') decreased dopaminergic D(2) binding and increased selectivity for the 5-HT(1A) receptor. Agonistic 5-HT(1A) receptor activity was confirmed in vivo in the ultrasonic vocalization test, and the results suggest that the introduction of the carboxamide residue leads to better bioavailability than the corresponding methyl ether. 3-[4-[4-(4-Carbamoylphenyl)piperazin-1-yl]butyl]-1H-indole-5-carboxamide 54 was identified as a highly selective 5-HT(1A) receptor agonist [GTPgammaS, ED(50) = 4.7 nM] with nanomolar 5-HT(1A) affinity [IC(50) = 0.9 nM] and selectivity [D(2), IC(50) > 850 nM]. 3-[4-[4-(4-Methoxyphenyl)piperazin-1-yl]butyl]-1H-indole-5-carboxamide 45 is one of the most potent and selective 5-HT(1A) agonists known [5-HT(1A), IC(50) = 0.09 nM; D(2), IC(50) = 140 nM].

Synthesis and structure--activity relationship in a class of indolebutylpiperazines as dual 5-HT(1A) receptor agonists and serotonin reuptake inhibitors.

Systematic structural modifications of indolealkylphenylpiperazines led to improved selectivity and affinity within this class of 5-HT(1A) receptor agonists. Introduction of electron-withdrawing groups in position 5 on the indole raises serotonin transporter affinity, and the cyano group proved to be the best substituent here. 5-Fluoro and 5-cyano substituted indoles show comparable results in in vitro and in vivo tests, and bioisosterism between these substituents was supported by calculation of the molecular electrostatic potentials and dipole moments. Compounds showing promising in vitro data were further examined in ex vivo (p-chloroamphetamine assay) and in vivo (ultrasonic vocalization) tests. Optimization of the arylpiperazine moiety indicated that the 5-benzofuranyl-2-carboxamide was best suited to increase 5-HT transporter and 5-HT(1A) receptor affinity and to suppress D(2) receptor binding. 5-[4-[4-(5-Cyano-3-indolyl)butyl]-1-piperazinyl]benzofuran-2-carboxamide 29 (vilazodone, EMD 68843) was identified as a highly selective 5-HT(1A) receptor agonist [GTPgammaS, ED(50) = 1.1 nM] with subnanomolar 5-HT(1A) affinity [IC(50) = 0.2 nM] and as a subnanomolar 5-HT reuptake inhibitor [RUI = 0.5 nM] showing a great selectivity to other GPCRs (e.g., D(2), IC(50) = 666 nM).

Palladium-catalyzed carbonylation of haloindoles: no need for protecting groups.

[reaction: see text] For the first time, palladium-catalyzed carbonylations of unprotected bromoindoles have been performed successfully with different N- and O-nucleophiles. Various indole carboxylic acid derivatives are accessible in excellent yield. For example, aminocarbonylation of 4-, 5-, 6-, or 7-bromoindole with arylethylpiperazines provides a direct one-step synthesis for CNS active amphetamine derivatives.