Cryo-EM Structures of CRAF2/14-3-32 and CRAF2/14-3-32/MEK12 Complexes.

RAF protein kinases are essential effectors in the MAPK pathway and are important cancer drug targets. Structural understanding of RAF activation is so far based on cryo-electron microscopy (cryo-EM) and X-ray structures of BRAF in different conformational states as inactive or active complexes with KRAS, 14-3-3 and MEK1. In this study, we have solved the first cryo-EM structures of CRAF2/14-3-32 at 3.4 Å resolution and CRAF2/14-3-32/MEK12 at 4.2 Å resolution using CRAF kinase domain expressed as constitutively active Y340D/Y341D mutant in insect cells. The overall architecture of our CRAF2/14-3-32 and CRAF2/14-3-32/MEK12 cryo-EM structures is highly similar to corresponding BRAF structures in complex with 14-3-3 or 14-3-3/MEK1 and represent the activated dimeric RAF conformation. Our CRAF cryo-EM structures provide additional insights into structural understanding of the activated CRAF2/14-3-32/MEK12 complex.

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.

Biophysical and structural characterization of the impacts of MET phosphorylation on tepotinib binding.

The receptor tyrosine kinase MET is activated by hepatocyte growth factor binding, followed by phosphorylation of the intracellular kinase domain (KD) mainly within the activation loop (A-loop) on Y1234 and Y1235. Dysregulation of MET can lead to both tumor growth and metastatic progression of cancer cells. Tepotinib is a highly selective, potent type Ib MET inhibitor and approved for treatment of non-small cell lung cancer harboring METex14 skipping alterations. Tepotinib binds to the ATP site of unphosphorylated MET with critical π-stacking contacts to Y1230 of the A-loop, resulting in a high residence time. In our study, we combined protein crystallography, biophysical methods (surface plasmon resonance, differential scanning fluorimetry), and mass spectrometry to clarify the impacts of A-loop conformation on tepotinib binding using different recombinant MET KD protein variants. We solved the first crystal structures of MET mutants Y1235D, Y1234E/1235E, and F1200I in complex with tepotinib. Our biophysical and structural data indicated a linkage between reduced residence times for tepotinib and modulation of A-loop conformation either by mutation (Y1235D), by affecting the overall Y1234/Y1235 phosphorylation status (L1195V and F1200I) or by disturbing critical π-stacking interactions with tepotinib (Y1230C). We corroborated these data with target engagement studies by fluorescence cross-correlation spectroscopy using KD constructs in cell lysates or full-length receptors from solubilized cellular membranes as WT or activated mutants (Y1235D and Y1234E/1235E). Collectively, our results provide further insight into the MET A-loop structural determinants that affect the binding of the selective inhibitor tepotinib.

Identification of M4205─A Highly Selective Inhibitor of KIT Mutations for Treatment of Unresectable Metastatic or Recurrent Gastrointestinal Stromal Tumors.

The treatment of gastrointestinal stromal tumors (GISTs) driven by activating mutations in the KIT gene is a prime example of targeted therapy for treatment of cancer. The approval of the tyrosine kinase inhibitor imatinib has significantly improved patient survival, but emerging resistance under treatment and relapse is observed. Several additional KIT inhibitors have been approved; still, there is a high unmet need for KIT inhibitors with high selectivity and broad coverage of all clinically relevant KIT mutants. An imidazopyridine hit featuring excellent kinase selectivity was identified in a high-throughput screen (HTS) and optimized to the clinical candidate M4205 (IDRX-42). This molecule has a superior profile compared to approved drugs, suggesting a best-in-class potential for recurrent and metastatic GISTs driven by KIT mutations.

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/).

Nitric Oxide Synthase Inhibitors into the Clinic at Last.

The 1998 Nobel Prize in Medicine and Physiology for the discovery of nitric oxide, a nitrogen containing reactive oxygen species (also termed reactive nitrogen or reactive nitrogen/oxygen species) stirred great hopes. Clinical applications, however, have so far pertained exclusively to the downstream signaling of cGMP enhancing drugs such as phosphodiesterase inhibitors and soluble guanylate cyclase stimulators. All clinical attempts, so far, to inhibit NOS have failed even though preclinical models were strikingly positive and clinical biomarkers correlated perfectly. This rather casts doubt on our current way of target identification in drug discovery in general and our way of patient stratification based on correlating but not causal biomarkers or symptoms. The opposite, NO donors, nitrite and enhancing NO synthesis by eNOS/NOS3 recoupling in situations of NO deficiency, are rapidly declining in clinical relevance or hold promise but need yet to enter formal therapeutic guidelines, respectively. Nevertheless, NOS inhibition in situations of NO overproduction often jointly with enhanced superoxide (or hydrogen peroxide production) still holds promise, but most likely only in acute conditions such as neurotrauma (Stover et al., J Neurotrauma 31(19):1599-1606, 2014) and stroke (Kleinschnitz et al., J Cereb Blood Flow Metab 1508-1512, 2016; Casas et al., Proc Natl Acad Sci U S A 116(14):7129-7136, 2019). Conversely, in chronic conditions, long-term inhibition of NOS might be too risky because of off-target effects on eNOS/NOS3 in particular for patients with cardiovascular risks or metabolic and renal diseases. Nitric oxide synthases (NOS) and their role in health (green) and disease (red). Only neuronal/type 1 NOS (NOS1) has a high degree of clinical validation and is in late stage development for traumatic brain injury, followed by a phase II safety/efficacy trial in ischemic stroke. The pathophysiology of NOS1 (Kleinschnitz et al., J Cereb Blood Flow Metab 1508-1512, 2016) is likely to be related to parallel superoxide or hydrogen peroxide formation (Kleinschnitz et al., J Cereb Blood Flow Metab 1508-1512, 2016; Casas et al., Proc Natl Acad Sci U S A 114(46):12315-12320, 2017; Casas et al., Proc Natl Acad Sci U S A 116(14):7129-7136, 2019) leading to peroxynitrite and protein nitration, etc. Endothelial/type 3 NOS (NOS3) is considered protective only and its inhibition should be avoided. The preclinical evidence for a role of high-output inducible/type 2 NOS (NOS2) isoform in sepsis, asthma, rheumatic arthritis, etc. was high, but all clinical development trials in these indications were neutral despite target engagement being validated. This casts doubt on the role of NOS2 in humans in health and disease (hence the neutral, black coloring).

Biochemical, cellular and structural characterization of novel and selective ERK3 inhibitors.

Triazolo[4,5-d]pyrimidin-5-amines were identified from kinase selectivity screening as novel ERK3 inhibitors with sub-100 nanomolar potencies in a biochemical assay using MK5 as substrate and with an attractive kinase selectivity profile. ERK3 crystal structures clarified the inhibitor binding mode in the ATP pocket with impact on A-loop, GC-loop and αC-helix conformations suggesting a potential structural link towards MK5 interaction via the FHIEDE motif. The inhibitors also showed sub-100 nM potencies in a cellular ERK3 NanoBRET assay and with excellent correlation to the biochemical IC50s. This novel series provides valuable tool compounds to further investigate the biological function and activation mechanism of ERK3.

Large-Scale Assessment of Binding Free Energy Calculations in Active Drug Discovery Projects.

Accurate ranking of compounds with regards to their binding affinity to a protein using computational methods is of great interest to pharmaceutical research. Physics-based free energy calculations are regarded as the most rigorous way to estimate binding affinity. In recent years, many retrospective studies carried out both in academia and industry have demonstrated its potential. Here, we present the results of large-scale prospective application of the FEP+ method in active drug discovery projects in an industry setting at Merck KGaA, Darmstadt, Germany. We compare these prospective data to results obtained on a new diverse, public benchmark of eight pharmaceutically relevant targets. Our results offer insights into the challenges faced when using free energy calculations in real-life drug discovery projects and identify limitations that could be tackled by future method development. The new public data set we provide to the community can support further method development and comparative benchmarking of free energy calculations.

Discovery of novel Cyclophilin D inhibitors starting from three dimensional fragments with millimolar potencies.

Fragment-based screening by SPR enabled the discovery of chemical diverse fragment hits with millimolar binding affinities to the peptidyl-prolyl isomerase Cyclophilin D (CypD). The CypD protein crystal structures of 6 fragment hits provided the basis for subsequent medicinal chemistry optimization by fragment merging and linking yielding three different chemical series with either urea, oxalyl or amide linkers connecting millimolar fragments in the S1' and S2 pockets. We successfully improved the in vitro CypD potencies in the biochemical FP and PPIase assays and in the biophysical SPR binding assay from millimolar towards the low micromolar and submicromolar range by >1000-fold for some fragment derivatives. The initial SAR together with the protein crystal structures of our novel CypD inhibitors provide a suitable basis for further hit-to-lead optimization.

Estimation of Drug-Target Residence Times by τ-Random Acceleration Molecular Dynamics Simulations.

Drug-target residence time (τ), one of the main determinants of drug efficacy, remains highly challenging to predict computationally and, therefore, is usually not considered in the early stages of drug design. Here, we present an efficient computational method, τ-random acceleration molecular dynamics (τRAMD), for the ranking of drug candidates by their residence time and obtaining insights into ligand-target dissociation mechanisms. We assessed τRAMD on a data set of 70 diverse drug-like ligands of the N-terminal domain of HSP90α, a pharmaceutically important target with a highly flexible binding site, obtaining computed relative residence times with an accuracy of about 2.3τ for 78% of the compounds and less than 2.0τ within congeneric series. Analysis of dissociation trajectories reveals features that affect ligand unbinding rates, including transient polar interactions and steric hindrance. These results suggest that τRAMD will be widely applicable as a computationally efficient aid to improving drug residence times during lead optimization.

Ligand Desolvation Steers On-Rate and Impacts Drug Residence Time of Heat Shock Protein 90 (Hsp90) Inhibitors.

Residence time and more recently the association rate constant kon are increasingly acknowledged as important parameters for in vivo efficacy and safety of drugs. However, their broader consideration in drug development is limited by a lack of knowledge of how to optimize these parameters. In this study on a set of 176 heat shock protein 90 inhibitors, structure-kinetic relationships, X-ray crystallography, and molecular dynamics simulations were combined to retrieve a concrete scheme of how to rationally slow down on-rates. We discovered that an increased ligand desolvation barrier by introducing polar substituents resulted in a significant kon decrease. The slowdown was accomplished by introducing polar moieties to those parts of the ligand that point toward a hydrophobic cavity. We validated this scheme by increasing polarity of three Hsp90 inhibitors and observed a 9-, 13-, and 45-fold slowdown of on-rates and a 9-fold prolongation in residence time. This prolongation was driven by transition state destabilization rather than ground state stabilization.

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.

Identification and optimization of pyridazinones as potent and selective c-Met kinase inhibitors.

In a high-throughput screening campaign for c-Met kinase inhibitors, a thiadiazinone derivative with a carbamate group was identified as a potent in vitro inhibitor. Subsequent optimization guided by c-Met-inhibitor X-ray structures furnished new compound classes with excellent in vitro and in vivo profiles. The thiadiazinone ring of the HTS hit was first replaced by a pyridazinone followed by an exchange of the carbamate hinge binder with a 1,5-disubstituted pyrimidine. Finally an optimized compound, 22 (MSC2156119), with excellent in vitro potency, high kinase selectivity, long half-life after oral administration and in vivo anti-tumor efficacy at low doses, was selected as a candidate for clinical development.

Structure-based optimization of non-peptidic Cathepsin D inhibitors.

We discovered a novel series of non-peptidic acylguanidine inhibitors of Cathepsin D as target for osteoarthritis. The initial HTS-hits were optimized by structure-based design using CatD X-ray structures resulting in single digit nanomolar potency in the biochemical CatD assay. However, the most potent analogues showed only micromolar activities in an ex vivo glycosaminoglycan (GAG) release assay in bovine cartilage together with low cellular permeability and suboptimal microsomal stability. This new scaffold can serve as a starting point for further optimization towards in vivo efficacy.

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.

EMD 1214063 and EMD 1204831 constitute a new class of potent and highly selective c-Met inhibitors.

PURPOSE: The mesenchymal-epithelial transition factor (c-Met) receptor, also known as hepatocyte growth factor receptor (HGFR), controls morphogenesis, a process that is physiologically required for embryonic development and tissue repair. Aberrant c-Met activation is associated with a variety of human malignancies including cancers of the lung, kidney, stomach, liver, and brain. In this study, we investigated the properties of two novel compounds developed to selectively inhibit the c-Met receptor in antitumor therapeutic interventions. EXPERIMENTAL DESIGN: The pharmacologic properties, c-Met inhibitory activity, and antitumor effects of EMD 1214063 and EMD 1204831 were investigated in vitro and in vivo, using human cancer cell lines and mouse xenograft models. RESULTS: EMD 1214063 and EMD 1204831 selectively suppressed the c-Met receptor tyrosine kinase activity. Their inhibitory activity was potent [inhibitory 50% concentration (IC50), 3 nmol/L and 9 nmol/L, respectively] and highly selective, when compared with their effect on a panel of 242 human kinases. Both EMD 1214063 and EMD 1204831 inhibited c-Met phosphorylation and downstream signaling in a dose-dependent fashion, but differed in the duration of their inhibitory activity. In murine xenograft models, both compounds induced regression of human tumors, regardless of whether c-Met activation was HGF dependent or independent. Both drugs were well tolerated and induced no substantial weight loss after more than 3 weeks of treatment. CONCLUSIONS: Our results indicate selective c-Met inhibition by EMD 1214063 and EMD 1204831 and strongly support clinical testing of these compounds in the context of molecularly targeted anticancer strategies.

Fragment-based discovery of new highly substituted 1H-pyrrolo[2,3-b]- and 3H-imidazolo[4,5-b]-pyridines as focal adhesion kinase inhibitors.

Focal adhesion kinase (FAK) is considered as an attractive target for oncology, and small-molecule inhibitors are reported to be in clinical testing. In a surface plasmon resonance (SPR)-mediated fragment screening campaign, we discovered bicyclic scaffolds like 1H-pyrazolo[3,4-d]pyrimidines binding to the hinge region of FAK. By an accelerated knowledge-based fragment growing approach, essential pharmacophores were added. The establishment of highly substituted unprecedented 1H-pyrrolo[2,3-b]pyridine derivatizations provided compounds with submicromolar cellular FAK inhibition potential. The combination of substituents on the bicyclic templates and the nature of the core structure itself have a significant impact on the compounds FAK selectivity. Structural analysis revealed that the appropriately substituted pyrrolo[2,3-b]pyridine induced a rare helical DFG-loop conformation. The discovered synthetic route to introduce three different substituents independently paves the way for versatile applications of the 7-azaindole core.

Structural and biophysical characterization of the Syk activation switch.

Syk is an essential non-receptor tyrosine kinase in intracellular immunological signaling, and the control of Syk kinase function is considered as a valuable target for pharmacological intervention in autoimmune or inflammation diseases. Upon immune receptor stimulation, the kinase activity of Syk is regulated by binding of phosphorylated immune receptor tyrosine-based activating motifs (pITAMs) to the N-terminal tandem Src homology 2 (tSH2) domain and by autophosphorylation with consequences for the molecular structure of the Syk protein. Here, we present the first crystal structures of full-length Syk (fl-Syk) as wild type and as Y348F,Y352F mutant forms in complex with AMP-PNP revealing an autoinhibited conformation. The comparison with the crystal structure of the truncated Syk kinase domain in complex with AMP-PNP taken together with ligand binding studies by surface plasmon resonance (SPR) suggests conformational differences in the ATP sites of autoinhibited and activated Syk forms. This hypothesis was corroborated by studying the thermodynamic and kinetic interaction of three published Syk inhibitors with isothermal titration calorimetry and SPR, respectively. We further demonstrate the modulation of inhibitor binding affinities in the presence of pITAM and discuss the observed differences of thermodynamic and kinetic signatures. The functional relevance of pITAM binding to fl-Syk was confirmed by a strong stimulation of in vitro autophosphorylation. A structural feedback mechanism on the kinase domain upon pITAM binding to the tSH2 domain is discussed in analogy of the related family kinase ZAP-70 (Zeta-chain-associated protein kinase 70). Surprisingly, we observed distinct conformations of the tSH2 domain and the activation switch including Tyr348 and Tyr352 in the interdomain linker of Syk in comparison to ZAP-70.

Fragment-based discovery of hydroxy-indazole-carboxamides as novel small molecule inhibitors of Hsp90.

Inhibitors of the Hsp90 molecular chaperone are showing considerable promise as potential molecular therapeutic agents for the treatment of cancer. Here we describe the identification of novel small molecular weight inhibitors of Hsp90 using a fragment based approach. Fragments were selected by docking, tested in a biochemical assay and the confirmed hits were crystallized. Information gained from X-ray structures of these fragments and other chemotypes was used to drive the fragment evolution process. Optimization of these high µM binders resulted in 3-benzylindazole derivatives with significantly improved affinity and anti-proliferative effects in different human cancer cell lines.