A Novel NAMPT Inhibitor-Based Antibody-Drug Conjugate Payload Class for Cancer Therapy.

Inhibition of intracellular nicotinamide phosphoribosyltransferase (NAMPT) represents a new mode of action for cancer-targeting antibody-drug conjugates (ADCs) with activity also in slowly proliferating cells. To extend the repertoire of available effector chemistries, we have developed a novel structural class of NAMPT inhibitors as ADC payloads. A structure-activity relationship-driven approach supported by protein structural information was pursued to identify a suitable attachment point for the linker to connect the NAMPT inhibitor with the antibody. Optimization of scaffolds and linker structures led to highly potent effector chemistries which were conjugated to antibodies targeting C4.4a (LYPD3), HER2 (c-erbB2), or B7H3 (CD276) and tested on antigen-positive and -negative cancer cell lines. Pharmacokinetic studies, including metabolite profiling, were performed to optimize the stability and selectivity of the ADCs and to evaluate potential bystander effects. Optimized NAMPTi-ADCs demonstrated potent in vivo antitumor efficacy in target antigen-expressing xenograft mouse models. This led to the development of highly potent NAMPT inhibitor ADCs with a very good selectivity profile compared with the corresponding isotype control ADCs. Moreover, we demonstrate─to our knowledge for the first time─the generation of NAMPTi payload metabolites from the NAMPTi-ADCs in vitro and in vivo. In conclusion, NAMPTi-ADCs represent an attractive new payload class designed for use in ADCs for the treatment of solid and hematological cancers.

Selective covalent targeting of GPX4 using masked nitrile-oxide electrophiles.

We recently described glutathione peroxidase 4 (GPX4) as a promising target for killing therapy-resistant cancer cells via ferroptosis. The onset of therapy resistance by multiple types of treatment results in a stable cell state marked by high levels of polyunsaturated lipids and an acquired dependency on GPX4. Unfortunately, all existing inhibitors of GPX4 act covalently via a reactive alkyl chloride moiety that confers poor selectivity and pharmacokinetic properties. Here, we report our discovery that masked nitrile-oxide electrophiles, which have not been explored previously as covalent cellular probes, undergo remarkable chemical transformations in cells and provide an effective strategy for selective targeting of GPX4. The new GPX4-inhibiting compounds we describe exhibit unexpected proteome-wide selectivity and, in some instances, vastly improved physiochemical and pharmacokinetic properties compared to existing chloroacetamide-based GPX4 inhibitors. These features make them superior tool compounds for biological interrogation of ferroptosis and constitute starting points for development of improved inhibitors of GPX4.

Discovery of potent SOS1 inhibitors that block RAS activation via disruption of the RAS-SOS1 interaction.

Since the late 1980s, mutations in the RAS genes have been recognized as major oncogenes with a high occurrence rate in human cancers. Such mutations reduce the ability of the small GTPase RAS to hydrolyze GTP, keeping this molecular switch in a constitutively active GTP-bound form that drives, unchecked, oncogenic downstream signaling. One strategy to reduce the levels of active RAS is to target guanine nucleotide exchange factors, which allow RAS to cycle from the inactive GDP-bound state to the active GTP-bound form. Here, we describe the identification of potent and cell-active small-molecule inhibitors which efficiently disrupt the interaction between KRAS and its exchange factor SOS1, a mode of action confirmed by a series of biophysical techniques. The binding sites, mode of action, and selectivity were elucidated using crystal structures of KRASG12C-SOS1, SOS1, and SOS2. By preventing formation of the KRAS-SOS1 complex, these inhibitors block reloading of KRAS with GTP, leading to antiproliferative activity. The final compound 23 (BAY-293) selectively inhibits the KRAS-SOS1 interaction with an IC50 of 21 nM and is a valuable chemical probe for future investigations.

Mechanism of inhibition of human glucose transporter GLUT1 is conserved between cytochalasin B and phenylalanine amides.

Cancerous cells have an acutely increased demand for energy, leading to increased levels of human glucose transporter 1 (hGLUT1). This up-regulation suggests hGLUT1 as a target for therapeutic inhibitors addressing a multitude of cancer types. Here, we present three inhibitor-bound, inward-open structures of WT-hGLUT1 crystallized with three different inhibitors: cytochalasin B, a nine-membered bicyclic ring fused to a 14-membered macrocycle, which has been described extensively in the literature of hGLUTs, and two previously undescribed Phe amide-derived inhibitors. Despite very different chemical backbones, all three compounds bind in the central cavity of the inward-open state of hGLUT1, and all binding sites overlap the glucose-binding site. The inhibitory action of the compounds was determined for hGLUT family members, hGLUT1-4, using cell-based assays, and compared with homology models for these hGLUT members. This comparison uncovered a probable basis for the observed differences in inhibition between family members. We pinpoint regions of the hGLUT proteins that can be targeted to achieve isoform selectivity, and show that these same regions are used for inhibitors with very distinct structural backbones. The inhibitor cocomplex structures of hGLUT1 provide an important structural insight for the design of more selective inhibitors for hGLUTs and hGLUT1 in particular.

Pan-mutant IDH1 inhibitor BAY 1436032 for effective treatment of IDH1 mutant astrocytoma in vivo.

Mutations in codon 132 of isocitrate dehydrogenase (IDH) 1 are frequent in diffuse glioma, acute myeloid leukemia, chondrosarcoma and intrahepatic cholangiocarcinoma. These mutations result in a neomorphic enzyme specificity which leads to a dramatic increase of intracellular D-2-hydroxyglutarate (2-HG) in tumor cells. Therefore, mutant IDH1 protein is a highly attractive target for inhibitory drugs. Here, we describe the development and properties of BAY 1436032, a pan-inhibitor of IDH1 protein with different codon 132 mutations. BAY 1436032 strongly reduces 2-HG levels in cells carrying IDH1-R132H, -R132C, -R132G, -R132S and -R132L mutations. Cells not carrying IDH mutations were unaffected. BAY 1436032 did not exhibit toxicity in vitro or in vivo. The pharmacokinetic properties of BAY 1436032 allow for oral administration. In two independent experiments, BAY 1436032 has been shown to significantly prolong survival of mice intracerebrally transplanted with human astrocytoma carrying the IDH1R132H mutation. In conclusion, we developed a pan-inhibitor targeting tumors with different IDH1R132 mutations.

Optimization of allosteric MEK inhibitors. Part 2: Taming the sulfamide group balances compound distribution properties.

Recently, we had identified an unexplored pocket adjacent to the known binding site of allosteric MEK inhibitors which allowed us to design highly potent and in vivo efficacious novel inhibitors. We now report that our initial preclinical candidate, featuring a phenoxy side chain with a sulfamide capping group, displayed human carbonic anhydrase off-target activity and species-dependent blood cell accumulation, which prevented us from advancing this candidate further. Since this sulfamide MEK inhibitor displayed an exceptionally favorable PK profile with low brain penetration potential despite being highly oral bioavailable, we elected to keep the sulfamide capping group intact while taming its unwanted off-target activity by optimizing the structural surroundings. Introduction of a neighboring fluorine atom or installation of a methylene linker reduced hCA potency sufficiently, at the cost of MEK target potency. Switching to a higher fluorinated central core reinstated high MEK potency, leading to two new preclinical candidates with long half-lives, high bioavailabilities, low brain penetration potential and convincing efficacy in a K-Ras-mutated A549 xenograft model.

Discovery and Characterization of BAY-184: A New Potent and Selective Acylsulfonamide-Benzofuran In Vivo-Active KAT6AB Inhibitor.

KAT6A and KAT6B genes are two closely related lysine acetyltransferases that transfer an acetyl group from acetyl coenzyme A (AcCoA) to lysine residues of target histone substrates, hence playing a key role in chromatin regulation. KAT6A and KAT6B genes are frequently amplified in various cancer types. In breast cancer, the 8p11-p12 amplicon occurs in 12-15% of cases, resulting in elevated copy numbers and expression levels of chromatin modifiers like KAT6A. Here, we report the discovery of a new acylsulfonamide-benzofuran series as a novel structural class for KAT6A/B inhibition. These compounds were identified through high-throughput screening and subsequently optimized using molecular modeling and cocrystal structure determination. The final tool compound, BAY-184 (29), was successfully validated in an in vivo proof-of-concept study.

Optimization of allosteric MEK inhibitors. Part 1: Venturing into underexplored SAR territories.

Using PD325901 as a starting point for identifying novel allosteric MEK inhibitors with high cell potency and long-lasting target inhibition in vivo, truncation of its hydroxamic ester headgroup was combined with incorporation of alkyl and aryl ethers at the neighboring ring position. Whereas alkoxy side chains did not yield sufficient levels of cell potency, specifically substituted aryloxy groups allowed for high enzymatic and cellular potencies. Sulfamide 28 was identified as a highly potent MEK inhibitor with nanomolar cell potency against B-RAF (V600E) as well as Ras-mutated cell lines, high metabolic stability and resulting long half-lives. It was efficacious against B-RAF as well as K-Ras driven xenograft models and showed-despite being orally bioavailable and not a P-glycoprotein substrate-much lower brain/plasma exposure ratios than PD325901.

Identification of the Highly Active, Species Cross-Reactive Complex I Inhibitor BAY-179.

Mitochondria are key regulators of energy supply and cell death. Generation of ATP within mitochondria occurs through oxidative phosphorylation (OXPHOS), a process which utilizes the four complexes (complex I-IV) of the electron transport chain and ATP synthase. Certain oncogenic mutations (e.g., LKB1 or mIDH) can further enhance the reliance of cancer cells on OXPHOS for their energetic requirements, rendering cells sensitive to complex I inhibition and highlighting the potential value of complex I as a therapeutic target. Herein, we describe the discovery of a potent, selective, and species cross-reactive complex I inhibitor. A high-throughput screen of the Bayer compound library followed by hit triaging and initial hit-to-lead activities led to a lead structure which was further optimized in a comprehensive lead optimization campaign. Focusing on balancing potency and metabolic stability, this program resulted in the identification of BAY-179, an excellent in vivo suitable tool with which to probe the biological relevance of complex I inhibition in cancer indications.

BAY-069, a Novel (Trifluoromethyl)pyrimidinedione-Based BCAT1/2 Inhibitor and Chemical Probe.

The branched-chain amino acid transaminases (BCATs) are enzymes that catalyze the first reaction of catabolism of the essential branched-chain amino acids to branched-chain keto acids to form glutamate. They are known to play a key role in different cancer types. Here, we report a new structural class of BCAT1/2 inhibitors, (trifluoromethyl)pyrimidinediones, identified by a high-throughput screening campaign and subsequent optimization guided by a series of X-ray crystal structures. Our potent dual BCAT1/2 inhibitor BAY-069 displays high cellular activity and very good selectivity. Along with a negative control (BAY-771), BAY-069 was donated as a chemical probe to the Structural Genomics Consortium.

A crystallographic fragment screen identifies cinnamic acid derivatives as starting points for potent Pim-1 inhibitors.

A crystallographic fragment screen was carried out to identify starting points for the development of inhibitors of protein kinase Pim-1, a potential target for tumour therapy. All fragment hits identified via soaking in this study turned out to bind to the unusually hydrophobic pocket at the hinge region. The most potent fragments, two cinnamic acid derivatives (with a best IC(50) of 130 µM), additionally form a well defined hydrogen bond. The balance between hydrophobic and polar interactions makes these molecules good starting points for further optimization. Pim-2 inhibitors from a recently reported high-throughput screening campaign also feature a cinnamic acid moiety. Two of these Pim-2 inhibitors were synthesized, their potencies against Pim-1 were determined and their cocrystal structures were elucidated in order to determine to what degree the binding modes identified by fragment screening are conserved in optimized inhibitors. The structures show that the cinnamic acid moieties indeed adopt the same binding mode. Fragment screening thus correctly identified binding modes which are maintained when fragments are grown into larger and higher affinity inhibitors. The high-throughput screening-derived compound (E)-3-{3-[6-(4-aminocyclohexylamino)-pyrazin-2-yl]phenyl}acrylic acid (compound 1) is the most potent inhibitor of the cinnamic acid series for which the three-dimensional binding mode is known (IC(50) = 17 nM, K(d) = 28 nM). The structure reveals the molecular basis for the large gain in potency between the initial fragment hit and this optimized inhibitor.

Crystal structures of the selenoprotein glutathione peroxidase 4 in its apo form and in complex with the covalently bound inhibitor ML162.

Wild-type human glutathione peroxidase 4 (GPX4) was co-expressed with SBP2 (selenocysteine insertion sequence-binding protein 2) in human HEK cells to achieve efficient production of this selenocysteine-containing enzyme on a preparative scale for structural biology. The protein was purified and crystallized, and the crystal structure of the wild-type form of GPX4 was determined at 1.0 Šresolution. The overall fold and the active site are conserved compared with previously determined crystal structures of mutated forms of GPX4. A mass-spectrometry-based approach was developed to monitor the reaction of the active-site selenocysteine Sec46 with covalent inhibitors. This, together with the introduction of a surface mutant (Cys66Ser), enabled the crystal structure determination of GPX4 in complex with the covalent inhibitor ML162 [(S)-enantiomer]. The mass-spectrometry-based approach described here opens the path to further co-complex crystal structures of this potential cancer drug target in complex with covalent inhibitors.

Design, Synthesis, and Pharmacological Characterization of a Neutral, Non-Prodrug Thrombin Inhibitor with Good Oral Pharmacokinetics.

Despite extensive research on small molecule thrombin inhibitors for oral application in the past decades, only a single double prodrug with very modest oral bioavailability has reached human therapy as a marketed drug. We have undertaken major efforts to identify neutral, non-prodrug inhibitors. Using a holistic analysis of all available internal data, we were able to build computational models and apply these for the selection of a lead series with the highest possibility of achieving oral bioavailability. In our design, we relied on protein structure knowledge to address potency and identified a small window of favorable physicochemical properties to balance absorption and metabolic stability. Protein structure information on the pregnane X receptor helped in overcoming a persistent cytochrome P450 3A4 induction problem. The selected compound series was optimized to a highly potent, neutral, non-prodrug thrombin inhibitor by designing, synthesizing, and testing derivatives. The resulting optimized compound, BAY1217224, has reached first clinical trials, which have confirmed the desired pharmacokinetic properties.

Computationally Empowered Workflow Identifies Novel Covalent Allosteric Binders for KRASG12C.

Due to its frequent mutations in multiple lethal cancers, KRAS is one of the most-studied anticancer targets nowadays. Since the discovery of the druggable allosteric binding site containing a G12C mutation, KRASG12C has been the focus of attention in oncology research. We report here a computationally driven approach aimed at identifying novel and selective KRASG12C covalent inhibitors. The workflow involved initial enumeration of virtual molecules tailored for the KRAS allosteric binding site. Tools such as pharmacophore modeling, docking, and free-energy perturbations were deployed to prioritize the compounds with the best profiles. The synthesized naphthyridinone scaffold showed the ability to react with G12C and inhibit KRASG12C . Analogues were prepared to establish structure-activity relationships, while molecular dynamics simulations and crystallization of the inhibitor-KRASG12C complex highlighted an unprecedented binding mode.

Discovery and Characterization of the Potent and Selective P2X4 Inhibitor N-[4-(3-Chlorophenoxy)-3-sulfamoylphenyl]-2-phenylacetamide (BAY-1797) and Structure-Guided Amelioration of Its CYP3A4 Induction Profile.

The P2X4 receptor is a ligand-gated ion channel that is expressed on a variety of cell types, especially those involved in inflammatory and immune processes. High-throughput screening led to a new class of P2X4 inhibitors with substantial CYP 3A4 induction in human hepatocytes. A structure-guided optimization with respect to decreased pregnane X receptor (PXR) binding was started. It was found that the introduction of larger and more polar substituents on the ether linker led to less PXR binding while maintaining the P2X4 inhibitory potency. This translated into significantly reduced CYP 3A4 induction for compounds 71 and 73. Unfortunately, the in vivo pharmacokinetic (PK) profiles of these compounds were insufficient for the desired profile in humans. However, BAY-1797 (10) was identified and characterized as a potent and selective P2X4 antagonist. This compound is suitable for in vivo studies in rodents, and the anti-inflammatory and anti-nociceptive effects of BAY-1797 were demonstrated in a mouse complete Freund's adjuvant (CFA) inflammatory pain model.

Structural basis for a high affinity inhibitor bound to protein kinase MK2.

The Ser/Thr protein kinase MAPKAP kinase 2 (MK2) plays a crucial role in inflammation. We determined the structure of the kinase domain of MK2 in complex with a low molecular mass inhibitor in two different crystal forms, obtained from soaking and co-crystallization. To our knowledge, these are the first structures of MK2 showing the binding mode of an inhibitor with high binding affinity (IC50 8.5 nM). The two crystal forms revealed conformational flexibility in the binding site and extend the experimental basis for rational drug design. Crystal form-1 contained one MK2 molecule per asymmetric unit. Form-2 contained 12 molecules, which arrange into two different types of MK2 trimers. One of them may serve as a model for an intermediate state during substrate phosphorylation, as each MK2 monomer places its activation segment into the substrate peptide binding groove of the trimer neighbor.

Crystallization and molecular-replacement solution of a diagnostic fluorescent dye in complex with a specific Fab fragment.

Tetrasulfocyanine (TSC) has been described as a fluorescent probe for tumour imaging. The complex of TSC and the Fab antibody fragment MOR03268 has been crystallized in three different crystal forms. MOR03268 was identified from the HuCAL GOLD library and further optimized to bind TSC with high affinity (Kd = 0.6 nM). For two of the three crystal forms (forms 1 and 2), data sets could be collected to 2.8 and 2.85 A resolution, respectively. Form 1 belongs to space group I222, with unit-cell parameters a = 72, b = 99, c = 154 A. Form 2 belongs to space group P4(3)2(1)2, with unit-cell parameters a = b = 77, c = 379 A. Form 3 only diffracted to 8 A and was not analyzed further. Molecular-replacement solutions for forms 1 and 2 were found and rebuilding and refinement is in progress. Form 1 contains one Fab molecule per asymmetric unit, while form 2 harbours two. Judging from the green colour of the crystals, both forms contain the Fab molecule bound to the green TSC dye and in both the hydrolysis-sensitive dye molecule is protected from degradation for several weeks to months. The structures should reveal the molecular basis of the high-affinity recognition of TSC by the Fab molecule MOR03268.

Identifying protein construct variants with increased crystallization propensity--a case study.

This study describes an efficient multiparallel automated workflow of cloning, expression, purification, and crystallization of a large set of construct variants for isolated protein domains aimed at structure determination by X-ray crystallography. This methodology is applied to MAPKAP kinase 2, a key enzyme in the inflammation pathway and thus an attractive drug target. The study reveals a distinct subset of truncation variants with improved crystallization properties. These constructs distinguish themselves by increased solubility and stability during a parallel automated multistep purification process including removal of the recombinant tag. High-throughput protein melting point analysis characterizes this subset of constructs as particularly thermostable. Both parallel purification screening and melting point determination clearly identify residue 364 as the optimal C terminus for the kinase domain. Moreover, all three constructs that ultimately crystallized feature this C terminus. At the N terminus, only three amino acids differentiate a noncrystallizing from a crystallizing construct. This study addresses the very common issues associated with difficult to crystallize proteins, those of solubility and stability, and the crucial importance of particular residues in the formation of crystal contacts. A methodology is suggested that includes biophysical measurements to efficiently identify and produce construct variants of isolated protein domains which exhibit higher crystallization propensity.

Discovery and Characterization of a Highly Potent and Selective Aminopyrazoline-Based in Vivo Probe (BAY-598) for the Protein Lysine Methyltransferase SMYD2.

Protein lysine methyltransferases have recently emerged as a new target class for the development of inhibitors that modulate gene transcription or signaling pathways. SET and MYND domain containing protein 2 (SMYD2) is a catalytic SET domain containing methyltransferase reported to monomethylate lysine residues on histone and nonhistone proteins. Although several studies have uncovered an important role of SMYD2 in promoting cancer by protein methylation, the biology of SMYD2 is far from being fully understood. Utilization of highly potent and selective chemical probes for target validation has emerged as a concept which circumvents possible limitations of knockdown experiments and, in particular, could result in an improved exploration of drug targets with a complex underlying biology. Here, we report the development of a potent, selective, and cell-active, substrate-competitive inhibitor of SMYD2, which is the first reported inhibitor suitable for in vivo target validation studies in rodents.

Modular Assembly of Allosteric MEK Inhibitor Structural Elements Unravels Potency and Feedback-Modulation Handles.

Having recently identified a so-far unexplored area adjacent to the known binding site of allosteric mitogen-activated protein kinase kinase (MEK) inhibitors, we now report an extension of these studies by combining our new side chains with different MEK inhibitor cores in a modular manner. Replacement of the amide headgroup with inverse sulfonamides resulted in the identification of new MEK inhibitors with at least 10-fold higher cellular potency against K-Ras-mutated tumor cells. A selected inhibitor from this new series retained the favorable pharmacokinetic profile of its predecessor in rodent and non-rodent species and displayed significant in vivo efficacy at once-daily oral doses of 0.25-1 mg kg(-1) in a K-Ras-mutated xenograft model. The brain penetration potential of this analogue was significantly attenuated relative to PD325901. In a second series, the central fluorophenyl core was replaced by a pyridine moiety which gave rise to a similar boost in cellular potency. Most notably, analogues from this second series do not show MEK feedback phosphorylation in K-Ras-mutated A549 cells. Our results complement recent reports on the structural intricacies of MEK-Raf feedback interactions.