Discovery of Small Molecule Interleukin 17A Inhibitors with Novel Binding Mode and Stoichiometry: Optimization of DNA-Encoded Chemical Library Hits to In Vivo Active Compounds.

Dysregulation of IL17A drives numerous inflammatory and autoimmune disorders with inhibition of IL17A using antibodies proven as an effective treatment. Oral anti-IL17 therapies are an attractive alternative option, and several preclinical small molecule IL17 inhibitors have previously been described. Herein, we report the discovery of a novel class of small molecule IL17A inhibitors, identified via a DNA-encoded chemical library screen, and their subsequent optimization to provide in vivo efficacious inhibitors. These new protein-protein interaction (PPI) inhibitors bind in a previously undescribed mode in the IL17A protein with two copies binding symmetrically to the central cavities of the IL17A homodimer.

Inhibition of MALT1 and BCL2 Induces Synergistic Antitumor Activity in Models of B-Cell Lymphoma.

The activated B cell (ABC) subset of diffuse large B-cell lymphoma (DLBCL) is characterized by chronic B-cell receptor signaling and associated with poor outcomes when treated with standard therapy. In ABC-DLBCL, MALT1 is a core enzyme that is constitutively activated by stimulation of the B-cell receptor or gain-of-function mutations in upstream components of the signaling pathway, making it an attractive therapeutic target. We discovered a novel small-molecule inhibitor, ABBV-MALT1, that potently shuts down B-cell signaling selectively in ABC-DLBCL preclinical models leading to potent cell growth and xenograft inhibition. We also identified a rational combination partner for ABBV-MALT1 in the BCL2 inhibitor, venetoclax, which when combined significantly synergizes to elicit deep and durable responses in preclinical models. This work highlights the potential of ABBV-MALT1 monotherapy and combination with venetoclax as effective treatment options for patients with ABC-DLBCL.

New Platform for Label-Free, Proximal Cellular Pharmacodynamic Assays: Identification of Glutaminase Inhibitors Using Infrared Matrix-Assisted Laser Desorption Electrospray Ionization Mass Spectrometry.

Cellular pharmacodynamic assays are crucial aspects of lead optimization programs in drug discovery. These assays are sometimes difficult to develop, oftentimes distal from the target and frequently low throughput, which necessitates their incorporation in the drug discovery funnel later than desired. The earlier direct pharmacodynamic modulation of a target can be established, the fewer resources are wasted on compounds that are acting via an off-target mechanism. Mass spectrometry is a versatile tool that is often used for direct, proximal cellular pharmacodynamic assay analysis, but liquid chromatography-mass spectrometry methods are low throughput and are unable to fully support structure-activity relationship efforts in early medicinal chemistry programs. Infrared matrix-assisted laser desorption electrospray ionization (IR-MALDESI) is an ambient ionization method amenable to high-throughput cellular assays, capable of diverse analyte detection, ambient and rapid laser sampling processes, and low cross-contamination. Here, we demonstrate the capability of IR-MALDESI for the detection of diverse analytes directly from cells and report the development of a high-throughput, label-free, proximal cellular pharmacodynamic assay using IR-MALDESI for the discovery of glutaminase inhibitors and a biochemical assay for hit confirmation. We demonstrate the throughput with a ∼100,000-compound cellular screen. Hits from the screening were confirmed by retesting in dose-response with mass spectrometry-based cellular and biochemical assays. A similar workflow can be applied to other targets with minimal modifications, which will speed up the discovery of cell active lead series and minimize wasted chemistry resources on off-target mechanisms.

Discovery of a Potent Chloroacetamide GPX4 Inhibitor with Bioavailability to Enable Target Engagement in Mice, a Potential Tool Compound for Inducing Ferroptosis In Vivo.

Compounds that inhibit glutathione peroxidase 4 (GPX4) hold promise as cancer therapeutics in their ability to induce a form of nonapoptotic cell death called ferroptosis. Our research identified 24, a structural analog of the potent GPX4 inhibitor RSL3, that has much better plasma stability (t1/2 > 5 h in mouse plasma). The bioavailability of 24 provided efficacious plasma drug concentrations with IP dosing, thus enabling in vivo studies to assess tolerability and efficacy. An efficacy study in mouse using a GPX4-sensitive tumor model found that doses of 24 up to 50 mg/kg were tolerated for 20 days but had no effect on tumor growth, although partial target engagement was observed in tumor homogenate.

Identification and structure-based drug design of cell-active inhibitors of interleukin 17A at a novel C-terminal site.

Anti-IL17A therapies have proven effective for numerous inflammatory diseases including psoriasis, axial spondylitis and psoriatic arthritis. Modulating and/or antagonizing protein-protein interactions of IL17A cytokine binding to its cell surface receptors with oral therapies offers the promise to bring forward biologics-like efficacy in a pill to patients. We used an NMR-based fragment screen of recombinant IL17A to uncover starting points for small molecule IL17A antagonist discovery. By examining chemical shift perturbations in 2D [1H, 13C-HSQC] spectra of isotopically labeled IL17A, we discovered fragments binding the cytokine at a previously undescribed site near the IL17A C-terminal region, albeit with weak affinity (> 250 µM). Importantly this binding location was distinct from previously known chemical matter modulating cytokine responses. Subsequently through analog screening, we identified related compounds that bound symmetrically in this novel site with two copies. From this observation we employed a linking strategy via structure-based drug design and obtained compounds with increased binding affinity (< 50 nM) and showed functional inhibition of IL17A-induced cellular signaling (IC50~1 µM). We also describe a fluorescence-based probe molecule suitable to discern/screen for additional molecules binding in this C-terminal site.

The HPK1 Inhibitor A-745 Verifies the Potential of Modulating T Cell Kinase Signaling for Immunotherapy.

Hematopoietic progenitor kinase 1 (HPK1) is an MAP4K family member within the Ste20-like serine/threonine branch of the kinome. HPK1 expression is limited to hematopoietic cells and has a predominant role as a negative regulator of T cell function. Because of the central/dominant role in negatively regulating T cell function, HPK1 has long been in the center of interest as a potential pharmacological target for immune therapy. The development of a small molecule HPK1 inhibitor remains challenging because of the need for high specificity relative to other kinases, including additional MAP4K family members, that are required for efficient immune cell activation. Here, we report the identification of the selective and potent HPK1 chemical probe, A-745. In unbiased cellular kinase-binding assays, A-745 demonstrates an excellent cellular selectivity binding profile within pharmacologically relevant concentrations. This HPK1 selectivity translates to an in vitro immune cell activation phenotype reminiscent of Hpk1-deficient and Hpk1-kinase-dead T cells, including augmented proliferation and cytokine production. The results from this work give a path forward for further developmental efforts to generate additional selective and potent small molecule HPK1 inhibitors with the pharmacological properties for immunotherapy.

Development of Orally Efficacious Allosteric Inhibitors of TNFα via Fragment-Based Drug Design.

Tumor necrosis factor α (TNFα) is a soluble cytokine that is directly involved in systemic inflammation through the regulation of the intracellular NF-κB and MAPK signaling pathways. The development of biologic drugs that inhibit TNFα has led to improved clinical outcomes for patients with rheumatoid arthritis and other chronic autoimmune diseases; however, TNFα has proven to be difficult to drug with small molecules. Herein, we present a two-phase, fragment-based drug discovery (FBDD) effort in which we first identified isoquinoline fragments that disrupt TNFα ligand-receptor binding through an allosteric desymmetrization mechanism as observed in high-resolution crystal structures. The second phase of discovery focused on the de novo design and optimization of fragments with improved binding efficiency and drug-like properties. The 3-indolinone-based lead presented here displays oral, in vivo efficacy in a mouse glucose-6-phosphate isomerase (GPI)-induced paw swelling model comparable to that seen with a TNFα antibody.

Selective inhibition of the BD2 bromodomain of BET proteins in prostate cancer.

Proteins of the bromodomain and extra-terminal (BET) domain family are epigenetic readers that bind acetylated histones through their bromodomains to regulate gene transcription. Dual-bromodomain BET inhibitors (DbBi) that bind with similar affinities to the first (BD1) and second (BD2) bromodomains of BRD2, BRD3, BRD4 and BRDt have displayed modest clinical activity in monotherapy cancer trials. A reduced number of thrombocytes in the blood (thrombocytopenia) as well as symptoms of gastrointestinal toxicity are dose-limiting adverse events for some types of DbBi1-5. Given that similar haematological and gastrointestinal defects were observed after genetic silencing of Brd4 in mice6, the platelet and gastrointestinal toxicities may represent on-target activities associated with BET inhibition. The two individual bromodomains in BET family proteins may have distinct functions7-9 and different cellular phenotypes after pharmacological inhibition of one or both bromodomains have been reported10,11, suggesting that selectively targeting one of the bromodomains may result in a different efficacy and tolerability profile compared with DbBi. Available compounds that are selective to individual domains lack sufficient potency and the pharmacokinetics properties that are required for in vivo efficacy and tolerability assessment10-13. Here we carried out a medicinal chemistry campaign that led to the discovery of ABBV-744, a highly potent and selective inhibitor of the BD2 domain of BET family proteins with drug-like properties. In contrast to the broad range of cell growth inhibition induced by DbBi, the antiproliferative activity of ABBV-744 was largely, but not exclusively, restricted to cell lines of acute myeloid leukaemia and prostate cancer that expressed the full-length androgen receptor (AR). ABBV-744 retained robust activity in prostate cancer xenografts, and showed fewer platelet and gastrointestinal toxicities than the DbBi ABBV-07514. Analyses of RNA expression and chromatin immunoprecipitation followed by sequencing revealed that ABBV-744 displaced BRD4 from AR-containing super-enhancers and inhibited AR-dependent transcription, with less impact on global transcription compared with ABBV-075. These results underscore the potential value of selectively targeting the BD2 domain of BET family proteins for cancer therapy.

Fragment-Based Discovery of an Apolipoprotein E4 (apoE4) Stabilizer.

Apolipoprotein E is a 299-residue lipid carrier protein produced in both the liver and the brain. The protein has three major isoforms denoted apoE2, apoE3, and apoE4 which differ at positions 112 and 158 and which occur at different frequencies in the human population. Genome-wide association studies indicate that the possession of two apoE4 alleles is a strong genetic risk factor for late-onset Alzheimer's disease (LOAD). In an attempt to identify a small molecule stabilizer of apoE4 function that may have utility as a therapy for Alzheimer's disease, we carried out an NMR-based fragment screen on the N-terminal domain of apoE4 and identified a benzyl amidine based fragment binder. In addition to NMR, binding was characterized using various other biophysical techniques, and a crystal structure of the bound core was obtained. Core elaboration ultimately yielded a compound that showed activity in an IL-6 and IL-8 cytokine release assay.

PARP1 Trapping by PARP Inhibitors Drives Cytotoxicity in Both Cancer Cells and Healthy Bone Marrow.

PARP inhibitors have recently been approved as monotherapies for the treatment of recurrent ovarian cancer and metastatic BRCA-associated breast cancer, and ongoing studies are exploring additional indications and combinations with other agents. PARP inhibitors trap PARP onto damaged chromatin when combined with temozolomide and methyl methanesulfonate, but the clinical relevance of these findings remains unknown. PARP trapping has thus far been undetectable in cancer cells treated with PARP inhibitors alone. Here, we evaluate the contribution of PARP trapping to the tolerability and efficacy of PARP inhibitors in the monotherapy setting. We developed a novel implementation of the proximity ligation assay to detect chromatin-trapped PARP1 at single-cell resolution with higher sensitivity and throughput than previously reported methods. We further demonstrate that the PARP inhibitor-induced trapping appears to drive single-agent cytotoxicity in healthy human bone marrow, indicating that the toxicity of trapped PARP complexes is not restricted to cancer cells with homologous recombination deficiency. Finally, we show that PARP inhibitors with dramatically different trapping potencies exhibit comparable tumor growth inhibition at MTDs in a xenograft model of BRCA1-mutant triple-negative breast cancer. These results are consistent with emerging clinical data and suggest that the inverse relationship between trapping potency and tolerability may limit the potential therapeutic advantage of potent trapping activity. IMPLICATIONS: PARP trapping contributes to single-agent cytotoxicity of PARP inhibitors in both cancer cells and healthy bone marrow, and the therapeutic advantage of potent trapping activity appears to be limited.

ABT-165, a Dual Variable Domain Immunoglobulin (DVD-Ig) Targeting DLL4 and VEGF, Demonstrates Superior Efficacy and Favorable Safety Profiles in Preclinical Models.

Antiangiogenic therapy is a clinically validated modality in cancer treatment. To date, all approved antiangiogenic drugs primarily inhibit the VEGF pathway. Delta-like ligand 4 (DLL4) has been identified as a potential drug target in VEGF-independent angiogenesis and tumor-initiating cell (TIC) survival. A dual-specific biologic targeting both VEGF and DLL4 could be an attractive strategy to improve the effectiveness of anti-VEGF therapy. ABT-165 was uniquely engineered using a proprietary dual-variable domain immunoglobulin (DVD-Ig) technology based on its ability to bind and inhibit both DLL4 and VEGF. In vivo, ABT-165 induced significant tumor growth inhibition compared with either parental antibody treatment alone, due, in part, to the disruption of functional tumor vasculature. In combination with chemotherapy agents, ABT-165 also induced greater antitumor response and outperformed anti-VEGF treatment. ABT-165 displayed nonlinear pharmacokinetic profiles in cynomolgus monkeys, with an apparent terminal half-life > 5 days at a target saturation dose. In a GLP monkey toxicity study, ABT-165 was well-tolerated at doses up to 200 mg/kg with non-adverse treatment-related histopathology findings limited to the liver and thymus. In summary, ABT-165 represents a novel antiangiogenic strategy that potently inhibits both DLL4 and VEGF, demonstrating favorable in vivo efficacy, pharmacokinetic, and safety profiles in preclinical models. Given these preclinical attributes, ABT-165 has progressed to a phase I study. Mol Cancer Ther; 17(5); 1039-50. ©2018 AACR.

Characterization of ABBV-221, a Tumor-Selective EGFR-Targeting Antibody Drug Conjugate.

Depatuxizumab mafodotin (depatux-m, ABT-414) is a tumor-selective antibody drug conjugate (ADC) comprised of the anti-EGFR antibody ABT-806 and the monomethyl auristatin F (MMAF) warhead. Depatux-m has demonstrated promising clinical activity in glioblastoma multiforme (GBM) patients and is currently being evaluated in clinical trials in first-line and recurrent GBM disease settings. Depatux-m responses have been restricted to patients with amplified EGFR, highlighting the need for therapies with activity against tumors with nonamplified EGFR overexpression. In addition, depatux-m dosing has been limited by corneal side effects common to MMAF conjugates. We hypothesized that a monomethyl auristatin E (MMAE) ADC utilizing an EGFR-targeting antibody with increased affinity may have broader utility against tumors with more modest EGFR overexpression while mitigating the risk of corneal side effects. We describe here preclinical characterization of ABBV-221, an EGFR-targeting ADC comprised of an affinity-matured ABT-806 conjugated to MMAE. ABBV-221 binds to a similar EGFR epitope as depatux-m and retains tumor selectivity with increased binding to EGFR-positive tumor cells and greater in vitro potency. ABBV-221 displays increased tumor uptake and antitumor activity against wild-type EGFR-positive xenografts with a greatly reduced incidence of corneal side effects relative to depatux-m. ABBV-221 has similar activity as depatux-m against an EGFR-amplified GBM patient derived xenograft (PDX) model and is highly effective alone and in combination with standard-of-care temozolomide in an EGFRvIII-positive GBM xenograft model. Based on these results, ABBV-221 has advanced to a phase I clinical trial in patients with advanced solid tumors associated with elevated levels of EGFR. Mol Cancer Ther; 17(4); 795-805. ©2018 AACR.

Fragment-based discovery of a potent NAMPT inhibitor.

NAMPT expression is elevated in many cancers, making this protein a potential target for anticancer therapy. We have carried out both NMR based and TR-FRET based fragment screens against human NAMPT and identified six novel binders with a range of potencies. Co-crystal structures were obtained for two of the fragments bound to NAMPT while for the other four fragments force-field driven docking was employed to generate a bound pose. Based on structural insights arising from comparison of the bound fragment poses to that of bound FK866 we were able to synthetically elaborate one of the fragments into a potent NAMPT inhibitor.

Are We There Yet? Applying Thermodynamic and Kinetic Profiling on Embryonic Ectoderm Development (EED) Hit-to-Lead Program.

It is advocated that kinetic and thermodynamic profiling of bioactive compounds should be incorporated and utilized as complementary tools for hit and lead optimizations in drug discovery. To assess their applications in the EED hit-to-lead optimization process, large amount of thermodynamic and kinetic data were collected and analyzed via isothermal titration calorimetry (ITC) and surface plasmon resonance (SPR), respectively. Slower dissociation rates (koff) of the lead compounds were observed as the program progressed. Analysis of the kinetic data indicated that compound cellular activity correlated with both Ki and koff. Our analysis revealed that ITC data should be interpreted in the context of chiral purity of the compounds. The thermodynamic signatures of the EED aminopyrrolidine compounds were found to be mainly enthalpy driven with improved enthalpic contributions as the program progressed. Our study also demonstrated that significant challenges still exist in utilizing kinetic and thermodynamic parameters for hit selection.

Fragment-Based, Structure-Enabled Discovery of Novel Pyridones and Pyridone Macrocycles as Potent Bromodomain and Extra-Terminal Domain (BET) Family Bromodomain Inhibitors.

Members of the BET family of bromodomain containing proteins have been identified as potential targets for blocking proliferation in a variety of cancer cell lines. A two-dimensional NMR fragment screen for binders to the bromodomains of BRD4 identified a phenylpyridazinone fragment with a weak binding affinity (1, Ki = 160 µM). SAR investigation of fragment 1, aided by X-ray structure-based design, enabled the synthesis of potent pyridone and macrocyclic pyridone inhibitors exhibiting single digit nanomolar potency in both biochemical and cell based assays. Advanced analogs in these series exhibited high oral exposures in rodent PK studies and demonstrated significant tumor growth inhibition efficacy in mouse flank xenograft models.

Methylpyrrole inhibitors of BET bromodomains.

An NMR fragment screen for binders to the bromodomains of BRD4 identified 2-methyl-3-ketopyrroles 1 and 2. Elaboration of these fragments guided by structure-based design provided lead molecules with significant activity in a mouse tumor model. Further modifications to the methylpyrrole core provided compounds with improved properties and enhanced activity in a mouse model of multiple myeloma.

The EED protein-protein interaction inhibitor A-395 inactivates the PRC2 complex.

Polycomb repressive complex 2 (PRC2) is a regulator of epigenetic states required for development and homeostasis. PRC2 trimethylates histone H3 at lysine 27 (H3K27me3), which leads to gene silencing, and is dysregulated in many cancers. The embryonic ectoderm development (EED) protein is an essential subunit of PRC2 that has both a scaffolding function and an H3K27me3-binding function. Here we report the identification of A-395, a potent antagonist of the H3K27me3 binding functions of EED. Structural studies demonstrate that A-395 binds to EED in the H3K27me3-binding pocket, thereby preventing allosteric activation of the catalytic activity of PRC2. Phenotypic effects observed in vitro and in vivo are similar to those of known PRC2 enzymatic inhibitors; however, A-395 retains potent activity against cell lines resistant to the catalytic inhibitors. A-395 represents a first-in-class antagonist of PRC2 protein-protein interactions (PPI) for use as a chemical probe to investigate the roles of EED-containing protein complexes.

Mechanistic Dissection of PARP1 Trapping and the Impact on In Vivo Tolerability and Efficacy of PARP Inhibitors.

UNLABELLED: Poly(ADP-ribose) polymerases (PARP1, -2, and -3) play important roles in DNA damage repair. As such, a number of PARP inhibitors are undergoing clinical development as anticancer therapies, particularly in tumors with DNA repair deficits and in combination with DNA-damaging agents. Preclinical evidence indicates that PARP inhibitors potentiate the cytotoxicity of DNA alkylating agents. It has been proposed that a major mechanism underlying this activity is the allosteric trapping of PARP1 at DNA single-strand breaks during base excision repair; however, direct evidence of allostery has not been reported. Here the data reveal that veliparib, olaparib, niraparib, and talazoparib (BMN-673) potentiate the cytotoxicity of alkylating agents. Consistent with this, all four drugs possess PARP1 trapping activity. Using biochemical and cellular approaches, we directly probe the trapping mechanism for an allosteric component. These studies indicate that trapping is due to catalytic inhibition and not allostery. The potency of PARP inhibitors with respect to trapping and catalytic inhibition is linearly correlated in biochemical systems but is nonlinear in cells. High-content imaging of γH2Ax levels suggests that this is attributable to differential potentiation of DNA damage in cells. Trapping potency is inversely correlated with tolerability when PARP inhibitors are combined with temozolomide in mouse xenograft studies. As a result, PARP inhibitors with dramatically different trapping potencies elicit comparable in vivo efficacy at maximum tolerated doses. Finally, the impact of trapping on tolerability and efficacy is likely to be context specific. IMPLICATIONS: Understanding the context-specific relationships of trapping and catalytic inhibition with both tolerability and efficacy will aid in determining the suitability of a PARP inhibitor for inclusion in a particular clinical regimen.

Cracking the molecular weight barrier: fragment screening of an aminotransferase using an NMR-based functional assay.

NMR-based screening of protein targets has become a well established part of the drug discovery process especially with respect to fragments. However, as target size increases the two-dimensional spectra typically used for such screening become more crowded due to the increased number of signals, and the individual signals broaden due to the decreased rotational correlation time of the protein. Here we present an NMR-based functional assay for the branched-chain aminotransferase BCATc, a dimer with a total molecular weight of 88 kDa, which overcomes the limitations of the typical protein-based NMR screening method. BCATc is involved in glutamate production in the brain and is a therapeutic target for neuronal disorders involving a glutamatergic mechanism. Several fragments which inhibit BCATc were discovered using this assay and these may serve as novel cores for the development of potent BCATc inhibitors.