Mechanistic insights into a heterobifunctional degrader-induced PTPN2/N1 complex.

PTPN2 (protein tyrosine phosphatase non-receptor type 2, or TC-PTP) and PTPN1 are attractive immuno-oncology targets, with the deletion of Ptpn1 and Ptpn2 improving response to immunotherapy in disease models. Targeted protein degradation has emerged as a promising approach to drug challenging targets including phosphatases. We developed potent PTPN2/N1 dual heterobifunctional degraders (Cmpd-1 and Cmpd-2) which facilitate efficient complex assembly with E3 ubiquitin ligase CRL4CRBN, and mediate potent PTPN2/N1 degradation in cells and mice. To provide mechanistic insights into the cooperative complex formation introduced by degraders, we employed a combination of structural approaches. Our crystal structure reveals how PTPN2 is recognized by the tri-substituted thiophene moiety of the degrader. We further determined a high-resolution structure of DDB1-CRBN/Cmpd-1/PTPN2 using single-particle cryo-electron microscopy (cryo-EM). This structure reveals that the degrader induces proximity between CRBN and PTPN2, albeit the large conformational heterogeneity of this ternary complex. The molecular dynamic (MD)-simulations constructed based on the cryo-EM structure exhibited a large rigid body movement of PTPN2 and illustrated the dynamic interactions between PTPN2 and CRBN. Together, our study demonstrates the development of PTPN2/N1 heterobifunctional degraders with potential applications in cancer immunotherapy. Furthermore, the developed structural workflow could help to understand the dynamic nature of degrader-induced cooperative ternary complexes.

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.

The PTPN2/PTPN1 inhibitor ABBV-CLS-484 unleashes potent anti-tumour immunity.

Immune checkpoint blockade is effective for some patients with cancer, but most are refractory to current immunotherapies and new approaches are needed to overcome resistance1,2. The protein tyrosine phosphatases PTPN2 and PTPN1 are central regulators of inflammation, and their genetic deletion in either tumour cells or immune cells promotes anti-tumour immunity3-6. However, phosphatases are challenging drug targets; in particular, the active site has been considered undruggable. Here we present the discovery and characterization of ABBV-CLS-484 (AC484), a first-in-class, orally bioavailable, potent PTPN2 and PTPN1 active-site inhibitor. AC484 treatment in vitro amplifies the response to interferon and promotes the activation and function of several immune cell subsets. In mouse models of cancer resistant to PD-1 blockade, AC484 monotherapy generates potent anti-tumour immunity. We show that AC484 inflames the tumour microenvironment and promotes natural killer cell and CD8+ T cell function by enhancing JAK-STAT signalling and reducing T cell dysfunction. Inhibitors of PTPN2 and PTPN1 offer a promising new strategy for cancer immunotherapy and are currently being evaluated in patients with advanced solid tumours (ClinicalTrials.gov identifier NCT04777994 ). More broadly, our study shows that small-molecule inhibitors of key intracellular immune regulators can achieve efficacy comparable to or exceeding that of antibody-based immune checkpoint blockade in preclinical models. Finally, to our knowledge, AC484 represents the first active-site phosphatase inhibitor to enter clinical evaluation for cancer immunotherapy and may pave the way for additional therapeutics that target this important class of enzymes.

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.

CRISPR Screen Reveals BRD2/4 Molecular Glue-like Degrader via Recruitment of DCAF16.

Molecular glues (MGs) are monovalent small molecules that induce an interaction between proteins (native or non-native partners) by altering the protein-protein interaction (PPI) interface toward a higher-affinity state. Enhancing the PPI between a protein and E3 ubiquitin ligase can lead to degradation of the partnering protein. Over the past decade, retrospective studies of clinical drugs identified that immunomodulatory drugs (e.g., thalidomide and analogues) and indisulam exhibit a molecular glue effect by driving the interaction between non-native substrates to CRBN and DCAF15 ligases, respectively. Ensuing reports of phenotypic screens focused on MG discovery have suggested that these molecules may be more common than initially anticipated. However, prospective discovery of MGs remains challenging. Thus, expanding the repertoire of MGs will enhance our understanding of principles for prospective design. Herein, we report the results of a CRISPR/Cas9 knockout screen of over 1000 ligases and ubiquitin proteasome system components in a BRD4 degradation assay with a JQ1-based monovalent degrader, compound 1a. We identified DCAF16, a substrate recognition component of the Cul4 ligase complex, as essential for compound activity, and we demonstrate that compound 1a drives the interaction between DCAF16 and BRD2/4 to promote target degradation. Taken together, our data suggest that compound 1a functions as an MG degrader between BRD2/4 and DCAF16 and provides a foundation for further mechanistic dissection to advance prospective MG discovery.

Implications of the Conformationally Flexible, Macrocyclic Structure of the First-Generation, Direct-Acting Anti-Viral Paritaprevir on Its Solid Form Complexity and Chameleonic Behavior.

Direct-acting antiviral regimens have transformed therapeutic management of hepatitis C across all prevalent genotypes. Most of the chemical matter in these regimens comprises molecules well outside the traditional drug development chemical space and presents significant challenges. Herein, the implications of high conformational flexibility and the presence of a 15-membered macrocyclic ring in paritaprevir are studied through a combination of advanced computational and experimental methods with focus on molecular chameleonicity and crystal form complexity. The ability of the molecule to toggle between high and low 3D polar surface area (PSA) conformations is underpinned by intramolecular hydrogen bonding (IMHB) interactions and intramolecular steric effects. Computational studies consequently show a very significant difference of over 75 Å2 in 3D PSA between polar and apolar environments and provide the structural basis for the perplexingly favorable passive permeability of the molecule. Crystal packing and protein binding resulting in strong intermolecular interactions disrupt these intramolecular interactions. Crystalline Form I benefits from strong intermolecular interactions, whereas the weaker intermolecular interactions in Form II are partially compensated by the energetic advantage of an IMHB. Like Form I, no IMHB is observed within the receptor-bound conformation; instead, an intermolecular H-bond contributes to the potency of the molecule. The choice of metastable Form II is derisked through strategies accounting for crystal surface and packing features to manage higher form specific solid-state chemical reactivity and specific processing requirements. Overall, the results show an unambiguous link between structural features and derived properties from crystallization to dissolution, permeation, and docking into the protein pocket.

Novel, potent, selective and brain penetrant vasopressin 1b receptor antagonists.

Herein we report the discovery of a novel oxindole-based series of vasopressin 1b (V1b) receptor antagonists. Introducing a substituted piperazine moiety and optimizing the southern and the northern aromatic rings resulted in potent, selective and brain penetrant V1b receptor antagonists. Compound 9c was found to be efficacious in a rat model of anti-depressant activity (3 mg/kg, ip). Interestingly, both moderate terminal half-life and moderate bioavailability could be achieved despite sub-optimal microsomal stability.

Discovery and optimization of novel constrained pyrrolopyridone BET family inhibitors.

Novel conformationally constrained BET bromodomain inhibitors have been developed. These inhibitors were optimized in two similar, yet distinct chemical series, the 6-methyl-1H-pyrrolo[2,3-c]pyridin-7(6H)-ones (A) and the 1-methyl-1H-pyrrolo[2,3-c]pyridin-7(6H)-ones (B). Each series demonstrated excellent activity in binding and cellular assays, and lead compounds from each series demonstrated significant efficacy in in vivo tumor xenograft models.

Discovery of N-(4-(2,4-Difluorophenoxy)-3-(6-methyl-7-oxo-6,7-dihydro-1H-pyrrolo[2,3-c]pyridin-4-yl)phenyl)ethanesulfonamide (ABBV-075/Mivebresib), a Potent and Orally Available Bromodomain and Extraterminal Domain (BET) Family Bromodomain Inhibitor.

The development of bromodomain and extraterminal domain (BET) bromodomain inhibitors and their examination in clinical studies, particularly in oncology settings, has garnered substantial recent interest. An effort to generate novel BET bromodomain inhibitors with excellent potency and drug metabolism and pharmacokinetics (DMPK) properties was initiated based upon elaboration of a simple pyridone core. Efforts to develop a bidentate interaction with a critical asparagine residue resulted in the incorporation of a pyrrolopyridone core, which improved potency by 9-19-fold. Additional structure-activity relationship (SAR) efforts aimed both at increasing potency and improving pharmacokinetic properties led to the discovery of the clinical candidate 63 (ABBV-075/mivebresib), which demonstrates excellent potency in biochemical and cellular assays, advantageous exposures and half-life both in animal models and in humans, and in vivo efficacy in mouse models of cancer progression and inflammation.

Discovery of Novel and Highly Selective Inhibitors of Calpain for the Treatment of Alzheimer's Disease: 2-(3-Phenyl-1H-pyrazol-1-yl)-nicotinamides.

Calpain overactivation has been implicated in a variety of pathological disorders including ischemia/reperfusion injury, cataract formation, and neurodegenerative diseases such as Alzheimer's disease (AD). Herein we describe our efforts leading to the identification of ketoamide-based 2-(3-phenyl-1H-pyrazol-1-yl)nicotinamides as potent and reversible inhibitors of calpain with high selectivity versus related cysteine protease cathepsins, other proteases, and receptors. Broad efficacy in a set of preclinical models relevant to AD suggests that inhibition of calpain represents an attractive approach with potential benefit for the treatment of AD.

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.

Turning a Substrate Peptide into a Potent Inhibitor for the Histone Methyltransferase SETD8.

SETD8 is a histone H4-K20 methyltransferase that plays an essential role in the maintenance of genomic integrity during mitosis and in DNA damage repair, making it an intriguing target for cancer research. While some small molecule inhibitors for SETD8 have been reported, the structural binding modes for these inhibitors have not been revealed. Using the complex structure of the substrate peptide bound to SETD8 as a starting point, different natural and unnatural amino acid substitutions were tested, and a potent (Ki 50 nM, IC50 0.33 µM) and selective norleucine containing peptide inhibitor has been obtained.

Current status and future prospects for enabling chemistry technology in the drug discovery process.

This review covers recent advances in the implementation of enabling chemistry technologies into the drug discovery process. Areas covered include parallel synthesis chemistry, high-throughput experimentation, automated synthesis and purification methods, flow chemistry methodology including photochemistry, electrochemistry, and the handling of "dangerous" reagents. Also featured are advances in the "computer-assisted drug design" area and the expanding application of novel mass spectrometry-based techniques to a wide range of drug discovery activities.

Ligand association rates to the inner-variable-domain of a dual-variable-domain immunoglobulin are significantly impacted by linker design.

The DVD-Ig (TM) protein is a dual-specific immunoglobulin. Each of the two arms of the molecule contains two variable domains, an inner variable domain and an outer variable domain linked in tandem, each with binding specificity for different targets or epitopes. One area of on-going research involves determining how the proximity of the outer variable domain affects the binding of ligands to the inner variable domain. To explore this area, we prepared a series of DVD-Ig proteins with binding specificities toward TNFα and an alternate therapeutic target. Kinetic measurements of TNFα binding to this series of DVD-Ig proteins were used to probe the effects of variable domain position and linker design on ligand on- and off-rates. We found that affinities for TNFα are generally lower when binding to the inner domain than to the outer domain and that this loss of affinity is primarily due to reduced association rate. This effect could be mitigated, to some degree, by linker design. We show several linker sequences that mitigate inner domain affinity losses in this series of DVD-Ig proteins. Moreover, we show that single chain proteolytic cleavage between the inner and outer domains, or complete outer domain removal, can largely restore inner domain TNFα affinity to that approaching the reference antibody. Taken together, these results suggest that a loss of affinity for inner variable domains in this set of DVD-Ig proteins may be largely driven by simple steric hindrance effects and can be reduced by careful linker design.

Analgesic potential of TRPV1 antagonists.

The discovery of TRPV1 antagonists as a new class of analgesic agents for the treatment of chronic pathological pain has been pursued aggressively across the pharmaceutical industry. This effort has led to the identification of several TRPV1 antagonists that have entered clinical trials, including ABT-102 (Abbott), SB-705498 (GSK), AMG-517 (Amgen), MK2295 (Merck/Neurogen), and GRC-6211 (Lilly/Glenmark). Using the published structures for ABT-102, SB-705498, AMG-517, and lead compounds representing six additional TRPV1 antagonist chemotypes, a pharmacophore model that describes the common structural features found in potent TRPV1 antagonists was established. The TRPV1 antagonist pharmacophore fits within the pore region of a TRPV1 receptor homology model, with critical hydrogen bond interactions proposed between the TRPV1 antagonist pharmacophore and Tyr 667 on helix six. In spite of the putative common binding site for all TRPV1 antagonists included in this particular TRPV1 pharmacophore, these ligands have demonstrated that they can still offer distinct pharmacological profiles, likely due to differences in their pharmacokinetic profiles. This is highlighted by differences in temperature elevation observed when comparing the clinical candidates ABT-102 and AMG-517.

Preparation and characterization of N-(3-pyridinyl) spirocyclic diamines as ligands for nicotinic acetylcholine receptors.

Several N-pyridin-3-yl spirobicyclic diamines, designed as conformationally restricted analogs of tebanicline (ABT-594), were synthesized as novel ligands for nicotinic acetylcholine receptors (nAChR). The spirocyclic compounds exhibited weaker binding affinity, than other constrained analogs in accord with a pharmacophore model. Nevertheless, some (1a, 1b) possessed (partial) agonist potencies comparable to nicotine at the alpha4beta2 subtype, but with greatly improved selectivity relative to the alpha3beta4* nAChR.

Molecular determinants of species-specific activation or blockade of TRPA1 channels.

TRPA1 is an excitatory, nonselective cation channel implicated in somatosensory function, pain, and neurogenic inflammation. Through covalent modification of cysteine and lysine residues, TRPA1 can be activated by electrophilic compounds, including active ingredients of pungent natural products (e.g., allyl isothiocyanate), environmental irritants (e.g., acrolein), and endogenous ligands (4-hydroxynonenal). However, how covalent modification leads to channel opening is not understood. Here, we report that electrophilic, thioaminal-containing compounds [e.g., CMP1 (4-methyl-N-[2,2,2-trichloro-1-(4-nitro-phenylsulfanyl)-ethyl]-benzamide)] covalently modify cysteine residues but produce striking species-specific effects [i.e., activation of rat TRPA1 (rTRPA1) and blockade of human TRPA1 (hTRPA1) activation by reactive and nonreactive agonists]. Through characterizing rTRPA1 and hTRPA1 chimeric channels and point mutations, we identified several residues in the upper portion of the S6 transmembrane domains as critical determinants of the opposite channel gating: Ala-946 and Met-949 of rTRPA1 determine channel activation, whereas equivalent residues of hTRPA1 (Ser-943 and Ile-946) determine channel block. Furthermore, side-chain replacements at these critical residues profoundly affect channel function. Therefore, our findings reveal a molecular basis of species-specific channel gating and provide novel insights into how TRPA1 respond to stimuli.

1,4-Dihydroindeno[1,2-c]pyrazoles with acetylenic side chains as novel and potent multitargeted receptor tyrosine kinase inhibitors with low affinity for the hERG ion channel.

The synthesis of a novel series of 1,4-dihydroindeno[1,2-c]pyrazoles with acetylene-type side chains is described. Optimization of those compounds as KDR kinase inhibitors identified 8, which displayed an oral activity in an estradiol-induced murine uterine edema model (ED50 = 3 mg/kg) superior to Sutent (ED50 = 9 mg/kg) and showed potent antitumor efficacy in an MX-1 human breast carcinoma xenograft tumor growth model (tumor growth inhibition = 90% at 25 mg/kg.day po). The compound was docked into a homology model of the homo-tetrameric pore domain of the hERG potassium channel to identify strategies to improve its cardiac safety profile. Systematic interruption of key binding interactions between 8 and Phe656, Tyr652, and Ser624 yielded 90, which only showed an IC50 of 11.6 microM in the hERG patch clamp assay. The selectivity profile for 8 and 90 revealed that both compounds are multitargeted receptor tyrosine kinase inhibitors with low nanomolar potencies against the members of the VEGFR and PDGFR kinase subfamilies.

Discovery and pharmacological evaluation of growth hormone secretagogue receptor antagonists.

The discovery and pharmacological evaluation of potent, selective, and orally bioavailable growth hormone secretagogue receptor (GHS-R) antagonists are reported. Previously, 2,4-diaminopyrimidine-based GHS-R antagonists reported from our laboratories have been shown to be dihydrofolate reductase (DHFR) inhibitors. By comparing the X-ray crystal structure of DHFR docked with our GHS-R antagonists and GHS-R modeling, we designed and synthesized a series of potent and DHFR selective GHS-R antagonists with good pharmacokinetic (PK) profiles. An amide derivative 13d (Ca2+ flux IC50 = 188 nM, [brain]/[plasma] = 0.97 @ 8 h in rat) showed a 10% decrease in 24 h food intake in rats, and over 5% body weight reduction after 14-day oral treatment in diet-induced obese (DIO) mice. In comparison, a urea derivative 14c (Ca2+ flux IC50 = 7 nM, [brain]/[plasma] = 0.0 in DIO) failed to show significant effect on food intake in the acute feeding DIO model. These observations demonstrated for the first time that peripheral GHS-R blockage with small molecule GHS-R antagonists might not be sufficient for suppressing appetite and inducing body weight reduction.

Aminopyridine-based c-Jun N-terminal kinase inhibitors with cellular activity and minimal cross-kinase activity.

The c-Jun N-terminal kinases (JNK-1, -2, and -3) are members of the mitogen activated protein (MAP) kinase family of enzymes. They are activated in response to certain cytokines, as well as by cellular stresses including chemotoxins, peroxides, and irradiation. They have been implicated in the pathology of a variety of different diseases with an inflammatory component including asthma, stroke, Alzheimer's disease, and type 2 diabetes mellitus. In this work, high-throughput screening identified a JNK inhibitor with an excellent kinase selectivity profile. Using X-ray crystallography and biochemical screening to guide our lead optimization, we prepared compounds with inhibitory potencies in the low-double-digit nanomolar range, activity in whole cells, and pharmacokinetics suitable for in vivo use. The new compounds were over 1,000-fold selective for JNK-1 and -2 over other MAP kinases including ERK2, p38alpha, and p38delta and showed little inhibitory activity against a panel of 74 kinases.