SAR towards indoline and 3-azaindoline classes of IDO1 inhibitors.

A novel series of IDO1 inhibitors have been identified with good IDO1 Hela cell and human whole blood activity. These inhibitors contain an indoline or a 3-azaindoline scaffold. Their structure-activity-relationship studies have been explored. Compounds 37 and 41 stood out as leads due to their good potency in IDO1 Hela assay, good IDO1 unbound hWB IC50s, reasonable unbound clearance, and good MRT in rat and dog PK studies.

A reevaluation of the spleen tyrosine kinase (SYK) activation mechanism.

Spleen tyrosine kinase (SYK) is a signaling node in many immune pathways and comprises two tandem Src homology (SH) 2 domains, an SH2-kinase linker, and a C-terminal tyrosine kinase domain. Two prevalent models of SYK activation exist. The "OR-gate" model contends that SYK can be fully activated by phosphorylation or binding of its SH2 domains to a dual-phosphorylated immune-receptor tyrosine-based activation motif (ppITAM). An alternative model proposes that SYK activation requires ppITAM binding and phosphorylation of the SH2-kinase linker by a SRC family kinase such as LYN proto-oncogene, SRC family tyrosine kinase (LYN). To evaluate these two models, we generated directly comparable unphosphorylated (upSYK) and phosphorylated (pSYK) proteins with or without an N-terminal glutathione S-transferase (GST) tag, resulting in monomeric or obligatory dimeric SYK, respectively. We assessed the ability of a ppITAM peptide and LYN to activate these SYK proteins. The ppITAM peptide strongly activated GST-SYK but was less effective in activating upSYK untagged with GST. LYN alone activated untagged upSYK to a greater extent than did ppITAM, and inclusion of both proteins rapidly and fully activated upSYK. Using immunoblot and phosphoproteomic approaches, we correlated the kinetics and order of site-specific SYK phosphorylation. Our results are consistent with the alternative model, indicating that ppITAM binding primes SYK for rapid LYN-mediated phosphorylation of Tyr-352 and then Tyr-348 of the SH2-kinase linker, which facilitates activation loop phosphorylation and full SYK activation. This gradual activation mechanism may also explain how SYK maintains ligand-independent tonic signaling, important for B-cell development and survival.

Characterization of indoleamine-2,3-dioxygenase 1, tryptophan-2,3-dioxygenase, and Ido1/Tdo2 knockout mice.

Indoleamine-2,3-dioxygenase 1 (IDO1) and tryptophan-2,3-dioxygenase 2 (TDO2) degrade tryptophan (Trp) to kynurenine (Kyn), and these enzymes have promise as therapeutic targets. A comprehensive characterization of potential safety liabilities of IDO1 and TDO2 inhibitors using knockout (KO) mice has not been assessed, nor has the dual Ido1/Tdo2 KO been reported. Here we characterized male and female mice with KOs for Ido1, Tdo2, and Ido1/Tdo2 and compared findings to the wild type (WT) mouse strain, evaluated for 14 days, using metabolomics, transcriptional profiling, behavioral analysis, spleen immunophenotyping, comprehensive histopathological analysis, and serum clinical chemistry. Multiple metabolomic changes were seen in KO mice. For catabolism of Trp to Kyn and anthranilic acid, both substrates were decreased in liver of Tdo2 and dual KO mice. Metabolism of Trp to serotonin and its metabolites resulted in an increase in 5-Hydroxyindole-3-acetic acid in the Tdo2 and dual KO mice. Ido1 and dual KO mice displayed a Kyn reduction in plasma but not in liver. Nicotinamide synthesis and conversion of glucose to lactic acid were not impacted. A slight decrease in serum alkaline phosphatase was seen in all KOs, and small changes in liver gene expression of genes unrelated to tryptophan metabolism were observed. Regarding other parameters, no genotype-specific changes were observed. In summary, this work shows metabolomic pathway changes for metabolites downstream of tryptophan in these KO mice, and suggests that inhibition of the IDO1 and TDO2 enzymes would be well tolerated whether inhibited individually or in combination since no safety liabilities were uncovered.

Structural basis for effectiveness of siderophore-conjugated monocarbams against clinically relevant strains of Pseudomonas aeruginosa.

Pseudomonas aeruginosa is an opportunistic Gram-negative pathogen that causes nosocomial infections for which there are limited treatment options. Penicillin-binding protein PBP3, a key therapeutic target, is an essential enzyme responsible for the final steps of peptidoglycan synthesis and is covalently inactivated by ß-lactam antibiotics. Here we disclose the first high resolution cocrystal structures of the P. aeruginosa PBP3 with both novel and marketed ß-lactams. These structures reveal a conformational rearrangement of Tyr532 and Phe533 and a ligand-induced conformational change of Tyr409 and Arg489. The well-known affinity of the monobactam aztreonam for P. aeruginosa PBP3 is due to a distinct hydrophobic aromatic wall composed of Tyr503, Tyr532, and Phe533 interacting with the gem-dimethyl group. The structure of MC-1, a new siderophore-conjugated monocarbam complexed with PBP3 provides molecular insights for lead optimization. Importantly, we have identified a novel conformation that is distinct to the high-molecular-weight class B PBP subfamily, which is identifiable by common features such as a hydrophobic aromatic wall formed by Tyr503, Tyr532, and Phe533 and the structural flexibility of Tyr409 flanked by two glycine residues. This is also the first example of a siderophore-conjugated triazolone-linked monocarbam complexed with any PBP. Energetic analysis of tightly and loosely held computed hydration sites indicates protein desolvation effects contribute significantly to PBP3 binding, and analysis of hydration site energies allows rank ordering of the second-order acylation rate constants. Taken together, these structural, biochemical, and computational studies provide a molecular basis for recognition of P. aeruginosa PBP3 and open avenues for future design of inhibitors of this class of PBPs.

Evaluation of pharmacokinetic/pharmacodynamic relationships of PD-0162819, a biotin carboxylase inhibitor representing a new class of antibacterial compounds, using in vitro infection models.

The present study investigated the pharmacokinetic/pharmacodynamic (PK/PD) relationships of a prototype biotin carboxylase (BC) inhibitor, PD-0162819, against Haemophilus influenzae 3113 in static concentration time-kill (SCTK) and one-compartment chemostat in vitro infection models. H. influenzae 3113 was exposed to PD-0162819 concentrations of 0.5 to 16× the MIC (MIC = 0.125 µg/ml) and area-under-the-curve (AUC)/MIC ratios of 1 to 1,100 in SCTK and chemostat experiments, respectively. Serial samples were collected over 24 h. For efficacy driver analysis, a sigmoid maximum-effect (E(max)) model was fitted to the relationship between bacterial density changes over 24 h and corresponding PK/PD indices. A semimechanistic PK/PD model describing the time course of bacterial growth and death was developed. The AUC/MIC ratio best explained efficacy (r(2) = 0.95) compared to the peak drug concentration (C(max))/MIC ratio (r(2) = 0.76) and time above the MIC (T>MIC) (r(2) = 0.88). Static effects and 99.9% killing were achieved at AUC/MIC values of 500 and 600, respectively. For time course analysis, the net bacterial growth rate constant, maximum bacterial density, and maximum kill rate constant were similar in SCTK and chemostat studies, but PD-0162819 was more potent in SCTK than in the chemostat (50% effective concentration [EC(50)] = 0.046 versus 0.34 µg/ml). In conclusion, basic PK/PD relationships for PD-0162819 were established using in vitro dynamic systems. Although the bacterial growth parameters and maximum drug effects were similar in SCTK and the chemostat system, PD-0162819 appeared to be more potent in SCTK, illustrating the importance of understanding the differences in preclinical models. Additional studies are needed to determine the in vivo relevance of these results.

A class of selective antibacterials derived from a protein kinase inhibitor pharmacophore.

As the need for novel antibiotic classes to combat bacterial drug resistance increases, the paucity of leads resulting from target-based antibacterial screening of pharmaceutical compound libraries is of major concern. One explanation for this lack of success is that antibacterial screening efforts have not leveraged the eukaryotic bias resulting from more extensive chemistry efforts targeting eukaryotic gene families such as G protein-coupled receptors and protein kinases. Consistent with a focus on antibacterial target space resembling these eukaryotic targets, we used whole-cell screening to identify a series of antibacterial pyridopyrimidines derived from a protein kinase inhibitor pharmacophore. In bacteria, the pyridopyrimidines target the ATP-binding site of biotin carboxylase (BC), which catalyzes the first enzymatic step of fatty acid biosynthesis. These inhibitors are effective in vitro and in vivo against fastidious gram-negative pathogens including Haemophilus influenzae. Although the BC active site has architectural similarity to those of eukaryotic protein kinases, inhibitor binding to the BC ATP-binding site is distinct from the protein kinase-binding mode, such that the inhibitors are selective for bacterial BC. In summary, we have discovered a promising class of potent antibacterials with a previously undescribed mechanism of action. In consideration of the eukaryotic bias of pharmaceutical libraries, our findings also suggest that pursuit of a novel inhibitor leads for antibacterial targets with active-site structural similarity to known human targets will likely be more fruitful than the traditional focus on unique bacterial target space, particularly when structure-based and computational methodologies are applied to ensure bacterial selectivity.

Oxetane Promise Delivered: Discovery of Long-Acting IDO1 Inhibitors Suitable for Q3W Oral or Parenteral Dosing.

3,3-Disubstituted oxetanes have been utilized as bioisosteres for gem-dimethyl and cyclobutane functionalities. We report the discovery of a novel class of oxetane indole-amine 2,3-dioxygenase (IDO1) inhibitors suitable for Q3W (once every 3 weeks) oral and parenteral dosing. A diamide class of IDO inhibitors was discovered through an automated ligand identification system (ALIS). Installation of an oxetane and fluorophenyl dramatically improved the potency. Identification of a biaryl moiety as an unconventional amide isostere addressed the metabolic liability of amide hydrolysis. Metabolism identification (Met-ID)-guided target design and the introduction of polarity resulted in the discovery of potent IDO inhibitors with excellent pharmacokinetic (PK) profiles in multiple species. To enable rapid synthesis of the key oxetane intermediate, a novel oxetane ring cyclization was also developed, as well as optimization of a literature route on kg scale. These IDO inhibitors may enable unambiguous proof-of-concept testing for the IDO1 inhibition mechanism for oncology.

Identification of Potent Reverse Indazole Inhibitors for HPK1.

Hematopoietic progenitor kinase (HPK1), a negative regulator of TCR-mediated T-cell activation, has been recognized as a novel antitumor immunotherapy target. Structural optimization of kinase inhibitor 4 through a systematic two-dimensional diversity screen of pyrazolopyridines led to the identification of potent and selective compounds. Crystallographic studies with HPK1 revealed a favorable water-mediated interaction with Asp155 and a salt bridge to Asp101 with optimized heterocyclic solvent fronts that were critical for enhanced potency and selectivity. Computational studies of model systems revealed differences in torsional profiles that allowed for these beneficial protein-ligand interactions. Further optimization of molecular properties led to identification of potent and selective reverse indazole inhibitor 36 that inhibited phosphorylation of adaptor protein SLP76 in human PBMC and exhibited low clearance with notable bioavailability in in vivo rat studies.

Cryo-EM structures of inhibitory antibodies complexed with arginase 1 provide insight into mechanism of action.

Human Arginase 1 (hArg1) is a metalloenzyme that catalyzes the hydrolysis of L-arginine to L-ornithine and urea, and modulates T-cell-mediated immune response. Arginase-targeted therapies have been pursued across several disease areas including immunology, oncology, nervous system dysfunction, and cardiovascular dysfunction and diseases. Currently, all published hArg1 inhibitors are small molecules usually less than 350 Da in size. Here we report the cryo-electron microscopy structures of potent and inhibitory anti-hArg antibodies bound to hArg1 which form distinct macromolecular complexes that are greater than 650 kDa. With local resolutions of 3.5 Å or better we unambiguously mapped epitopes and paratopes for all five antibodies and determined that the antibodies act through orthosteric and allosteric mechanisms. These hArg1:antibody complexes present an alternative mechanism to inhibit hArg1 activity and highlight the ability to utilize antibodies as probes in the discovery and development of peptide and small molecule inhibitors for enzymes in general.

Discovery of Diaminopyrimidine Carboxamide HPK1 Inhibitors as Preclinical Immunotherapy Tool Compounds.

Hematopoietic progenitor kinase 1 (HPK1), a serine/threonine kinase, is a negative immune regulator of T cell receptor (TCR) and B cell signaling that is primarily expressed in hematopoietic cells. Accordingly, it has been reported that HPK1 loss-of-function in HPK1 kinase-dead syngeneic mouse models shows enhanced T cell signaling and cytokine production as well as tumor growth inhibition in vivo, supporting its value as an immunotherapeutic target. Herein, we present the structurally enabled discovery of novel, potent, and selective diaminopyrimidine carboxamide HPK1 inhibitors. The key discovery of a carboxamide moiety was essential for enhanced enzyme inhibitory potency and kinome selectivity as well as sustained elevation of cellular IL-2 production across a titration range in human peripheral blood mononuclear cells. The elucidation of structure-activity relationships using various pendant amino ring systems allowed for the identification of several small molecule type-I inhibitors with promising in vitro profiles.

Development of High-Throughput Assays for Evaluation of Hematopoietic Progenitor Kinase 1 Inhibitors.

Hematopoietic progenitor kinase 1 (HPK1), also referred to as mitogen-activated protein kinase kinase kinase kinase 1 (MAP4K1), is a serine/threonine kinase that negatively regulates T-cell signaling by phosphorylating Ser376 of Src homology 2 (SH2) domain-containing leukocyte protein of 76 kDa (SLP-76), a critical mediator of T-cell receptor activation. HPK1 loss of function mouse models demonstrated enhanced immune cell activation and beneficial antitumor activity. To enable discovery and functional characterization of high-affinity small-molecule HPK1 inhibitors, we have established high-throughput biochemical, cell-based, and novel pharmacodynamic (PD) assays. Kinase activity-based time-resolved fluorescence energy transfer (TR-FRET) assays were established as the primary biochemical approach to screen for potent inhibitors and assess selectivity against members of MAP4K and other closely related kinases. A proximal target engagement (TE) assay quantifying pSLP-76 levels as a readout and a distal assay measuring IL-2 secretion as a functional response were established using human peripheral blood mononuclear cells (PBMCs) from two healthy donors. Significant correlations between biochemical and cellular assays as well as excellent correlation between the two donors for the cellular assays were observed. pSLP-76 levels were further used as a PD marker in the preclinical murine model. This effort required the development of a novel ultrasensitive single-molecule array (SiMoA) assay to monitor pSLP-76 changes in mouse spleen.

Utilization of Metabolite Identification and Structural Data to Guide Design of Low-Dose IDO1 Inhibitors.

Herein the discovery of potent IDO1 inhibitors with low predicted human dose is discussed. Metabolite identification (MetID) and structural data were used to strategically incorporate cyclopropane rings into this tetrahydronaphthyridine series of IDO1 inhibitors to improve their metabolic stability and potency. Enabling synthetic chemistry was developed to construct these unique fused cyclopropyl compounds, leading to inhibitors with improved pharmacokinetics and human whole blood potency and a predicted human oral dose as low as 9 mg once daily (QD).

Structure-Based Discovery of Proline-Derived Arginase Inhibitors with Improved Oral Bioavailability for Immuno-Oncology.

Recent data suggest that the inhibition of arginase (ARG) has therapeutic potential for the treatment of a number of indications ranging from pulmonary and vascular disease to cancer. Thus, high demand exists for selective small molecule ARG inhibitors with favorable druglike properties and good oral bioavailability. In light of the significant challenges associated with the unique physicochemical properties of previously disclosed ARG inhibitors, we use structure-based drug design combined with a focused optimization strategy to discover a class of boronic acids featuring a privileged proline scaffold with superior potency and oral bioavailability. These compounds, exemplified by inhibitors 4a, 18, and 27, demonstrated a favorable overall profile, and 4a was well tolerated following multiple days of dosing at concentrations that exceed those required for serum arginase inhibition and concomitant arginine elevation in a syngeneic mouse carcinoma model.

Comprehensive Strategies to Bicyclic Prolines: Applications in the Synthesis of Potent Arginase Inhibitors.

Comprehensive synthetic strategies afforded a diverse set of structurally unique bicyclic proline-containing arginase inhibitors with a high degree of three-dimensionality. The analogs that favored the Cγ-exo conformation of the proline improved the arginase potency over the initial lead. The novel synthetic strategies reported here not only enable access to previously unknown stereochemically complex proline derivatives but also provide a foundation for the future synthesis of bicyclic proline analogs, which incorporate inherent three-dimensional character into building blocks, medicine, and catalysts and could have a profound impact on the conformation of proline-containing peptides and macrocycles.

Carbamate and N-Pyrimidine Mitigate Amide Hydrolysis: Structure-Based Drug Design of Tetrahydroquinoline IDO1 Inhibitors.

Indoleamine-2,3-dioxygenase-1 (IDO1) has emerged as an attractive target for cancer immunotherapy. An automated ligand identification system screen afforded the tetrahydroquinoline class of novel IDO1 inhibitors. Potency and pharmacokinetic (PK) were key issues with this class of compounds. Structure-based drug design and strategic incorporation of polarity enabled the rapid improvement on potency, solubility, and oxidative metabolic stability. Metabolite identification studies revealed that amide hydrolysis in the D-pocket was the key clearance mechanism for this class. Strategic survey of amide isosteres revealed that carbamates and N-pyrimidines, which maintained exquisite potencies, mitigated the amide hydrolysis issue and led to an improved rat PK profile. The lead compound 28 is a potent IDO1 inhibitor, with clean off-target profiles and the potential for quaque die dosing in humans.

Discovery and Optimization of Rationally Designed Bicyclic Inhibitors of Human Arginase to Enhance Cancer Immunotherapy.

The action of arginase, a metalloenzyme responsible for the hydrolysis of arginine to urea and ornithine, is hypothesized to suppress immune-cell activity within the tumor microenvironment, and thus its inhibition may constitute a means by which to potentiate the efficacy of immunotherapeutics such as anti-PD-1 checkpoint inhibitors. Taking inspiration from reported enzyme-inhibitor cocrystal structures, we designed and synthesized novel inhibitors of human arginase possessing a fused 5,5-bicyclic ring system. The prototypical member of this class, 3, when dosed orally, successfully demonstrated serum arginase inhibition and concomitant arginine elevation in a syngeneic mouse carcinoma model, despite modest oral bioavailability. Structure-based design strategies to improve the bioavailability of this class, including scaffold modification, fluorination, and installation of active-transport recognition motifs were explored.

Cancer-Associated Fibroblasts Promote Immunosuppression by Inducing ROS-Generating Monocytic MDSCs in Lung Squamous Cell Carcinoma.

Cancer-associated fibroblasts (CAF) represent a functionally heterogeneous population of activated fibroblasts that constitutes a major component of tumor stroma. Although CAFs have been shown to promote tumor growth and mediate resistance to chemotherapy, the mechanisms by which they may contribute to immune suppression within the tumor microenvironment (TME) in lung squamous cell carcinoma (LSCC) remain largely unexplored. Here, we identified a positive correlation between CAF and monocytic myeloid cell abundances in 501 primary LSCCs by mining The Cancer Genome Atlas data sets. We further validated this finding in an independent cohort using imaging mass cytometry and found a significant spatial interaction between CAFs and monocytic myeloid cells in the TME. To delineate the interplay between CAFs and monocytic myeloid cells, we used chemotaxis assays to show that LSCC patient-derived CAFs promoted recruitment of CCR2+ monocytes via CCL2, which could be reversed by CCR2 inhibition. Using a three-dimensional culture system, we found that CAFs polarized monocytes to adopt a myeloid-derived suppressor cell (MDSC) phenotype, characterized by robust suppression of autologous CD8+ T-cell proliferation and IFNγ production. We further demonstrated that inhibiting IDO1 and NADPH oxidases, NOX2 and NOX4, restored CD8+ T-cell proliferation by reducing reactive oxygen species (ROS) generation in CAF-induced MDSCs. Taken together, our study highlights a pivotal role of CAFs in regulating monocyte recruitment and differentiation and demonstrated that CCR2 inhibition and ROS scavenging abrogate the CAF-MDSC axis, illuminating a potential therapeutic path to reversing the CAF-mediated immunosuppressive microenvironment.

Discovery of Potent and Orally Available Bicyclo[1.1.1]pentane-Derived Indoleamine-2,3-dioxygenase 1 (IDO1) Inhibitors.

Indoleamine-2,3-dioxygenase 1 (IDO1) inhibition and its combination with immune checkpoint inhibitors like pembrolizumab have drawn considerable attention from both academia and the pharmaceutical industry. Here, we describe the discovery of a novel class of highly potent IDO1 heme-displacing inhibitors featuring a unique bicyclo[1.1.1]pentane motif. Compound 1, evolving from an ALIS (automated ligand identification system) hit, exhibited excellent potency but lacked the desired pharmacokinetic profile due to extensive amide hydrolysis of the benzamide moiety. Replacing the central phenyl ring in 1 with a bicyclo[1.1.1]pentane bioisostere effectively circumvented the amide hydrolysis issue, resulting in the discovery of compound 2 with a favorable overall profile such as excellent potency, selectivity, pharmacokinetics, and a low predicted human dose.

Discovery of N-(Indazol-3-yl)piperidine-4-carboxylic Acids as RORγt Allosteric Inhibitors for Autoimmune Diseases.

The clinical success of anti-IL-17 monoclonal antibodies (i.e., Cosentyx and Taltz) has validated Th17 pathway modulation for the treatment of autoimmune diseases. The nuclear hormone receptor RORγt is a master regulator of Th17 cells and affects the production of a host of cytokines, including IL-17A, IL-17F, IL-22, IL-26, and GM-CSF. Substantial interest has been spurred across both academia and industry to seek small molecules suitable for RORγt inhibition. A variety of RORγt inhibitors have been reported in the past few years, the majority of which are orthosteric binders. Here we disclose the discovery and optimization of a class of inhibitors, which bind differently to an allosteric binding pocket. Starting from a weakly active hit 1, a tool compound 14 was quickly identified that demonstrated superior potency, selectivity, and off-target profile. Further optimization focused on improving metabolic stability. Replacing the benzoic acid moiety with piperidinyl carboxylate, modifying the 4-aza-indazole core in 14 to 4-F-indazole, and incorporating a key hydroxyl group led to the discovery of 25, which possesses exquisite potency and selectivity, as well as an improved pharmacokinetic profile suitable for oral dosing.