Identification of highly selective SIK1/2 inhibitors that modulate innate immune activation and suppress intestinal inflammation.

The salt-inducible kinases (SIK) 1-3 are key regulators of pro- versus anti-inflammatory cytokine responses during innate immune activation. The lack of highly SIK-family or SIK isoform-selective inhibitors suitable for repeat, oral dosing has limited the study of the optimal SIK isoform selectivity profile for suppressing inflammation in vivo. To overcome this challenge, we devised a structure-based design strategy for developing potent SIK inhibitors that are highly selective against other kinases by engaging two differentiating features of the SIK catalytic site. This effort resulted in SIK1/2-selective probes that inhibit key intracellular proximal signaling events including reducing phosphorylation of the SIK substrate cAMP response element binding protein (CREB) regulated transcription coactivator 3 (CRTC3) as detected with an internally generated phospho-Ser329-CRTC3-specific antibody. These inhibitors also suppress production of pro-inflammatory cytokines while inducing anti-inflammatory interleukin-10 in activated human and murine myeloid cells and in mice following a lipopolysaccharide challenge. Oral dosing of these compounds ameliorates disease in a murine colitis model. These findings define an approach to generate highly selective SIK1/2 inhibitors and establish that targeting these isoforms may be a useful strategy to suppress pathological inflammation.

Preclinical efficacy of the potent, selective menin-KMT2A inhibitor JNJ-75276617 (bleximenib) in KMT2A- and NPM1-altered leukemias.

ABSTRACT: The interaction between menin and histone-lysine N-methyltransferase 2A (KMT2A) is a critical dependency for KMT2A- or nucleophosmin 1 (NPM1)-altered leukemias and an emerging opportunity for therapeutic development. JNJ-75276617 (bleximenib) is a novel, orally bioavailable, potent, and selective protein-protein interaction inhibitor of the binding between menin and KMT2A. In KMT2A-rearranged (KMT2A-r) and NPM1-mutant (NPM1c) acute myeloid leukemia (AML) cells, JNJ-75276617 inhibited the association of the menin-KMT2A complex with chromatin at target gene promoters, resulting in reduced expression of several menin-KMT2A target genes, including MEIS1 and FLT3. JNJ-75276617 displayed potent antiproliferative activity across several AML and acute lymphoblastic leukemia (ALL) cell lines and patient samples harboring KMT2A or NPM1 alterations in vitro. In xenograft models of AML and ALL, JNJ-75276617 reduced leukemic burden and provided a significant dose-dependent survival benefit accompanied by expression changes of menin-KMT2A target genes. JNJ-75276617 demonstrated synergistic effects with gilteritinib in vitro in AML cells harboring KMT2A-r. JNJ-75276617 further exhibited synergistic effects with venetoclax and azacitidine in AML cells bearing KMT2A-r in vitro, and significantly increased survival in mice. Interestingly, JNJ-75276617 showed potent antiproliferative activity in cell lines engineered with recently discovered mutations (MEN1M327I or MEN1T349M) that developed in patients refractory to the menin-KMT2A inhibitor revumenib. A cocrystal structure of menin in complex with JNJ-75276617 indicates a unique binding mode distinct from other menin-KMT2A inhibitors, including revumenib. JNJ-75276617 is being clinically investigated for acute leukemias harboring KMT2A or NPM1 alterations, as a monotherapy for relapsed/refractory acute leukemia (NCT04811560), or in combination with AML-directed therapies (NCT05453903).

Identification of isoquinolinone DHODH inhibitor isosteres.

DHODH inhibition represents an attractive approach to overcome differentiation blockade for the treatment of AML. In a previous communication, we described our efforts leading to the discovery of compound 3 (JNJ-74856665), an orally bioavailable, potent, and selective DHODH inhibitor for clinical development. Guided by the co-crystal structures bound to human DHODH, other fused six-membered constructs were explored as isosteric replacements of the isoquinolinone central core. The correct positioning of the nitrogen in these core systems proved to be essential in modulating potency. Herein is described the synthesis of these complexly functionalized cores and their profiling, leading to DHODH inhibitors that possess favorable properties suitable for further development.

Di-fluoro azepane HBV capsid assembly modulators.

The protein that forms the inner shell of the HBV virus, known as the capsid core protein, plays a crucial role in allowing chronic HBV infections to persist. Studies have shown that disrupting the assembly of the capsid can effectively combat the virus, and small molecule drugs that target the HBV capsid assembly modulator (CAM) process have been successful in clinical trials. Herein is described a distinct series of di-fluoro azepane CAMs with exceptional potency, pharmacokinetic, and solubility properties.

Modulation of Ligand-Gated Glycine Receptors Via Functional Monoclonal Antibodies.

Ion channels are targets of considerable therapeutic interest to address a wide variety of neurologic indications, including pain perception. Current pharmacological strategies have focused mostly on small molecule approaches that can be limited by selectivity requirements within members of a channel family or superfamily. Therapeutic antibodies have been proposed, designed, and characterized to alleviate this selectivity limitation; however, there are no Food and Drug Administration-approved therapeutic antibody-based drugs targeting ion channels on the market to date. Here, in an effort to identify novel classes of engineered ion channel modulators for potential neurologic therapeutic applications, we report the generation and characterization of six (EC50 < 25nM) Cys-loop receptor family monoclonal antibodies with modulatory function against rat and human glycine receptor alpha 1 (GlyRα1) and/or GlyRα3. These antibodies have activating (i.e., positive modulator) or inhibiting (i.e., negative modulator) profiles. Moreover, GlyRα3 selectivity was successfully achieved for two of the three positive modulators identified. When dosed intravenously, the antibodies achieved sufficient brain exposure to cover their calculated in vitro EC50 values. When compared head-to-head at identical exposures, the GlyRα3-selective antibody showed a more desirable safety profile over the nonselective antibody, thus demonstrating, for the first time, an advantage for GlyRα3-selectivity. Our data show that ligand-gated ion channels of the glycine receptor family within the central nervous system can be functionally modulated by engineered biologics in a dose-dependent manner and that, despite high protein homology between the alpha subunits, selectivity can be achieved within this receptor family, resulting in future therapeutic candidates with more desirable drug safety profiles. SIGNIFICANCE STATEMENT: This study presents immunization and multiplatform screening approaches to generate a diverse library of functional antibodies (agonist, potentiator, or inhibitory) raised against human glycine receptors (GlyRs). This study also demonstrates the feasibility of acquiring alpha subunit selectivity, a desirable therapeutic profile. When tested in vivo, these tool molecules demonstrated an increased safety profile in favor of GlyRα3-selectivity. These are the first reported functional GlyR antibodies that may open new avenues to treating central nervous system diseases with subunit selective biologics.

Diazepinone HBV capsid assembly modulators.

The HBV capsid core protein serves a number of important functions in the viral life cycle enabling chronic HBV infection to persist, and therefore is a promising drug target. Interfering with capsid assembly has shown efficacy in clinical trials with small molecule capsid assembly modulators (CAMs). Herein is described the further optimization of a progressive series of diazepinone HBV CAMs.

Crystal structure of human glycine receptor-α3 bound to antagonist strychnine.

Neurotransmitter-gated ion channels of the Cys-loop receptor family are essential mediators of fast neurotransmission throughout the nervous system and are implicated in many neurological disorders. Available X-ray structures of prokaryotic and eukaryotic Cys-loop receptors provide tremendous insights into the binding of agonists, the subsequent opening of the ion channel, and the mechanism of channel activation. Yet the mechanism of inactivation by antagonists remains unknown. Here we present a 3.0 Å X-ray structure of the human glycine receptor-α3 homopentamer in complex with a high affinity, high-specificity antagonist, strychnine. Our structure allows us to explore in detail the molecular recognition of antagonists. Comparisons with previous structures reveal a mechanism for antagonist-induced inactivation of Cys-loop receptors, involving an expansion of the orthosteric binding site in the extracellular domain that is coupled to closure of the ion pore in the transmembrane domain.

A carboxylic acid isostere screen of the DHODH inhibitor Brequinar.

Dihydroorotate dehydrogenase (DHODH) enzymatic activity impacts many aspects critical to cell proliferation and survival. Recently, DHODH has been identified as a target for acute myeloid differentiation therapy. In preclinical models of AML, the DHODH inhibitor Brequinar (BRQ) demonstrated potent anti-leukemic activity. Herein we describe a carboxylic acid isostere study of Brequinar which revealed a more potent non-carboxylic acid derivative with improved cellular potency and good pharmacokinetic properties.

Discovery of Alternative Binding Poses through Fragment-Based Identification of DHODH Inhibitors.

Dihydroorotate dehydrogenase (DHODH) is a mitochondrial enzyme that affects many aspects essential to cell proliferation and survival. Recently, DHODH has been identified as a potential target for acute myeloid leukemia therapy. Herein, we describe the identification of potent DHODH inhibitors through a scaffold hopping approach emanating from a fragment screen followed by structure-based drug design to further improve the overall profile and reveal an unexpected novel binding mode. Additionally, these compounds had low P-gp efflux ratios, allowing for applications where exposure to the brain would be required.

Discovery of JNJ-74856665: A Novel Isoquinolinone DHODH Inhibitor for the Treatment of AML.

Acute myelogenous leukemia (AML), a heterogeneous disease of the blood and bone marrow, is characterized by the inability of myeloblasts to differentiate into mature cell types. Dihydroorotate dehydrogenase (DHODH) is an enzyme well-known in the pyrimidine biosynthesis pathway and preclinical findings demonstrated that DHODH is a metabolic vulnerability in AML as inhibitors can induce differentiation across multiple AML subtypes. As a result of virtual screening and structure-based drug design approaches, a novel series of isoquinolinone DHODH inhibitors was identified. Further lead optimization afforded JNJ-74856665 as an orally bioavailable, potent, and selective DHODH inhibitor with favorable physicochemical properties selected for clinical development in patients with AML and myelodysplastic syndromes (MDS).

Spiro-Azetidine Oxindoles as Long-Acting Injectables for Pre-Exposure Prophylaxis against Respiratory Syncytial Virus Infections.

Respiratory syncytial virus (RSV) is a major cause of hospitalization in infants, the elderly, and immune-compromised patients. While a half-life extended monoclonal antibody and 2 vaccines have recently been approved for infants and the elderly, respectively, options to prevent disease in immune-compromised patients are still needed. Here, we describe spiro-azetidine oxindoles as small molecule RSV entry inhibitors displaying favorable potency, developability attributes, and long-acting PK when injected as an aqueous suspension, suggesting their potential to prevent complications following RSV infection over a period of 3 to 6 months with 1 or 2 long-acting intramuscular (IM) or subcutaneous (SC) injections in these immune-compromised patients.

Rapid screening of membrane protein expression in transiently transfected insect cells.

Membrane proteins play critical roles in many biological processes and are the focus of intense biomedical research. One bottleneck for studying membrane proteins is the difficulty in expressing correctly folded and stable proteins, which often requires extensive protein engineering and multiple rounds of optimization, a time and resource intensive process. Here, we describe a method for rapidly screening membrane protein expression in insect cells. The method uses a green fluorescent protein (GFP) covalently fused to target membrane proteins and the resulting fusion proteins are then transiently expressed in insect cells. This approach enables us to dramatically reduce the time and resources required for expression screening by eliminating the need to create recombinant baculovirus. We show that transiently expressed membrane proteins can be directly monitored for their subcellular localizations by fluorescence microscopy. Moreover, their expression levels, approximate molecular mass, and stability can be evaluated with nanogram levels of unpurified proteins by ultrasensitive fluorescence-detection size exclusion chromatography (FSEC). We present our proof of principle studies using a homotrimeric ion channel (ASIC3) and a heterodimeric transporter (SLC7A5/SLC3A2) as examples, and demonstrate the utility of transient expression coupled with FSEC in optimizing membrane protein expression.

Discovery and optimization of potent and selective imidazopyridine and imidazopyridazine mTOR inhibitors.

mTOR is a critical regulator of cellular signaling downstream of multiple growth factors. The mTOR/PI3K/AKT pathway is frequently mutated in human cancers and is thus an important oncology target. Herein we report the evolution of our program to discover ATP-competitive mTOR inhibitors that demonstrate improved pharmacokinetic properties and selectivity compared to our previous leads. Through targeted SAR and structure-guided design, new imidazopyridine and imidazopyridazine scaffolds were identified that demonstrated superior inhibition of mTOR in cellular assays, selectivity over the closely related PIKK family and improved in vivo clearance over our previously reported benzimidazole series.

Integrative structural and functional analysis of human malic enzyme 3: A potential therapeutic target for pancreatic cancer.

Malic enzymes (ME1, ME2, and ME3) are involved in cellular energy regulation, redox homeostasis, and biosynthetic processes, through the production of pyruvate and reducing agent NAD(P)H. Recent studies have implicated the third and least well-characterized isoform, mitochondrial NADP+-dependent malic enzyme 3 (ME3), as a therapeutic target for pancreatic cancers. Here, we utilized an integrated structure approach to determine the structures of ME3 in various ligand-binding states at near-atomic resolutions. ME3 is captured in the open form existing as a stable tetramer and its dynamic Domain C is critical for activity. Catalytic assay results reveal that ME3 is a non-allosteric enzyme and does not require modulators for activity while structural analysis suggests that the inner stability of ME3 Domain A relative to ME2 disables allostery in ME3. With structural information available for all three malic enzymes, the foundation has been laid to understand the structural and biochemical differences of these enzymes and could aid in the development of specific malic enzyme small molecule drugs.

Structure of human spermine oxidase in complex with a highly selective allosteric inhibitor.

Human spermine oxidase (hSMOX) plays a central role in polyamine catabolism. Due to its association with several pathological processes, including inflammation and cancer, hSMOX has garnered interest as a possible therapeutic target. Therefore, determination of the structure of hSMOX is an important step to enable drug discovery and validate hSMOX as a drug target. Using insights from hydrogen/deuterium exchange mass spectrometry (HDX-MS), we engineered a hSMOX construct to obtain the first crystal structure of hSMOX bound to the known polyamine oxidase inhibitor MDL72527 at 2.4 Å resolution. While the overall fold of hSMOX is similar to its homolog, murine N1-acetylpolyamine oxidase (mPAOX), the two structures contain significant differences, notably in their substrate-binding domains and active site pockets. Subsequently, we employed a sensitive biochemical assay to conduct a high-throughput screen that identified a potent and selective hSMOX inhibitor, JNJ-1289. The co-crystal structure of hSMOX with JNJ-1289 was determined at 2.1 Å resolution, revealing that JNJ-1289 binds to an allosteric site, providing JNJ-1289 with a high degree of selectivity towards hSMOX. These results provide crucial insights into understanding the substrate specificity and enzymatic mechanism of hSMOX, and for the design of highly selective inhibitors.

N-Heterocyclic 3-Pyridyl Carboxamide Inhibitors of DHODH for the Treatment of Acute Myelogenous Leukemia.

Acute myelogenous leukemia (AML), a disease of the blood and bone marrow, is characterized by the inability of myeloblasts to differentiate into mature cell types. Dihydroorotate dehydrogenase (DHODH) is an enzyme well-known in the pyrimidine biosynthesis pathway; however, small molecule DHODH inhibitors were recently shown to induce differentiation in multiple AML subtypes. Using virtual screening and structure-based drug design approaches, a new series of N-heterocyclic 3-pyridyl carboxamide DHODH inhibitors were discovered. Two lead compounds, 19 and 29, have potent biochemical and cellular DHODH activity, favorable physicochemical properties, and efficacy in a preclinical model of AML.

Discovery of Potent and Orally Bioavailable Pyridine N-Oxide-Based Factor XIa Inhibitors through Exploiting Nonclassical Interactions.

Activated factor XI (FXIa) inhibitors are promising novel anticoagulants with low bleeding risk compared with current anticoagulants. The discovery of potent FXIa inhibitors with good oral bioavailability has been challenging. Herein, we describe our discovery effort, utilizing nonclassical interactions to improve potency, cellular permeability, and oral bioavailability by enhancing the binding while reducing polar atoms. Beginning with literature-inspired pyridine N-oxide-based FXIa inhibitor 1, the imidazole linker was first replaced with a pyrazole moiety to establish a polar C-H···water hydrogen-bonding interaction. Then, structure-based drug design was employed to modify lead molecule 2d in the P1' and P2' regions, with substituents interacting with key residues through various nonclassical interactions. As a result, a potent FXIa inhibitor 3f (Ki = 0.17 nM) was discovered. This compound demonstrated oral bioavailability in preclinical species (rat 36.4%, dog 80.5%, and monkey 43.0%) and displayed a dose-dependent antithrombotic effect in a rabbit arteriovenous shunt model of thrombosis.

Diazaspirononane Nonsaccharide Inhibitors of O-GlcNAcase (OGA) for the Treatment of Neurodegenerative Disorders.

O-GlcNAcylation is a post-translational modification of tau understood to lower the speed and yield of its aggregation, a pathological hallmark of Alzheimer's disease (AD). O-GlcNAcase (OGA) is the only enzyme that removes O-linked N-acetyl-d-glucosamine (O-GlcNAc) from target proteins. Therefore, inhibition of OGA represents a potential approach for the treatment of AD by preserving the O-GlcNAcylated tau protein. Herein, we report the multifactorial optimization of high-throughput screening hit 8 to a potent, metabolically stable, and orally bioavailable diazaspirononane OGA inhibitor (+)-56. The human OGA X-ray crystal structure has been recently solved, but bacterial hydrolases are still widely used as structural homologues. For the first time, we reveal how a nonsaccharide series of inhibitors binds bacterial OGA and discuss the suitability of two different bacterial orthologues as surrogates for human OGA. These breakthroughs enabled structure-activity relationships to be understood and provided context and boundaries for the optimization of druglike properties.

Relationship of opioid analgesic protocols to assessed pain and length of stay in the pediatric postanesthesia unit following tonsillectomy.

After tonsillectomy and adenoidectomy in children, postoperative pain management is an essential, yet often challenging, task. In addition to discomfort, lack of pain management can lead to delays in oral intake of patients, resulting in extended stays and increased costs. At one North Texas pediatric facility, postoperative coblation tonsillectomy and adenoidectomy pain management orders include the as-needed use of both intravenous fentanyl and intravenous morphine. Both drugs are effective and both have potential side effects that might prolong the recovery period. Nurses in the postanesthesia care unit retrospectively compared a fentanyl and morphine regimen with a morphine-only regimen to determine whether either protocol made a difference in length-of-stay or pain relief. Analysis of available data revealed no statistically significant differences in length of stay between the groups and trivial differences thought to be clinically irrelevant on other variables.

Crystal Structures of Human GlyRα3 Bound to Ivermectin.

Ivermectin acts as a positive allosteric modulator of several Cys-loop receptors including the glutamate-gated chloride channels (GluCls), γ-aminobutyric acid receptors (GABAARs), glycine receptors (GlyRs), and neuronal α7-nicotinic receptors (α7 nAChRs). The crystal structure of Caenorhabditis elegans GluCl complexed with ivermectin revealed the details of its ivermectin binding site. Although the electron microscopy structure of zebrafish GlyRα1 complexed with ivermectin demonstrated a similar binding orientation, detailed structural information on the ivermectin binding and pore opening for Cys-loop receptors in vertebrates has been elusive. Here we present the crystal structures of human GlyRα3 in complex with ivermectin at 2.85 and 3.08 Å resolution. Our structures allow us to explore in detail the molecular recognition of ivermectin by GlyRs, GABAARs, and α7 nAChRs. Comparisons with previous structures reveal how the ivermectin binding expands the ion channel pore. Our results hold promise in structure-based design of GlyR modulators for the treatment of neuropathic pain.