Advancing Biological Understanding and Therapeutics Discovery with Small-Molecule Probes.

Small-molecule probes can illuminate biological processes and aid in the assessment of emerging therapeutic targets by perturbing biological systems in a manner distinct from other experimental approaches. Despite the tremendous promise of chemical tools for investigating biology and disease, small-molecule probes were unavailable for most targets and pathways as recently as a decade ago. In 2005, the NIH launched the decade-long Molecular Libraries Program with the intent of innovating in and broadening access to small-molecule science. This Perspective describes how novel small-molecule probes identified through the program are enabling the exploration of biological pathways and therapeutic hypotheses not otherwise testable. These experiences illustrate how small-molecule probes can help bridge the chasm between biological research and the development of medicines but also highlight the need to innovate the science of therapeutic discovery.

In Vivo Pharmacokinetic and Pharmacodynamic Properties of the Antiarrhythmic Molecule ent-Verticilide.

The unnatural verticilide enantiomer (ent-verticilide) is a selective and potent inhibitor of cardiac ryanodine receptor (RyR2) calcium release channels and exhibits antiarrhythmic activity in a murine model of catecholaminergic polymorphic ventricular tachycardia (CPVT). To determine verticilide's pharmacokinetic and pharmacodynamic properties in vivo, we developed a bioassay to measure nat- and ent-verticilide in murine plasma and correlated plasma concentrations with antiarrhythmic efficacy in a mouse model of CPVT. nat-Verticilide rapidly degraded in plasma in vitro, showing >95% degradation within 5 minutes, whereas ent-verticilide showed <1% degradation over 6 hours. Plasma was collected from mice following intraperitoneal administration of ent-verticilide at two doses (3 mg/kg, 30 mg/kg). Peak C max and area under the plasma-concentration time curve (AUC) scaled proportionally to dose, and the half-life was 6.9 hours for the 3-mg/kg dose and 6.4 hours for the 30-mg/kg dose. Antiarrhythmic efficacy was examined using a catecholamine challenge protocol at time points ranging from 5 to 1440 minutes after intraperitoneal dosing. ent-Verticilide inhibited ventricular arrhythmias as early as 7 minutes after administration in a concentration-dependent manner, with an estimated potency (IC50) of 266 ng/ml (312 nM) and an estimated maximum inhibitory effect of 93.5%. Unlike the US Food and Drug Administration-approved pan-RyR blocker dantrolene, the RyR2-selective blocker ent-verticilide (30 mg/kg) did not reduce skeletal muscle strength in vivo. We conclude that ent-verticilide has favorable pharmacokinetic properties and reduces ventricular arrhythmias with an estimated potency in the nanomolar range, warranting further drug development. SIGNIFICANCE STATEMENT: ent-Verticilide has therapeutic potential to treat cardiac arrhythmias, but little is known about its pharmacological profile in vivo. The primary purpose of this study is to determine the systemic exposure and pharmacokinetics of ent-verticilide in mice and estimate its efficacy and potency in vivo. The current work suggests ent-verticilide has favorable pharmacokinetic properties and reduces ventricular arrhythmias with an estimated potency in the nanomolar range, warranting further drug development.

Discovery, synthesis and biological evaluation of a series of N-(phenylcarbamothioyl)-2-napthamides as inhibitors of Claudin-1.

Colorectal cancer (CRC) remains a leading cause of cancer-related deaths worldwide, despite advancements in diagnosis. The main reason for this is that many newly diagnosed CRC patients will suffer from metastasis to other organs. Thus, the development of new therapies is of critical importance. Claudin-1 protein is a component of tight junctions in epithelial cells, including those found in the lining of the colon. It plays a critical role in the formation and maintenance of tight junctions, which are essential for regulating the passage of molecules between cells. In CRC, claudin-1 is often overexpressed, leading to an increase in cell adhesion, which can contribute to the development and progression of the disease. Studies show that high levels of claudin-1 are associated with poor prognosis in CRC patients and targeting claudin-1 may have therapeutic potential for the treatment of CRC. Previously, we have identified a small molecule that inhibits claudin-1 dependent CRC progression. Reported herein are our lead optimization efforts around this scaffold to identify the key SAR components and the discovery of a key new compound that exhibits enhanced potency in SW620 cells.

Bidirectional influence of limbic GIRK channel activation on innate avoidance behavior.

Systemic administration of ML297, a selective activator of G protein-gated inwardly rectifying K+ (GIRK) channels, decreases innate avoidance behavior in male C57BL/6J mice. The cellular mechanisms mediating the ML297-induced suppression of avoidance behavior are unknown. Here, we show that systemic ML297 administration suppresses elevated plus maze (EPM)-induced neuronal activation in the ventral hippocampus (vHPC) and basolateral amygdala (BLA), and that ML297 activates GIRK1-containing GIRK channels in these limbic structures. While intracranial infusion of ML297 into the vHPC suppressed avoidance behavior in the EPM test, mirroring the effect of systemic ML297, intra-BLA administration of ML297 provoked the opposite effect. Using neuron-specific viral genetic and chemogenetic approaches, we found that the combined inhibition of excitatory neurons in CA3 and dentate gyrus (DG) sub-regions of the vHPC was sufficient to decrease innate avoidance behavior in the EPM, open-field, and light-dark tests in male C57BL/6J mice, while performance in the marble-burying test was not impacted. Furthermore, genetic ablation of GIRK channels in CA3/DG excitatory neurons precluded the suppression of avoidance behavior evoked by systemic ML297 in the EPM test. Thus, acute inhibition of excitatory neurons in the ventral CA3 and DG sub-regions of the vHPC is necessary for the apparent anxiolytic efficacy of systemic ML297 and is sufficient to decrease innate avoidance behavior in male C57BL/6J mice.SIGNIFICANT STATEMENTWe interrogated the cellular mechanisms underlying the apparent anxiolytic efficacy of ML297, a selective activator of GIRK channels and promising lead compound. Intracranial infusion of ML297 into the vHPC and BLA complex exerted opposing influence on innate avoidance behavior in male C57BL/6J mice, the former recapitulating the suppression of avoidance behavior evoked by systemic ML297. Using viral genetic and chemogenetic approaches, we showed that combined inhibition of excitatory neurons in CA3 and dentate gyrus sub-regions of the ventral hippocampus is sufficient to decrease innate avoidance behavior in male mice and mediates the decrease in avoidance behavior evoked by systemic ML297. These findings establish a foundation for future investigations into the therapeutic potential of GIRK channel modulation in anxiety disorders.

Discovery and characterization of benzyloxy piperidine based dopamine 4 receptor antagonists.

The dopamine receptor 4 (D4R) is highly expressed in both motor, associative and limbic subdivisions of the cortico-basal ganglia network. Due to the distribution in the brain, there is mounting evidence pointing to a role for the D4R in the modulation of this network and its subsequent involvement in l-DOPA induced dyskinesias in Parkinson's disease. As part of our continued effort in the discovery of novel D4R antagonists, we report the discovery and characterization of a new 3- or 4-benzyloxypiperidine scaffold as D4R antagonists. We report several D4R selective compounds (>30-fold vs. other dopamine receptor subtypes) with improved in vitro and in vivo stability over previously reported D4R antagonists.

Characterization of VU0468554, a New Selective Inhibitor of Cardiac G Protein-Gated Inwardly Rectifying K+ Channels.

G protein-gated inwardly rectifying K+ (GIRK) channels are critical mediators of excitability in the heart and brain. Enhanced GIRK-channel activity has been implicated in the pathogenesis of supraventricular arrhythmias, including atrial fibrillation. The lack of selective pharmacological tools has impeded efforts to investigate the therapeutic potential of cardiac GIRK-channel interventions in arrhythmias. Here, we characterize a recently identified GIRK-channel inhibitor, VU0468554. Using whole-cell electrophysiological approaches and primary cultures of sinoatrial nodal cells and hippocampal neurons, we show that VU0468554 more effectively inhibits the cardiac GIRK channel than the neuronal GIRK channel. Concentration-response experiments suggest that VU0468554 inhibits Gßγ-activated GIRK channels in noncompetitive and potentially uncompetitive fashion. In contrast, VU0468554 competitively inhibits GIRK-channel activation by ML297, a GIRK-channel activator containing the same chemical scaffold as VU0468554. In the isolated heart model, VU0468554 partially reversed carbachol-induced bradycardia in hearts from wild-type mice but not Girk4-/- mice. Collectively, these data suggest that VU0468554 represents a promising new pharmacological tool for targeting cardiac GIRK channels with therapeutic implications for relevant cardiac arrhythmias. SIGNIFICANCE STATEMENT: Although cardiac GIRK-channel inhibition shows promise for the treatment of supraventricular arrhythmias, the absence of subtype-selective channel inhibitors has hindered exploration into this therapeutic strategy. This study utilizes whole-cell patch-clamp electrophysiology to characterize the new GIRK-channel inhibitor VU0468554 in human embryonic kidney 293T cells and primary cultures. We report that VU0468554 exhibits a favorable pharmacodynamic profile for cardiac over neuronal GIRK channels and partially reverses GIRK-mediated bradycardia in the isolated mouse heart model.

Virtual screening and biological evaluation of PPARγ antagonists as potential anti-prostate cancer agents.

The peroxisome proliferator-activated receptor gamma (PPARγ) was identified as an oncogene and it plays a key role in prostate cancer (PC) development and progression. PPARγ antagonists have been shown to inhibit PC cell growth. Herein, we describe a virtual screening-based approach that led to the discovery of novel PPARγ antagonist chemotypes that bind at the allosteric pocket. Arg288, Lys367, and His449 appear to be important for PPARγ antagonist binding.

Discovery, synthesis and characterization of a series of 7-aryl-imidazo[1,2-a]pyridine-3-ylquinolines as activin-like kinase (ALK) inhibitors.

The activin-like kinases are a family of kinases that play important roles in a variety of disease states. Of this class of kinases, ALK2, has been shown by a gain-of-function to be the primary driver of the childhood skeletal disease fibrodysplasia ossificans progressiva (FOP) and more recently the pediatric cancer diffuse intrinsic pontine glioma (DIPG). Herein, we report our efforts to identify a novel imidazo[1,2-a]pyridine scaffold as potent inhibitors of ALK2 with good in vivo pharmacokinetic properties suitable for future animal studies.

Targeting human Mas-related G protein-coupled receptor X1 to inhibit persistent pain.

Human Mas-related G protein-coupled receptor X1 (MRGPRX1) is a promising target for pain inhibition, mainly because of its restricted expression in nociceptors within the peripheral nervous system. However, constrained by species differences across Mrgprs, drug candidates that activate MRGPRX1 do not activate rodent receptors, leaving no responsive animal model to test the effect on pain in vivo. Here, we generated a transgenic mouse line in which we replaced mouse Mrgprs with human MrgprX1 This humanized mouse allowed us to characterize an agonist [bovine adrenal medulla 8-22 (BAM8-22)] and a positive allosteric modulator (PAM), ML382, of MRGPRX1. Cellular studies suggested that ML382 enhances the ability of BAM8-22 to inhibit high-voltage-activated Ca2+ channels and attenuate spinal nociceptive transmission. Importantly, both BAM8-22 and ML382 effectively attenuated evoked, persistent, and spontaneous pain without causing obvious side effects. Notably, ML382 by itself attenuated both evoked pain hypersensitivity and spontaneous pain in MrgprX1 mice after nerve injury without acquiring coadministration of an exogenous agonist. Our findings suggest that humanized MrgprX1 mice provide a promising preclinical model and that activating MRGPRX1 is an effective way to treat persistent pain.

Design, synthesis and characterization of novel N-heterocyclic-1-benzyl-1H-benzo[d]imidazole-2-amines as selective TRPC5 inhibitors leading to the identification of the selective compound, AC1903.

The transient receptor potential cation channel 5 (TRPC5) has been previously shown to affect podocyte survival in the kidney. As such, inhibitors of TRPC5 are interesting candidates for the treatment of chronic kidney disease (CKD). Herein, we report the synthesis and biological characterization of a series of N-heterocyclic-1-benzyl-1H-benzo[d]imidazole-2-amines as selective TRPC5 inhibitors. Work reported here evaluates the benzimidazole scaffold and substituents resulting in the discovery of AC1903, a TRPC5 inhibitor that is active in multiple animal models of CKD.

Discovery, synthesis and characterization of a series of (1-alkyl-3-methyl-1H-pyrazol-5-yl)-2-(5-aryl-2H-tetrazol-2-yl)acetamides as novel GIRK1/2 potassium channel activators.

The present study describes the discovery and characterization of a series of 5-aryl-2H-tetrazol-3-ylacetamides as G protein-gated inwardly-rectifying potassium (GIRK) channels activators. Working from an initial hit discovered during a high-throughput screening campaign, we identified a tetrazole scaffold that shifts away from the previously reported urea-based scaffolds while remaining effective GIRK1/2 channel activators. In addition, we evaluated the compounds in Tier 1 DMPK assays and have identified a (3-methyl-1H-pyrazol-1-yl)tetrahydrothiophene-1,1-dioxide head group that imparts interesting and unexpected microsomal stability compared to previously-reported pyrazole head groups.

The discovery of VU0652957 (VU2957, Valiglurax): SAR and DMPK challenges en route to an mGlu4 PAM development candidate.

This letter describes the first account of the chemical optimization (SAR and DMPK profiling) of a new series of mGlu4 positive allosteric modulators (PAMs), leading to the identification of VU0652957 (VU2957, Valiglurax), a compound profiled as a preclinical development candidate. Here, we detail the challenges faced in allosteric modulator programs (e.g., steep SAR, as well as subtle structural changes affecting overall physiochemical/DMPK properties and CNS penetration).

Discovery, Characterization, and Effects on Renal Fluid and Electrolyte Excretion of the Kir4.1 Potassium Channel Pore Blocker, VU0134992.

The inward rectifier potassium (Kir) channel Kir4.1 (KCNJ10) carries out important physiologic roles in epithelial cells of the kidney, astrocytes in the central nervous system, and stria vascularis of the inner ear. Loss-of-function mutations in KCNJ10 lead to EAST/SeSAME syndrome, which is characterized by epilepsy, ataxia, renal salt wasting, and sensorineural deafness. Although genetic approaches have been indispensable for establishing the importance of Kir4.1 in the normal function of these tissues, the availability of pharmacological tools for acutely manipulating the activity of Kir4.1 in genetically normal animals has been lacking. We therefore carried out a high-throughput screen of 76,575 compounds from the Vanderbilt Institute of Chemical Biology library for small-molecule modulators of Kir4.1. The most potent inhibitor identified was 2-(2-bromo-4-isopropylphenoxy)-N-(2,2,6,6-tetramethylpiperidin-4-yl)acetamide (VU0134992). In whole-cell patch-clamp electrophysiology experiments, VU0134992 inhibits Kir4.1 with an IC50 value of 0.97 µM and is 9-fold selective for homomeric Kir4.1 over Kir4.1/5.1 concatemeric channels (IC50 = 9 µM) at -120 mV. In thallium (Tl+) flux assays, VU0134992 is greater than 30-fold selective for Kir4.1 over Kir1.1, Kir2.1, and Kir2.2; is weakly active toward Kir2.3, Kir6.2/SUR1, and Kir7.1; and is equally active toward Kir3.1/3.2, Kir3.1/3.4, and Kir4.2. This potency and selectivity profile is superior to Kir4.1 inhibitors amitriptyline, nortriptyline, and fluoxetine. Medicinal chemistry identified components of VU0134992 that are critical for inhibiting Kir4.1. Patch-clamp electrophysiology, molecular modeling, and site-directed mutagenesis identified pore-lining glutamate 158 and isoleucine 159 as critical residues for block of the channel. VU0134992 displayed a large free unbound fraction (fu) in rat plasma (fu = 0.213). Consistent with the known role of Kir4.1 in renal function, oral dosing of VU0134992 led to a dose-dependent diuresis, natriuresis, and kaliuresis in rats. Thus, VU0134992 represents the first in vivo active tool compound for probing the therapeutic potential of Kir4.1 as a novel diuretic target for the treatment of hypertension.

Discovery and characterization of N-(1,3-dialkyl-1H-indazol-6-yl)-1H-pyrazolo[4,3-b]pyridin-3-amine scaffold as mGlu4 positive allosteric modulators that mitigate CYP1A2 induction liability.

Previous reports from our laboratory disclosed the structure and activity of a novel 1H-pyrazolo[4,3-b]pyridine-3-amine scaffold (VU8506) which showed excellent potency, selectivity and in vivo efficacy in preclinical rodent models of Parkinson's disease. Unfortunately, this compound suffered from significant CYP1A2 induction as measured through upstream AhR activation (125-fold) and thus was precluded from further advancement in chronic studies. Herein, we report a new scaffold developed recently which was systematically studied in order to mitigate the CYP1A2 liabilities presented in the earlier scaffolds. We have identified a novel structure that maintains the potency and selectivity of other mGlu4 PAMs, leading to 9i (hmGlu4 EC50 = 43 nM; AhR activation = 2.3-fold).

Practical Strategies and Concepts in GPCR Allosteric Modulator Discovery: Recent Advances with Metabotropic Glutamate Receptors.

Allosteric modulation of GPCRs has initiated a new era of basic and translational discovery, filled with therapeutic promise yet fraught with caveats. Allosteric ligands stabilize unique conformations of the GPCR that afford fundamentally new receptors, capable of novel pharmacology, unprecedented subtype selectivity, and unique signal bias. This review provides a comprehensive overview of the basics of GPCR allosteric pharmacology, medicinal chemistry, drug metabolism, and validated approaches to address each of the major challenges and caveats. Then, the review narrows focus to highlight recent advances in the discovery of allosteric ligands for metabotropic glutamate receptor subtypes 1-5 and 7 (mGlu1-5,7) highlighting key concepts ("molecular switches", signal bias, heterodimers) and practical solutions to enable the development of tool compounds and clinical candidates. The review closes with a section on late-breaking new advances with allosteric ligands for other GPCRs and emerging data for endogenous allosteric modulators.

Activation of Metabotropic Glutamate Receptor 7 Is Required for Induction of Long-Term Potentiation at SC-CA1 Synapses in the Hippocampus.

Of the eight metabotropic glutamate (mGlu) receptor subtypes, only mGlu7 is expressed presynaptically at the Schaffer collateral (SC)-CA1 synapse in the hippocampus in adult animals. Coupled with the inhibitory effects of Group III mGlu receptor agonists on transmission at this synapse, mGlu7 is thought to be the predominant autoreceptor responsible for regulating glutamate release at SC terminals. However, the lack of mGlu7-selective pharmacological tools has hampered direct testing of this hypothesis. We used a novel, selective mGlu7-negative allosteric modulator (NAM), ADX71743, and a newly described Group III mGlu receptor agonist, LSP4-2022, to elucidate the role of mGlu7 in modulating transmission in hippocampal area CA1 in adult C57BL/6J male mice. Interestingly, although mGlu7 agonists inhibit SC-CA1 EPSPs, we found no evidence for activation of mGlu7 by stimulation of SC-CA1 afferents. However, LSP4-2022 also reduced evoked monosynaptic IPSCs in CA1 pyramidal cells and, in contrast to its effect on SC-CA1 EPSPs, ADX71743 reversed the ability of high-frequency stimulation of SC afferents to reduce IPSC amplitudes. Furthermore, blockade of mGlu7 prevented induction of LTP at the SC-CA1 synapse and activation of mGlu7 potentiated submaximal LTP. Together, these data suggest that mGlu7 serves as a heteroreceptor at inhibitory synapses in area CA1 and that the predominant effect of activation of mGlu7 by stimulation of glutamatergic afferents is disinhibition, rather than reduced excitatory transmission. Furthermore, this mGlu7-mediated disinhibition is required for induction of LTP at the SC-CA1 synapse, suggesting that mGlu7 could serve as a novel therapeutic target for treatment of cognitive disorders.

Validation of an improved scale for rating l-DOPA-induced dyskinesia in the mouse and effects of specific dopamine receptor antagonists.

Rodent models of l-DOPA-induced dyskinesia (LID) are essential to investigate pathophysiological mechanisms and treatment options. Ratings of abnormal involuntary movements (AIMs) are used to capture both qualitative and quantitative features of dyskinetic behaviors. Thus far, validated rating scales for the mouse have anchored the definition of severity to the time during which AIMs are present. Here we have asked whether the severity of axial, limb, and orolingual AIMs can be objectively assessed with scores based on movement amplitude. Mice sustained 6-OHDA lesions in the medial forebrain bundle and were treated with l-DOPA (3-6mg/kg/day) until they developed stable AIMs scores. Two independent investigators rated AIM severity using both the validated time-based scale and a novel amplitude scale, evaluating the degree of deviation of dyskinetic body parts relative to their resting position. The amplitude scale yielded a high degree of consistency both within- and between raters. Thus, time-based scores, amplitude scores, and a combination of the two ('global AIM scores') were applied to compare antidyskinetic effects produced by amantadine and by the following subtype-specific DA receptor antagonists: SCH23390 (D1/D5), Raclopride (D2/D3), PG01037 (D3), L-745,870 (D4), and VU6004461 (D4). SCH23390 and Raclopride produced similarly robust reductions in both time-based scores and amplitude scores, while PG01037 and L-745,870 had more partial effects. Interestingly, a novel and highly brain penetrable D4 receptor antagonist (VU6004461) markedly attenuated both time-based and amplitude scores without diminishing the general motor stimulant effect of l-DOPA. In summary, our results show that a dyskinesia scale combining a time dimension with an amplitude dimension ('global AIMs') is more sensitive than unidimensional scales. Moreover, the antidyskinetic effects produced by two chemically distinct D4 antagonists identify the D4 receptor as a potential future target for the treatment of LID.

Discovery, characterization and biological evaluation of a novel (R)-4,4-difluoropiperidine scaffold as dopamine receptor 4 (D4R) antagonists.

Herein, we report the synthesis and structure-activity relationship of a novel series of (R)-4,4-difluoropiperidine core scaffold as dopamine receptor 4 (D4) antagonists. A series of compounds from this scaffold are highly potent against the D4 receptor and selective against the other dopamine receptors. In addition, we were able to confirm the active isomer as the (R)-enantiomer via an X-ray crystal structure.

Synthesis and characterization of a series of chiral alkoxymethyl morpholine analogs as dopamine receptor 4 (D4R) antagonists.

Herein, we report the synthesis and structure-activity relationship of a series of chiral alkoxymethyl morpholine analogs. Our efforts have culminated in the identification of (S)-2-(((6-chloropyridin-2-yl)oxy)methyl)-4-((6-fluoro-1H-indol-3-yl)methyl)morpholine as a novel potent and selective dopamine D4 receptor antagonist with selectivity against the other dopamine receptors tested (<10% inhibition at 1µM against D1, D2L, D2S, D3, and D5).

Discovery and characterization of a novel series of N-phenylsulfonyl-1H-pyrrole picolinamides as positive allosteric modulators of the metabotropic glutamate receptor 4 (mGlu4).

Herein we report the synthesis and characterization of a novel series of N-phenylsulfonyl-1H-pyrrole picolinamides as novel positive allosteric modulators of mGlu4. We detail our work towards finding phenyl replacements for the core scaffold of previously reported phenyl sulfonamides and phenyl sulfone compounds. Our efforts culminated in the identification of N-(1-((3,4-dimethylphenyl)sulfonyl)-1H-pyrrol-3-yl)picolinamide as a potent PAM of mGlu4.