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.

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.

Identification and characterization of a first-generation inhibitor of claudin-1 in colon cancer progression and metastasis.

Colorectal cancer (CRC) is a leading cause of the cancer-related deaths worldwide. Thus, developing novel and targeted therapies for inhibiting CRC progression and metastasis is urgent. Several studies, including ours, have reported a causal role for an upregulated claudin-1 expression in promoting CRC metastasis through the activation of the Src and ß-catenin-signaling. In murine models of colon tumorigenesis, claudin-1 overexpression promotes oncogenic properties such as transformation and invasiveness. Conversely, the downregulation of claudin-1 inhibits colon tumorigenesis. Despite being a desirable target for cancer treatment, there are currently no known claudin-1 inhibitors with antitumor efficacy. Using a rigorous analytical design and implementing in- vitro and in-vivo testing and a brief medicinal chemistry campaign, we identified a claudin-1-specific inhibitor and named it I-6. Despite its high potency, I-6 was rapidly cleared in human liver microsomes. We, therefore, synthesized I-6 analogs and discovered a novel small molecule, PDS-0330. We determined that PDS0330 inhibits claudin-1-dependent CRC progression without exhibiting toxicity in in-vitro and in-vivo models of CRC and that it binds directly and specifically to claudin-1 with micromolar affinity. Further analyses revealed that PDS-0330 exhibits antitumor and chemosensitizer activities with favorable pharmacokinetic properties by inhibiting the association with metastatic oncogene Src. Overall, our data propose that PDS-0330 interferes with claudin-1/Src association to inhibit CRC progression and metastasis. Our findings are of direct clinical relevance and may open new therapeutic opportunities in colon cancer treatment and/or management by targeting claudin-1.

Structure-Activity Relationships for the N-Me- Versus N-H-Amide Modification to Macrocyclic ent-Verticilide Antiarrhythmics.

The synthesis of all N-Me and N-H analogues of ent-verticilide is described, enabling a structure-activity relationship study based on cardiac ryanodine receptor (RyR2) calcium ion channel inhibition. The use of permeabilized cardiomyocytes allowed us to correlate the degree of N-methylation with activity without concern for changes in passive membrane permeability that these modifications can cause. A key hypothesis was that the minimal pharmacophore may be repeated in this cyclic oligomeric octadepsipeptide (a 24-membered macrocycle), opening the possibility that target engagement will not necessarily be lost with a single N-Me → N-H modification. The effect in the corresponding 18-membered ring oligomer (ent-verticilide B1) was also investigated. We report here that a high degree of N-methyl amide content is critical for activity in the ent-verticilide series but not entirely so for the ent-verticilide B1 series.

From dopamine 4 to sigma 1: Synthesis, SAR and biological characterization of a piperidine scaffold of σ1 modulators.

The sigma 1 receptor is a multifunctional receptor with wide distribution in the nervous system and its function has been implicated with a number of neurological disorders including dementia and Alzheimer's disease (AD) and other neurodegenerative disorders. In addition, modulators of σ1 have been advanced into clinical trials for the treatment of pain. Starting from our previously disclosed piperidine scaffold, we have identified a class of potent sigma 1 modulators. This work highlights the key SAR components that lead to the divergence in D4 and σ1 activity. In addition, we further profile lead compounds in a panel of off-target receptors, in vitro and in vivo pharmacokinetic studies. This has culminated in the discovery of multiple σ1 receptor modulators with properties that will allow for study in animal models.

Further Exploration of the Benzimidazole Scaffold as TRPC5 Inhibitors: Identification of 1-Alkyl-2-(pyrrolidin-1-yl)-1H-benzo[d]imidazoles as Potent and Selective Inhibitors.

The transient receptor potential cation channel 5 (TRPC5) plays an important role in numerous cellular processes. Due to this, it has gained considerable attention over the past few years as a potential therapeutic target. Recently, TRPC5 has been shown to be involved in the regulation of podocyte survival, indicating a potential treatment option for chronic kidney disease. In addition, a recent study has shown TRPC5 to be expressed in human sensory neurons and suggests that TRPC5 inhibition could be an effective treatment for spontaneous and tactile pain. To understand these processes more fully, potent and selective tool compounds are needed. Herein we report further exploration of the 2-aminobenzimidazole scaffold as a potent TRPC5 inhibitor, culminating in the discovery of 16 f as a potent and selective TRPC5 inhibitor.

Synthesis and Biological Characterization of a Series of 2-Sulfonamidebenzamides as Allosteric Modulators of MrgX1.

The present study describes our continued efforts in the discovery and characterization of a series of 2-sulfonamidebenzamides as allosteric modulators of MrgX1. MrgX1 has been shown to be an attractive target as a nonopioid receptor for the potential treatment of chronic pain. Working from our original compound, ML382, and utilizing iterative medicinal chemistry, we have identified key halogen substituents that improve MrgX1 potency by ∼8-fold. In addition, we have evaluated the compounds in Tier 1 drug metabolism and pharmacokinetics assays and have identified key compounds that impart improved potency and microsomal stability.

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.

TRPC5 Channel Inhibition Protects Podocytes in Puromycin-Aminonucleoside Induced Nephrosis Models.

Podocyte injury and the appearance of proteinuria are key features of several progressive kidney diseases. Genetic deletion or selective inhibition of TRPC5 channels with small-molecule inhibitors protects podocytes in rodent models of kidney disease, but less is known about the human relevance and translatability of TRPC5 inhibition. Here, we investigate the effect of TRPC5 inhibition in puromycin aminonucleoside (PAN)-treated rats, human iPSC-derived podocytes, and kidney organoids. We first established that systemic administration of the TRPC5 inhibitor AC1903 was sufficient to protect podocyte cytoskeletal proteins and suppress proteinuria in PAN-induced nephrosis rats, an established model of podocyte injury. TRPC5 current was recorded in the human iPSC-derived podocytes and was blocked by AC1903. PAN treatment caused podocyte injury in human iPSC-derived podocytes and kidney organoids. Inhibition of TRPC5 channels reversed the effects of PAN-induced injury in human podocytes in both 2D and 3D culture systems. Taken together, these results revealed the relevance of TRPC5 channel inhibition in puromycin-aminonucleoside induced nephrosis models, highlighting the potential of this therapeutic strategy for patients.

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 biological characterization of a series of N-(1-(1,1-dioxidotetrahydrothiophen-3-yl)-3-methyl-1H-pyrazol-5-yl)acetamide ethers as novel GIRK1/2 potassium channel activators.

The present study describes the discovery and characterization of a series of N-(1-(1,1-dioxidotetrahydrothiophen-3-yl)-3-methyl-1H-pyrazol-5-yl)acetamide ethers as G protein-gated inwardly-rectifying potassium (GIRK) channel activators. From our previous lead optimization efforts, we have identified a new ether-based scaffold and paired this with a novel sulfone-based head group to identify a potent and selective GIRK1/2 activator. In addition, we evaluated the compounds in tier 1 DMPK assays and have identified compounds that display nanomolar potency as GIRK1/2 activators with improved metabolic stability over the prototypical urea-based compounds.

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.

PIK3C3 Inhibition Promotes Sensitivity to Colon Cancer Therapy by Inhibiting Cancer Stem Cells.

Background: Despite recent advances in therapies, resistance to chemotherapy remains a critical problem in the clinical management of colorectal cancer (CRC). Cancer stem cells (CSCs) play a central role in therapy resistance. Thus, elimination of CSCs is crucial for effective CRC therapy; however, such strategies are limited. Autophagy promotes resistance to cancer therapy; however, whether autophagy protects CSCs to promote resistance to CRC-therapy is not well understood. Moreover, specific and potent autophagy inhibitors are warranted as clinical trials with hydroxychloroquine have not been successful. Methods: Colon cancer cells and tumoroids were used. Fluorescent reporter-based analysis of autophagy flux, spheroid and side population (SP) culture, and qPCR were done. We synthesized 36-077, a potent inhibitor of PIK3C3/VPS34 kinase, to inhibit autophagy. Combination treatments were done using 5-fluorouracil (5-FU) and 36-077. Results: The 5-FU treatment induced autophagy only in a subset of the treated colon cancer. These autophagy-enriched cells also showed increased expression of CSC markers. Co-treatment with 36-077 significantly improved efficacy of the 5-FU treatment. Mechanistic studies revealed that combination therapy inhibited GSK-3ß/Wnt/ß-catenin signaling to inhibit CSC population. Conclusion: Autophagy promotes resistance to CRC-therapy by specifically promoting GSK-3ß/Wnt/ß-catenin signaling to promote CSC survival, and 36-077, a PIK3C3/VPS34 inhibitor, helps promote efficacy of CRC therapy.

Further SAR on the (Phenylsulfonyl)piperazine Scaffold as Inhibitors of the Aedes aegypti Kir1 (AeKir) Channel and Larvicides.

Zika virus (ZIKV), dengue fever (DENV) and chikungunya (CHIKV) are arboviruses that are spread to humans from the bite of an infected adult female Aedes aegypti mosquito. As there are no effective vaccines or therapeutics for these diseases, the primary strategy for controlling the spread of these viruses is to prevent the mosquito from biting humans through the use of insecticides. Unfortunately, the commonly used classes of insecticides have seen a significant increase in resistance, thus complicating control efforts. Inhibiting the renal inward rectifier potassium (Kir) channel of the mosquito vector Aedes aegypti has been shown to be a promising target for the development of novel mosquitocides. We have shown that Kir1 channels play key roles in mosquito diuresis, hemolymph potassium homeostasis, flight, and reproduction. Previous work from our laboratories identified a novel (phenylsulfonyl)piperazine scaffold as potent AeKir channel inhibitors with activity against both adult and larval mosquitoes. Herein, we report further SAR work around this scaffold and have identified additional compounds with improved in vitro potency and mosquito larvae toxicity.

Synthesis and SAR Studies of 1H-Pyrrolo[2,3-b]pyridine-2-carboxamides as Phosphodiesterase 4B (PDE4B) Inhibitors.

Herein we report the synthesis, SAR, and biological evaluation of a series of 1H-pyrrolo[2,3-b]pyridine-2-carboxamide derivatives as selective and potent PDE4B inhibitors. Compound 11h is a PDE4B preferring inhibitor and exhibited acceptable in vitro ADME and significantly inhibited TNF-α release from macrophages exposed to pro-inflammatory stimuli (i.e., lipopolysaccharide and the synthetic bacterial lipopeptide Pam3Cys). In addition, 11h was selective against a panel of CNS receptors and represents an excellent lead for further optimization and preclinical testing in the setting of CNS diseases.

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.

KVA-D-88, a Novel Preferable Phosphodiesterase 4B Inhibitor, Decreases Cocaine-Mediated Reward Properties in Vivo.

Cocaine addiction remains a major public concern throughout the world especially in developed countries. In the last three decades, significant achievements have led to a greater understanding of the signaling pathways involved in the development of cocaine addiction; however, there are no FDA-approved treatments available to reverse or block this brain disease due to either the unsatisfactory therapeutic efficacy or severe side effects. Previous studies have demonstrated that chronic exposure to cocaine elevates levels of cyclic AMP (cAMP) as a neuroadaptative response in reward-related brain regions. Phosphodiesterase 4 (PDE4) inhibitors, which elevate cAMP levels, have been shown to block cocaine-mediated behavioral changes related to psychoactive and reinforcing properties. Unfortunately, previously studied PDE4 inhibitors induce severe side-effects, which limit their clinical usage. In this study, we identified a novel PDE4B inhibitor, KVA-D-88, with an improved selectivity profile compared to previous compounds (e.g., rolipram). Pharmacokinetic studies have shown that this compound is brain penetrant and preferably acts on PDE4B compared to PDE4D in vitro, alluding to less unwanted side effects with KVA-D-88 in vivo. Interestingly, pretreatment with KVA-D-88 significantly inhibited cocaine-induced hyperlocomotor activity. In cocaine self-administering mice with differential schedules, KVA-D-88 strikingly decreased the number of active nose-pokes and cocaine infusions and reduced the break point. Taken together, our findings demonstrate that this novel PDE4 inhibitor, KVA-D-88, could inhibit cocaine-mediated rewarding effects implying its potential clinical usage for cocaine addiction.

Selective α7 nicotinic receptor agonists and positive allosteric modulators for the treatment of schizophrenia - a review.

INTRODUCTION: Alpha 7 nicotinic acetylcholine receptor (α7 nAChR) partial agonists, agonists, and positive allosteric modulators (PAMs) have been in development for over a decade. The initial candidates were in clinical trials for a wide variety of diseases including schizophrenia, but there has yet to be a successful compound to make it to the market for any disorder. Although difficult to assess the cause of all the clinical failures, the lack of efficacy played a major role. The development of more selective compounds, may bring a successful compound to long-suffering schizophrenia patients. AREAS COVERED: This article examines investigational agonists and positive allosteric modulators of the α7 nicotinic receptor in preclinical studies as well as clinical trials. Our search included the use of SciFinder, Google, and clinicaltrials.gov with search dates of 2015 to the present. EXPERT OPINION: Researchers must rethink their approach should look more closely at the selectivity of new compounds and how to tackle the translational gap. Perhaps new positive allosteric modulators that can help minimize receptor desensitization and selectivity profiles can be a path forward for α7 nAChRs in schizophrenia.