Search

Background: Harmine is a component of the hallucinogenic brew, Ayahuasca, which also contains the psychoactive compound, N , N -dimethyltryptamine. Whether pharmaceutical-grade harmine hydrochloride (HCl) has psychoactive effects, the doses at which these might occur, and the dose-response relationship to side effects and safety in humans are unknown., Methods: We conducted a Phase 1, open-label single ascending dose trial in healthy adults with normal body mass index and no prior psychiatric illness. The primary goal was to determine the maximum tolerated dose (MTD) of oral pharmaceutical-grade harmine HCl and to characterize safety and tolerability. A secondary goal was to ascertain whether any oral dose has psychoactive effects., Results: Thirty-four adult participants, aged 18-55 years, were screened for study eligibility. Twenty-five participants met eligibility criteria and were randomized to a single dose of 100, 200, 300, or 500 mg of harmine HCl, respectively, using a continuous reassessment method. The most common adverse events (AEs) observed were gastrointestinal and/or neurological, dose-related, and of mild to moderate severity. The MTD was determined to be between 100 and 200 mg and is weight-based, with 90% of those participants receiving >2.7 mg/kg experiencing a dose-limiting toxicity. No serious AEs of harmine HCl were identified., Conclusions: Harmine HCl can be orally administered to healthy participants in doses <2.7 mg/kg with minimal or no AEs. Doses >2.7 mg/kg are associated with vomiting, drowsiness, and limited psychoactivity. This study is the first to systematically characterize the psychoactive effects of pharmaceutical quality harmine in healthy participants.

Protein monoaminylation is a class of posttranslational modification (PTM) that contributes to transcription, physiology and behavior. While recent analyses have focused on histones as critical substrates of monoaminylation, the broader repertoire of monoaminylated proteins in brain remains unclear. Here, we report the development/implementation of a chemical probe for the bioorthogonal labeling, enrichment and proteomics-based detection of dopaminylated proteins in brain. We identified 1,557 dopaminylated proteins - many synaptic - including γCaMKII, which mediates Ca 2+ -dependent cellular signaling and hippocampal-dependent memory. We found that γCaMKII dopaminylation is largely synaptic and mediates synaptic-to-nuclear signaling, neuronal gene expression and intrinsic excitability, and contextual memory. These results indicate a critical role for synaptic dopaminylation in adaptive brain plasticity, and may suggest roles for these phenomena in pathologies associated with altered monoaminergic signaling., Competing Interests: COMPETING INTERESTS The Authors declare no competing interests.

Glutaric Aciduria Type 1 (GA1) is a serious inborn error of metabolism with no pharmacological treatments. A novel strategy to treat this disease is to divert the toxic biochemical intermediates to less toxic or nontoxic metabolites. Here, we report a putative novel target, succinyl-CoA:glutarate-CoA transferase (SUGCT), which we hypothesize suppresses the GA1 metabolic phenotype through decreasing glutaryl-CoA and the derived 3-hydroxyglutaric acid. SUGCT is a type III CoA transferase that uses succinyl-CoA and glutaric acid as substrates. We report the structure of SUGCT, develop enzyme- and cell-based assays, and identify valsartan and losartan carboxylic acid as inhibitors of the enzyme in a high-throughput screen of FDA-approved compounds. The cocrystal structure of SUGCT with losartan carboxylic acid revealed a novel pocket in the active site and further validated the high-throughput screening approach. These results may form the basis for the future development of new pharmacological intervention to treat GA1.

Five hundred thirty-seven million people globally suffer from diabetes. Insulin-producing β cells are reduced in number in most people with diabetes, but most individuals still have some residual β cells. However, none of the many diabetes drugs in common use increases human β cell numbers. Recently, small molecules that inhibit dual tyrosine-regulated kinase 1A (DYRK1A) have been shown to induce immunohistochemical markers of human β cell replication, and this is enhanced by drugs that stimulate the glucagon-like peptide 1 (GLP1) receptor (GLP1R) on β cells. However, it remains to be demonstrated whether these immunohistochemical findings translate into an actual increase in human β cell numbers in vivo. It is also unknown whether DYRK1A inhibitors together with GLP1R agonists (GLP1RAs) affect human β cell survival. Here, using an optimized immunolabeling-enabled three-dimensional imaging of solvent-cleared organs (iDISCO + ) protocol in mouse kidneys bearing human islet grafts, we demonstrate that combination of a DYRK1A inhibitor with exendin-4 increases actual human β cell mass in vivo by a mean of four- to sevenfold in diabetic and nondiabetic mice over 3 months and reverses diabetes, without alteration in human α cell mass. The augmentation in human β cell mass occurred through mechanisms that included enhanced human β cell proliferation, function, and survival. The increase in human β cell survival was mediated, in part, by the islet prohormone VGF. Together, these findings demonstrate the therapeutic potential and favorable preclinical safety profile of the DYRK1A inhibitor-GLP1RA combination for diabetes treatment.

Multiple sclerosis, and its murine model experimental autoimmune encephalomyelitis (EAE), is a neurodegenerative autoimmune disease of the CNS characterized by T cell influx and demyelination. Similar to other autoimmune diseases, therapies can alleviate symptoms but often come with side effects, necessitating the exploration of new treatments. We recently demonstrated that the Cullin-RING E3 ubiquitin ligase 4b (CRL4b) aided in maintaining genome stability in proliferating T cells. In this study, we examined whether CRL4b was required for T cells to expand and drive EAE. Mice lacking Cul4b (Cullin 4b) in T cells had reduced EAE symptoms and decreased inflammation during the peak of the disease. Significantly fewer CD4+ and CD8+ T cells were found in the CNS, particularly among the CD4+ T cell population producing IL-17A, IFN-γ, GM-CSF, and TNF-α. Additionally, Cul4b-deficient CD4+ T cells cultured in vitro with their wild-type counterparts were less likely to expand and differentiate into IL-17A- or IFN-γ-producing effector cells. When wild-type CD4+ T cells were activated in vitro in the presence of the recently developed CRL4 inhibitor KH-4-43, they exhibited increased apoptosis and DNA damage. Treatment of mice with KH-4-43 following EAE induction resulted in stabilized clinical scores and significantly reduced numbers of T cells and innate immune cells in the CNS compared with control mice. Furthermore, KH-4-43 treatment resulted in elevated expression of p21 and cyclin E2 in T cells. These studies support that therapeutic inhibition of CRL4 and/or CRL4-related pathways could be used to treat autoimmune disease., (Copyright © 2024 by The American Association of Immunologists, Inc.)

Ubiquitination often generates lysine 48-linked polyubiquitin chains that signal proteolytic destruction of the protein target. A significant subset of ubiquitination proceeds by a priming/extending mechanism, in which a substrate is first monoubiquitinated with a priming E2-conjugating enzyme or a set of E3 ARIH/E2 enzymes specific for priming. This is then followed by ubiquitin (Ub) chain extension catalyzed by an E2 enzyme capable of elongation. This report provides further insights into the priming/extending mechanism. We employed reconstituted ubiquitination systems of substrates CK1α (casein kinase 1α) and β-catenin by Cullin-RING E3 Ub ligases (CRLs) CRL4 CRBN and CRL1 βTrCP , respectively, in the presence of priming E2 UbcH5c and elongating E2 Cdc34b (cell division cycle 34b). We have established a new "apyrase chase" strategy that uncouples priming from chain elongation, which allows accurate measurement of the decay rates of the ubiquitinated substrate with a defined chain length. Our work has revealed highly robust turnover of monoubiquitinated β-catenin that empowers efficient polyubiquitination. The results of competition experiments suggest that the interactions between the ubiquitinated β-catenin and CRL1 βTrCP are highly dynamic. Moreover, ubiquitination of the Ub-modified β-catenin appeared more resistant to inhibition by competitors than the unmodified substrate, suggesting tighter binding with CRL1 βTrCP . These findings support a role for conjugated Ub in enhancing interactions with E3., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Cullin (CUL)-RING (Really Interesting New Gene) E3 ubiquitin (Ub) ligases (CRLs) are the largest E3 family. The E3 CRL core ligase is a subcomplex formed by the CUL C-terminal domain bound with the ROC1/RBX1 RING finger protein, which acts as a hub that mediates and organizes multiple interactions with E2, Ub, Nedd8, and the ARIH family protein, thereby resulting in Ub transfer to the E3-bound substrate. This report describes the modulation of CRL-dependent ubiquitination by small molecule compounds including KH-4-43, #33, and suramin, which target the CRL core ligases. We show that both KH-4-43 and #33 inhibit the ubiquitination of CK1α by CRL4 CRBN . However, either compound's inhibitory effect on this reaction is significantly reduced when a neddylated form of CRL4 CRBN is used. On the other hand, both #33 and KH-4-43 inhibit the ubiquitination of β-catenin by CRL1 β-TrCP and Nedd8-CRL1 β-TrCP almost equally. Thus, neddylation of CRL1 β-TrCP does not negatively impact the sensitivity to inhibition by #33 and KH-4-43. These findings suggest that the effects of neddylation to alter the sensitivity of CRL inhibition by KH-4-43/#33 is dependent upon the specific CRL type. Suramin, a compound that targets CUL's basic canyon, can effectively inhibit CRL1/4-dependent ubiquitination regardless of neddylation status, in contrast to the results observed with KH-4-43/#33. This observed differential drug sensitivity of KH-4-43/#33 appears to echo CUL-specific Nedd8 effects on CRLs as revealed by recent high-resolution structural biology efforts. The highly diversified CRL core ligase structures may provide opportunities for specific targeting by small molecule modulators., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Toxicity of accumulating substrates is a significant problem in several disorders of valine and isoleucine degradation notably short-chain enoyl-CoA hydratase (ECHS1 or crotonase) deficiency, 3-hydroxyisobutyryl-CoA hydrolase (HIBCH) deficiency, propionic acidemia (PA), and methylmalonic aciduria (MMA). Isobutyryl-CoA dehydrogenase (ACAD8) and short/branched-chain acyl-CoA dehydrogenase (SBCAD, ACADSB) function in the valine and isoleucine degradation pathways, respectively. Deficiencies of these acyl-CoA dehydrogenase (ACAD) enzymes are considered biochemical abnormalities with limited or no clinical consequences. We investigated whether substrate reduction therapy through inhibition of ACAD8 and SBCAD can limit the accumulation of toxic metabolic intermediates in disorders of valine and isoleucine metabolism. Using analysis of acylcarnitine isomers, we show that 2-methylenecyclopropaneacetic acid (MCPA) inhibited SBCAD, isovaleryl-CoA dehydrogenase, short-chain acyl-CoA dehydrogenase and medium-chain acyl-CoA dehydrogenase, but not ACAD8. MCPA treatment of wild-type and PA HEK-293 cells caused a pronounced decrease in C3-carnitine. Furthermore, deletion of ACADSB in HEK-293 cells led to an equally strong decrease in C3-carnitine when compared to wild-type cells. Deletion of ECHS1 in HEK-293 cells caused a defect in lipoylation of the E2 component of the pyruvate dehydrogenase complex, which was not rescued by ACAD8 deletion. MCPA was able to rescue lipoylation in ECHS1 KO cells, but only in cells with prior ACAD8 deletion. SBCAD was not the sole ACAD responsible for this compensation, which indicates substantial promiscuity of ACADs in HEK-293 cells for the isobutyryl-CoA substrate. Substrate promiscuity appeared less prominent for 2-methylbutyryl-CoA at least in HEK-293 cells. We suggest that pharmacological inhibition of SBCAD to treat PA should be investigated further., (© 2023 SSIEM.)

In humans, a single enzyme 2-aminoadipic semialdehyde synthase (AASS) catalyses the initial two critical reactions in the lysine degradation pathway. This enzyme evolved to be a bifunctional enzyme with both lysine-2-oxoglutarate reductase (LOR) and saccharopine dehydrogenase domains (SDH). Moreover, AASS is a unique drug target for inborn errors of metabolism such as glutaric aciduria type 1 that arise from deficiencies downstream in the lysine degradation pathway. While work has been done to elucidate the SDH domain structurally and to develop inhibitors, neither has been done for the LOR domain. Here, we purify and characterize LOR and show that it is activated by alkylation of cysteine 414 by N-ethylmaleimide. We also provide evidence that AASS is rate-limiting upon high lysine exposure of mice. Finally, we present the crystal structure of the human LOR domain. Our combined work should enable future efforts to identify inhibitors of this novel drug target.

The macro- and microvascular complications of type 1 and 2 diabetes lead to increased disease severity and mortality. The receptor for advanced glycation end products (RAGE) can bind AGEs and multiple proinflammatory ligands that accumulate in diabetic tissues. Preclinical studies indicate that RAGE antagonists have beneficial effects on numerous complications of diabetes. However, these antagonists target the extracellular domains of RAGE, which bind distinct RAGE ligands at diverse sites in the immunoglobulin-like variable domain and two constant domains. The cytoplasmic tail of RAGE (ctRAGE) binds to the formin, Diaphanous-1 (DIAPH1), and this interaction is important for RAGE signaling. To comprehensively capture the breadth of RAGE signaling, we developed small-molecule antagonists of ctRAGE-DIAPH1 interaction, termed RAGE229. We demonstrated that RAGE229 is effective in suppressing RAGE-DIAPH1 binding, Förster resonance energy transfer, and biological activities in cellular assays. Using solution nuclear magnetic resonance spectroscopy, we defined the molecular underpinnings of the interaction of RAGE229 with RAGE. Through in vivo experimentation, we showed that RAGE229 assuaged short- and long-term complications of diabetes in both male and female mice, without lowering blood glucose concentrations. Last, the treatment with RAGE229 reduced plasma concentrations of TNF-α, IL-6, and CCL2/JE-MCP1 in diabetic mice, in parallel with reduced pathological and functional indices of diabetes-like kidney disease. Targeting ctRAGE-DIAPH1 interaction with RAGE229 mitigated diabetic complications in rodents by attenuating inflammatory signaling.

A quantitative deficiency of normally functioning insulin-producing pancreatic beta cells is a major contributor to all common forms of diabetes. This is the underlying premise for attempts to replace beta cells in people with diabetes by pancreas transplantation, pancreatic islet transplantation, and transplantation of beta cells or pancreatic islets derived from human stem cells. While progress is rapid and impressive in the beta cell replacement field, these approaches are expensive, and for transplant approaches, limited by donor organ availability. For these reasons, beta cell replacement will not likely become available to the hundreds of millions of people around the world with diabetes. Since the large majority of people with diabetes have some residual beta cells in their pancreata, an alternate approach to reversing diabetes would be developing pharmacologic approaches to induce these residual beta cells to regenerate and expand in a way that also permits normal function. Unfortunately, despite the broad availability of multiple classes of diabetes drugs in the current diabetes armamentarium, none has the ability to induce regeneration or expansion of human beta cells. Development of such drugs would be transformative for diabetes care around the world. This picture has begun to change. Over the past half-decade, a novel class of beta cell regenerative small molecules has emerged: the DYRK1A inhibitors. Their emergence has tremendous potential, but many areas of uncertainty and challenge remain. In this review, we summarize the accomplishments in the world of beta cell regenerative drug development and summarize areas in which most experts would agree. We also outline and summarize areas of disagreement or lack of unanimity, of controversy in the field, of obstacles to beta cell regeneration, and of challenges that will need to be overcome in order to establish human beta cell regenerative drug therapeutics as a clinically viable class of diabetes drugs., Competing Interests: The Icahn School of Medicine at Mount Sinai has filed patents on material discussed in this manuscript on behalf of some of the authors (PW, KK, RD, AG-O, AS). The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Wang, Karakose, Choleva, Kumar, DeVita, Garcia-Ocaña and Stewart.)

Both genetic predisposition and environmental factors appear to play a role in inflammatory bowel disease (IBD) development. Genetic studies in humans have linked the interleukin (IL)-23 signaling pathway with IBD, but the environmental factors contributing to disease have remained elusive. Here, we show that the azo dyes Red 40 and Yellow 6, the most abundant food colorants in the world, can trigger an IBD-like colitis in mice conditionally expressing IL-23, or in two additional animal models in which IL-23 expression was augmented. Increased IL-23 expression led to generation of activated CD4 + T cells that expressed interferon-γ and transferred disease to mice exposed to Red 40. Colitis induction was dependent on the commensal microbiota promoting the azo reduction of Red 40 and generation of a metabolite, 1-amino-2-naphthol-6-sulfonate sodium salt. Together these findings suggest that specific food colorants represent novel risk factors for development of colitis in mice with increased IL-23 signaling., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 Elsevier Inc. All rights reserved.)

According to the World Health Organization (WHO), 422 million people are suffering from diabetes worldwide. Current diabetes therapies are focused on optimizing blood glucose control to prevent long-term diabetes complications. Unfortunately, current therapies have failed to achieve glycemic targets in the majority of people with diabetes. In this context, regeneration of functional insulin-producing human β-cells in people with diabetes through the use of DYRK1A inhibitor drugs has recently received special attention. Several small molecule DYRK1A inhibitors have been identified that induce human β-cell proliferation in vitro and in vivo. Furthermore, DYRK1A inhibitors have also been shown to synergize β-cell proliferation with other classes of drugs, such as TGFβ inhibitors and GLP-1 receptor agonists. In this perspective, we review the status of DYRK1A as a therapeutic target for β-cell proliferation and provide perspectives on technical and scientific challenges for future translational development.

Cullin-RING (really intersting new gene) E3 ubiquitin ligases (CRLs) are the largest E3 family and direct numerous protein substrates for proteasomal degradation, thereby impacting a myriad of physiological and pathological processes including cancer. To date, there are no reported small-molecule inhibitors of the catalytic activity of CRLs. Here, we describe high-throughput screening and medicinal chemistry optimization efforts that led to the identification of two compounds, 33-11 and KH-4-43, which inhibit E3 CRL4 and exhibit antitumor potential. These compounds bind to CRL4's core catalytic complex, inhibit CRL4-mediated ubiquitination, and cause stabilization of CRL4's substrate CDT1 in cells. Treatment with 33-11 or KH-4-43 in a panel of 36 tumor cell lines revealed cytotoxicity. The antitumor activity was validated by the ability of the compounds to suppress the growth of human tumor xenografts in mice. Mechanistically, the compounds' cytotoxicity was linked to aberrant accumulation of CDT1 that is known to trigger apoptosis. Moreover, a subset of tumor cells was found to express cullin4 proteins at levels as much as 70-fold lower than those in other tumor lines. The low-cullin4-expressing tumor cells appeared to exhibit increased sensitivity to 33-11/KH-4-43, raising a provocative hypothesis for the role of low E3 abundance as a cancer vulnerability., Competing Interests: Competing interest statement: R.J.D., Z.-Q.P., K.W., K.Q.H., and M.M. are inventors on patent application 63/144,358 submitted by Icahn School of Medicine at Mount Sinai claiming inhibitors of Cullin-RING E3 ubiquitin ligase 4 (CRL4) to treat leukemia and other cancers., (Copyright © 2021 the Author(s). Published by PNAS.)

Cullin-RING E3 ubiquitin ligase 4 (CRL4) plays an essential role in cell cycle progression. Recent efforts using high throughput screening and follow up hit-to-lead studies have led to identification of small molecules 33-11 and KH-4-43 that inhibit E3 CRL4's core ligase complex and exhibit anticancer potential. This review provides: 1) an updated perspective of E3 CRL4, including structural organization, major substrate targets and role in cancer; 2) a discussion of the challenges and strategies for finding the CRL inhibitor; and 3) a summary of the properties of the identified CRL4 inhibitors as well as a perspective on their potential utility to probe CRL4 biology and act as therapeutic agents., Competing Interests: Competing Interest Statement R.J.D., Z.-Q.P., and K.W. are inventors on patent application 63/144,358 submitted by Icahn School of Medicine at Mount Sinai claiming inhibitors of Cullin-RING E3 ubiquitin ligase 4 (CRL4) to treat leukemia and other cancers.

Glutaric aciduria type 1 (GA1) is an inborn error of lysine degradation characterized by acute encephalopathy that is caused by toxic accumulation of lysine degradation intermediates. We investigated the efficacy of substrate reduction through inhibition of 2-aminoadipic semialdehyde synthase (AASS), an enzyme upstream of the defective glutaryl-CoA dehydrogenase (GCDH), in a cell line and mouse model of GA1. We show that loss of AASS function in GCDH-deficient HEK-293 cells leads to an approximately fivefold reduction in the established GA1 clinical biomarker glutarylcarnitine. In the GA1 mouse model, deletion of Aass leads to a 4.3-, 3.8-, and 3.2-fold decrease in the glutaric acid levels in urine, brain, and liver, respectively. Parallel decreases were observed in urine and brain 3-hydroxyglutaric acid levels, and plasma, urine, and brain glutarylcarnitine levels. These in vivo data demonstrate that the saccharopine pathway is the main source of glutaric acid production in the brain and periphery of a mouse model for GA1, and support the notion that pharmacological inhibition of AASS may represent an attractive strategy to treat GA1., (© 2020 SSIEM.)

Glutaric aciduria type 1 (GA1) is an inborn error of lysine degradation characterized by a specific encephalopathy that is caused by toxic accumulation of lysine degradation intermediates. Substrate reduction through inhibition of DHTKD1, an enzyme upstream of the defective glutaryl-CoA dehydrogenase, has been investigated as a potential therapy, but revealed the existence of an alternative enzymatic source of glutaryl-CoA. Here, we show that loss of DHTKD1 in glutaryl-CoA dehydrogenase-deficient HEK-293 cells leads to a 2-fold decrease in the established GA1 clinical biomarker glutarylcarnitine and demonstrate that oxoglutarate dehydrogenase (OGDH) is responsible for this remaining glutarylcarnitine production. We furthermore show that DHTKD1 interacts with OGDH, dihydrolipoyl succinyltransferase and dihydrolipoamide dehydrogenase to form a hybrid 2-oxoglutaric and 2-oxoadipic acid dehydrogenase complex. In summary, 2-oxoadipic acid is a substrate for DHTKD1, but also for OGDH in a cell model system. The classical 2-oxoglutaric dehydrogenase complex can exist as a previously undiscovered hybrid containing DHTKD1 displaying improved kinetics towards 2-oxoadipic acid., (© The Author(s) 2020. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Recently, we have shown that harmine induces β-cell proliferation both in vitro and in vivo, mediated via the DYRK1A-NFAT pathway. We explore structure-activity relationships of the 7-position of harmine for both DYRK1A kinase inhibition and β-cell proliferation based on our related previous structure-activity relationship studies of harmine in the context of diabetes and β-cell specific targeting strategies. 33 harmine analogs of the 7-position substituent were synthesized and evaluated for biological activity. Two novel inhibitors were identified which showed DYRK1A inhibition and human β-cell proliferation capability. The DYRK1A inhibitor, compound 1-2b , induced β-cell proliferation half that of harmine at three times higher concentration. From these studies we can draw the inference that 7-position modification is limited for further harmine optimization focused on β-cell proliferation and cell-specific targeting approach for diabetes therapeutics.

Recently, our group identified that harmine is able to induce β-cell proliferation both in vitro and in vivo, mediated via the DYRK1A-NFAT pathway. Since, harmine suffers from a lack of selectivity, both against other kinases and CNS off-targets, we therefore sought to expand structure-activity relationships for harmine's DYRK1A activity, to enhance selectivity for off-targets while retaining human β-cell proliferation activity. We carried out optimization of the 9- N -position of harmine to synthesize 29 harmine-based analogs. Several novel inhibitors showed excellent DYRK1A inhibition and human β-cell proliferation capability. An optimized DYRK1A inhibitor, 2-2c , was identified as a novel, efficacious in vivo lead candidate. 2-2c also demonstrates improved selectivity for kinases and CNS off-targets, as well as in vivo efficacy for β-cell proliferation and regeneration at lower doses than harmine. Collectively, these findings demonstrate that 2-2c is a much improved in vivo lead candidate as compared to harmine for the treatment of diabetes.

Small molecule inhibitors of dual specificity, tyrosine phosphorylation-regulated kinase 1A (DYRK1A), including harmine and others, are able to drive human β cell regeneration. While DYRK1A is certainly a target of this class, whether it is the only or the most important target is uncertain. Here, we employ a combined pharmacologic and genetic approach to refine the potential mitogenic targets of the DYRK1A inhibitor family in human islets. A combination of human β cell RNA sequencing, DYRK1A inhibitor kinome screens, pharmacologic inhibitors, and targeted silencing of candidate genes confirms that DYRK1A is a central target. Surprisingly, however, DYRK1B also proves to be an important target: silencing DYRK1A results in an increase in DYRK1B. Simultaneous silencing of both DYRK1A and DYRK1B yields greater β cell proliferation than silencing either individually. Importantly, other potential kinases, such as the CLK and the GSK3 families, are excluded as important harmine targets. Finally, we describe adenoviruses that are able to silence up to 7 targets simultaneously. Collectively, we report that inhibition of both DYRK1A and DYRK1B is required for induction of maximal rates of human β cell proliferation, and we provide clarity for future efforts in structure-based drug design for human β cell regenerative drugs.

Nonselective antagonists of voltage-gated sodium (Na V ) channels have been long used for the treatment of epilepsies. The efficacy of these drugs is thought to be due to the block of sodium channels on excitatory neurons, primarily Na V 1.6 and Na V 1.2. However, these currently marketed drugs require high drug exposure and suffer from narrow therapeutic indices. Selective inhibition of Na V 1.6, while sparing Na V 1.1, is anticipated to provide a more effective and better tolerated treatment for epilepsies. In addition, block of Na V 1.2 may complement the anticonvulsant activity of Na V 1.6 inhibition. We discovered a novel series of aryl sulfonamides as CNS-penetrant, isoform-selective Na V 1.6 inhibitors, which also displayed potent block of Na V 1.2. Optimization focused on increasing selectivity over Na V 1.1, improving metabolic stability, reducing active efflux, and addressing a pregnane X-receptor liability. We obtained compounds 30-32 , which produced potent anticonvulsant activity in mouse seizure models, including a direct current maximal electroshock seizure assay.

The derivatization of pharmaceuticals is a core activity in the discovery and development of new medicines. Late-stage functionalization via modern CH functionalization chemistry has emerged as a powerful technique with which to diversify advanced pharmaceutical intermediates. We report herein a case study in late-stage functionalization towards the development of a new class of indazole-based mineralocorticoid receptor antagonists (MRA). An effort to modify the electronics of the core indazole heterocycle inspired the use of modern CH borylation chemistry. New reactivity patterns were revealed and studied computationally. Ultimately, a de novo synthesis delivered a key 6-fluoroindazole compound 26, a potent MRA with excellent metabolic stability., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Previously disclosed benzimidazole-based DGAT1 inhibitors containing a cyclohexane carboxylic acid moiety suffer from isomerization at the alpha position of the carboxylic acid group, generating active metabolites which exhibit DGAT1 inhibition comparable to the corresponding parent compounds. In this report, we describe the design, synthesis and profiling of benzimidazole-based DGAT1 inhibitors with a [3.1.0] bicyclohexane carboxylic acid moiety. Our results show that single isomer 3A maintains in vitro and in vivo inhibition against DGAT1. In contrast to previous lead compounds, 3A does not undergo isomerization during in vitro hepatocyte incubation study or in vivo mouse study., (Copyright © 2019. Published by Elsevier Ltd.)

Covalent inhibitors can obtain optimal selectivity and extended residence time. In this issue of Cell Chemical Biology, Shraga et al. (2019) take a comprehensive computational and experimental approach to modulate the JNK-Jun pathway through design of MKK7 covalent inhibitors. This study highlights a promising and emerging strategy for therapeutic discovery., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

DYRK1A has been implicated as an important drug target in various therapeutic areas, including neurological disorders and oncology. DYRK1A has more recently been shown to be involved in pathways regulating human β-cell proliferation, thus making it a potential therapeutic target for both Type 1 and Type 2 diabetes. Our group, using a high-throughput phenotypic screen, identified harmine that is able to induce β-cell proliferation both in vitro and in vivo. Since harmine has suboptimal kinase selectivity, we sought to expand structure-activity relationships for harmine's DYRK1A activity, to enhance selectivity, while retaining human β-cell proliferation capability. We carried out the optimization of the 1-position of harmine and synthesized 15 harmine analogues. Six compounds showed excellent DYRK1A inhibition with IC 50 in the range of 49.5-264 nM. Two compounds, 2-2 and 2-8, exhibited excellent human β-cell proliferation at doses of 3-30 μM, and compound 2-2 showed improved kinase selectivity as compared to harmine.

The Dual-Specificity Tyrosine Phosphorylation-Regulated Kinase 1A (DYRK1A) is an enzyme that has been implicated as an important drug target in various therapeutic areas, including neurological disorders (Down syndrome, Alzheimer's disease), oncology, and diabetes (pancreatic β-cell expansion). Current small molecule DYRK1A inhibitors are ATP-competitive inhibitors that bind to the kinase in an active conformation. As a result, these inhibitors are promiscuous, resulting in pharmacological side effects that limit their therapeutic applications. None are in clinical trials at this time. In order to identify a new DYRK1A inhibitor scaffold, we constructed a homology model of DYRK1A in an inactive, DFG-out conformation. Virtual screening of 2.2 million lead-like compounds from the ZINC database, followed by in vitro testing of selected 68 compounds revealed 8 hits representing 5 different chemical classes. We chose to focus on one of the hits from the computational screen, thiadiazine 1 which was found to inhibit DYRK1A with IC 50 of 9.41 μM (K d  = 7.3 μM). Optimization of the hit compound 1, using structure-activity relationship (SAR) analysis and in vitro testing led to the identification of potent thiadiazine analogs with significantly improved binding as compared to the initial hit (K d  = 71-185 nM). Compound 3-5 induced human β-cell proliferation at 5 μM while showing selectivity for DYRK1A over DYRK1B and DYRK2 at 10 μM. This newly developed DYRK1A inhibitor scaffold with unique kinase selectivity profiles has potential to be further optimized as novel therapeutics for diabetes., (Copyright © 2018. Published by Elsevier Masson SAS.)

S119 was a top hit from an ultrahigh throughput screen performed to identify novel inhibitors of influenza virus replication. It showed a potent antiviral effect (50% inhibitory concentration, IC 50 = 20 nM) and no detectable cytotoxicity (50% cytotoxic concentration, CC 50 > 500 μM) to yield a selectivity index greater than 25 000. Upon investigation, we found that S119 selected for resistant viruses carrying mutations in the viral nucleoprotein (NP). These resistance mutations highlight a likely S119 binding site overlapping with but not identical to that found for the compound nucleozin. Mechanism of action studies revealed that S119 affects both the oligomerization state and cellular localization of the NP protein which has an impact on viral transcription, replication, and protein expression. Through a hit-to-lead structure-activity relationship (SAR) study, we found an analog of S119, named S119-8, which had increased breadth of inhibition against influenza A and B viruses accompanied by only a small loss in potency. Finally, in vitro viral inhibition assays showed a synergistic relationship between S119-8 and oseltamivir when they were combined, indicating the potential for future drug cocktails.

Miniaturization and parallel processing play an important role in the evolution of many technologies. We demonstrate the application of miniaturized high-throughput experimentation methods to resolve synthetic chemistry challenges on the frontlines of a lead optimization effort to develop diacylglycerol acyltransferase (DGAT1) inhibitors. Reactions were performed on ∼1 mg scale using glass microvials providing a miniaturized high-throughput experimentation capability that was used to study a challenging S N Ar reaction. The availability of robust synthetic chemistry conditions discovered in these miniaturized investigations enabled the development of structure-activity relationships that ultimately led to the discovery of soluble, selective, and potent inhibitors of DGAT1.

The receptor for advanced glycation endproducts (RAGE) binds diverse ligands linked to chronic inflammation and disease. NMR spectroscopy and x-ray crystallization studies of the extracellular domains of RAGE indicate that RAGE ligands bind by distinct charge- and hydrophobicity-dependent mechanisms. The cytoplasmic tail (ct) of RAGE is essential for RAGE ligand-mediated signal transduction and consequent modulation of gene expression and cellular properties. RAGE signaling requires interaction of ctRAGE with the intracellular effector, mammalian diaphanous 1 or DIAPH1. We screened a library of 58,000 small molecules and identified 13 small molecule competitive inhibitors of ctRAGE interaction with DIAPH1. These compounds, which exhibit in vitro and in vivo inhibition of RAGE-dependent molecular processes, present attractive molecular scaffolds for the development of therapeutics against RAGE-mediated diseases, such as those linked to diabetic complications, Alzheimer's disease, and chronic inflammation, and provide support for the feasibility of inhibition of protein-protein interaction (PPI).

Decreased activity and expression of the cardiac sarcoplasmic reticulum calcium ATPase (SERCA2a), a critical pump regulating calcium cycling in cardiomyocyte, are hallmarks of heart failure. We have previously described a role for the small ubiquitin-like modifier type 1 (SUMO-1) as a regulator of SERCA2a and have shown that gene transfer of SUMO-1 in rodents and large animal models of heart failure restores cardiac function. Here, we identify and characterize a small molecule, N106, which increases SUMOylation of SERCA2a. This compound directly activates the SUMO-activating enzyme, E1 ligase, and triggers intrinsic SUMOylation of SERCA2a. We identify a pocket on SUMO E1 likely to be responsible for N106's effect. N106 treatment increases contractile properties of cultured rat cardiomyocytes and significantly improves ventricular function in mice with heart failure. This first-in-class small-molecule activator targeting SERCA2a SUMOylation may serve as a potential therapeutic strategy for treatment of heart failure.

We report the discovery of a novel series of DGAT1 inhibitors in the benzimidazole class with a piperdinyl-oxy-cyclohexanecarboxylic acid moiety. This novel series possesses significantly improved selectivity against the A2A receptor, no ACAT1 off-target activity at 10 μM, and higher aqueous solubility and free fraction in plasma as compared to the previously reported pyridyl-oxy-cyclohexanecarboxylic acid series. In particular, 5B was shown to possess an excellent selectivity profile by screening it against a panel of more than 100 biological targets. Compound 5B significantly reduces lipid excursion in LTT in mouse and rat, demonstrates DGAT1 mediated reduction of food intake and body weight in mice, is negative in a 3-strain Ames test, and appears to distribute preferentially in the liver and the intestine in mice. We believe this lead series possesses significant potential to identify optimized compounds for clinical development.

We report herein the identification of MK-4409, a potent and selective fatty acid amide hydrolase (FAAH) inhibitor. Starting from a high throughput screening (HTS) hit, medicinal chemistry efforts focused on optimizing of FAAH inhibition in vitro potency, improving the pharmacokinetic (PK) profile, and increasing in vivo efficacy in rodent inflammatory and neuropathic pain assays.

Inhibition of Diacylglycerol O-acyltransferase 1 (DGAT1) has been a mechanism of interest for metabolic disorders. DGAT1 inhibition has been shown to be a key regulator in an array of metabolic pathways; however, based on the DGAT1 KO mouse phenotype the anticipation is that pharmacological inhibition of DGAT1 could potentially lead to skin related adverse effects. One of the aims in developing small molecule DGAT1 inhibitors that target key metabolic tissues is to avoid activity on skin-localized DGAT1 enzyme. In this report we describe a modeling-based approach to identify molecules with physical properties leading to differential exposure distribution. In addition, we demonstrate histological and RNA based biomarker approaches that can detect sebaceous gland atrophy pre-clinically that could be used as potential biomarkers in a clinical setting.

Diacylglycerol O-acyltransferase 1 (DGAT1) has recently become a highly interesting target for metabolic disorders as well as for hepatitis C virus (HCV). DGAT1 processes diacylglycerol to triglycerides in the final step of resynthesis for the absorption of fat across the intestine. Pharmaceutical companies have developed many novel inhibitors of DGAT1, several of which have reached the clinic. Proof of target engagement was achieved with the observation of reduced triglycerides upon treatment of humans with DGAT1 inhibitors; however, there were gastrointestinal adverse events such as nausea, diarrhea, and vomiting. These adverse events have been reported with multiple compounds and are possibly linked to the target because of the recent identification of a human cohort deficient in DGAT1. Clinical studies are continuing in a trial to treat patients with an orphan indication for familial chylomicronemia. The full potential of DGAT1 as a therapeutic target will need to overcome observed clinical adverse events, which are possibly mechanism based. The widespread use of DGAT1 inhibitors will ultimately depend upon a better understanding of how to improve the GI tolerability of these agents.

Hydroisoindoline 2 has been previously identified as a potent, brain-penetrant NK1 receptor antagonist with a long duration of action and improved profile of CYP3A4 inhibition and induction compared to aprepitant. However, compound 2 is predicted, based on data in preclinical species, to have a human half-life longer than 40 h and likely to have drug-drug-interactions (DDI), as 2 is a victim of CYP3A4 inhibition caused by its exclusive clearance pathway via CYP3A4 oxidation in humans. We now report 2-[(3aR,4R,5S,7aS)-5-{(1S)-1-[3,5-bis(trifluoromethyl)phenyl]-2-hydroxyethoxy}-4-(2-methylphenyl)octahydro-2H-isoindol-2-yl]-1,3-oxazol-4(5H)-one (3) as a next generation NK1 antagonist that possesses an additional clearance pathway through glucuronidation in addition to that via CYP3A4 oxidation. Compound 3 has a much lower propensity for drug-drug interactions and a reduced estimated human half-life consistent with once daily dosing. In preclinical species, compound 3 has demonstrated potency, brain penetration, and a safety profile similar to 2, as well as excellent pharmacokinetics.

We report the design and synthesis of a series of novel DGAT1 inhibitors in the benzimidazole class with a pyridyl-oxy-cyclohexanecarboxylic acid moiety. In particular, compound 11A is a potent DGAT1 inhibitor with excellent selectivity against ACAT1. Compound 11A significantly reduces triglyceride excursion in lipid tolerance tests (LTT) in both mice and dogs at low plasma exposure. An in vivo study in mice with des-fluoro analogue 10A indicates that this series of compounds appears to distribute in intestine preferentially over plasma. The propensity to target intestine over plasma could be advantageous in reducing potential side effects since lower circulating levels of drug are required for efficacy. However, in the preclinical species, compound 11A undergoes cis/trans epimerization in vivo, which could complicate further development due to the presence of an active metabolite.

We report herein the discovery of a fatty acid amide hydrolase (FAAH) positron emission tomography (PET) tracer. Starting from a pyrazole lead, medicinal chemistry efforts directed toward reducing lipophilicity led to the synthesis of a series of imidazole analogues. Compound 6 was chosen for further profiling due to its appropriate physical chemical properties and excellent FAAH inhibition potency across species. [(11)C]-6 (MK-3168) exhibited good brain uptake and FAAH-specific signal in rhesus monkeys and is a suitable PET tracer for imaging FAAH in the brain.

The use of stable isotopically labeled substrates and analysis by mass spectrometry have provided substantial insight into rates of synthesis, disposition, and utilization of lipids in vivo. The information to be gained from such studies is of particular benefit to therapeutic research where the underlying causes of disease may be related to the production and utilization of lipids. When studying biology through the use of isotope tracers, care must be exercised in interpreting the data to ensure that any response observed can truly be interpreted as biological and not as an artifact of the experimental design or a dilutional effect on the isotope. We studied the effects of dosing route and tracer concentration on the mass isotopomer distribution profile as well as the action of selective inhibitors of microsomal tri-glyceride transfer protein (MTP) in mice and diacylglycerol acyltransferase 1 (DGAT1) in nonhuman primates, using a stable-isotopically labeled approach. Subjects were treated with inhibitor and subsequently given a dose of uniformly ¹³C-labeled oleic acid. Samples were analyzed using a rapid LC-MS technique, allowing the effects of the intervention on the assembly and disposition of triglycerides, cholesteryl esters, and phospholipids to be determined in a single 3 min run from just 10 μl of plasma.

Ezetimibe (Zetia®), a cholesterol-absorption inhibitor (CAI) approved by the FDA for the treatment of hypercholesterolemia, is believed to target the intestine protein Niemann-Pick C1-Like 1 (NPC1L1) or its pathway. A spiroimidazolidinone NPC1L1 inhibitor identified by virtual screening showed moderate binding activity but was not efficacious in an in vivo rodent model of cholesterol absorption. Synthesis of analogs established the structure-activity relationships for binding activity, and resulted in compounds with in vivo efficacy, including 24, which inhibited plasma cholesterol absorption by 67% in the mouse, thereby providing proof-of-concept that non-β-lactams can be effective CAIs., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

Previously, we had disclosed a novel class of hNK(1) antagonists based on the 5,5-fused pyrrolidine core. These compounds displayed subnanomolar hNK(1) affinity along with good efficacy in a gerbil foot-tapping (GFT) model, but unfortunately they had low to moderate functional antagonist (IP-1) activity. To elaborate on the SAR of this class of hNK(1) compounds and to improve functional activity, we have designed and synthesized a new class of hNK(1) antagonist with a third fused ring. Compared to the 5,5-fused pyrrolidine class, these 5,5,5-fused tricyclic hNK(1) antagonists maintain subnanomolar hNK(1) binding affinity with highly improved functional IP-1 activity (<10% SP remaining). A fused tricyclic methyl, hydroxyl geminally substituted pyrrolizinone (compound 20) had excellent functional IP (<2% SP remaining), hNK(1) binding affinity, off-target selectivity, pharmacokinetic profile and in vivo activity. Complete inhibition of agonist activity was observed at both 0 and 24h in the gerbil foot-tapping model with an ID(50) of 0.02 mpk at both 0 and 24h, respectively., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

A new class of potent NK(1) receptor antagonists with a tetrahydroindolizinone core has been identified. This series of compounds demonstrated improved functional activities as compared to previously identified 5,5-fused pyrrolidine lead structures. SAR at the 7-position of the tetrahydroindolizinone core is discussed in detail. A number of compounds displayed high NK(1) receptor occupancy at both 1 h and 24 h in a gerbil foot tapping model. Compound 40 has high NK(1) binding affinity, good selectivity for other NK receptors and promising in vivo properties. It also has clean P(450) inhibition and hPXR induction profiles., (2010 Elsevier Ltd. All rights reserved.)

Previous work on human NK(1) (hNK(1)) antagonists in which the core of the structure is a 5,5-fused pyrrolizinone has been disclosed. The structural-activity-relationship studies on simple alpha- and beta-substituted compounds of this series provided several potent and bioavailable hNK(1) antagonists that displayed excellent brain penetration as observed by their good efficacy in the gerbil foot-tapping (GFT) model assay. Several of these compounds exhibited 100% inhibition of the foot-tapping response at 0.1 and 24h with ID(50)'s of less than 1 mpk. One particular alpha-substituted compound (2b) had an excellent pharmacokinetic profile across preclinical species with reasonable in vivo functional activity and minimal ancillary activity., (Copyright 2010 Elsevier Ltd. All rights reserved.)

During our effort to design a receptor binding assay to aid in the elucidation of the molecular mechanism of ezetimibe, we prepared a sulfur-35 containing radioligand which exhibits improved potency over the glucuronide conjugate of ezetimibe in both native enterocyte brush border membranes and membranes from cells expressing recombinant NPC1L1. Herein, we describe the different synthetic strategies which were used to obtain this compound as well as its effectiveness in the aforementioned assay.

A series of spiroimidazolidinone NPC1L1 inhibitors was discovered by virtual screening of the Merck corporate sample repository using 3D-similarity-based screening. Selection of 330 compounds for testing in an in vitro NPC1L1 binding assay yielded six hits in six distinct chemical series. Follow-up 2D similarity searching yielded several sub- to low-micromolar leads; among these was spiroimidazolidinone 10, with an IC(50) of 2.5 microM. Compound 10 provided a useful scaffold to initiate a medicinal chemistry campaign.

3-[(3aR,4R,5S,7aS)-5-{(1R)-1-[3,5-bis(trifluoromethyl)phenyl]ethoxy}-4-(4-fluorophenyl)octahydro-2H-isoindol-2-yl]cyclopent-2-en-1-one (17) is a high affinity, brain-penetrant, hydroisoindoline-based neurokinin-1 (NK(1)) receptor antagonist with a long central duration of action in preclinical species and a minimal drug-drug interaction profile. Positron emission tomography (PET) studies in rhesus showed that this compound provides 90% NK(1) receptor blockade in rhesus brain at a plasma level of 67 nM, which is about 10-fold more potent than aprepitant, an NK(1) antagonist marketed for the prevention of chemotherapy-induced and postoperative nausea and vomiting (CINV and PONV). The synthesis of this enantiomerically pure compound containing five stereocenters includes a Diels-Alder condensation, one chiral separation of the cyclohexanol intermediate, an ether formation using a trichloroacetimidate intermediate, and bis-alkylation to form the cyclic amine.