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Allosteric modulators of the metabotropic glutamate receptor subtype 5 (mGlu5) have exciting potential as therapeutic agents for multiple brain disorders. Translational studies with mGlu5 modulators have relied on mGlu5 allosteric site positron emission tomography (PET) radioligands to assess receptor occupancy in the brain. However, recent structural and modeling studies suggest that closely related mGlu5 allosteric modulators can bind to overlapping but not identical sites, which could complicate interpretation of in vivo occupancy data, even when PET ligands and drug leads are developed from the same chemical scaffold. We now report that systemic administration of the novel mGlu5 positive allosteric modulator VU0092273 displaced the structurally related mGlu5 PET ligand, [18F]FPEB, with measures of in vivo occupancy that closely aligned with its in vivo efficacy. In contrast, a close analog of VU0092273 and [18F]FPEB, VU0360172, provided robust efficacy in rodent models in the absence of detectable occupancy. Furthermore, a structurally unrelated mGlu5 negative allosteric modulator, VU0409106, displayed measures of in vivo occupancy that correlated well with behavioral effects, despite the fact that VU0409106 is structurally unrelated to [18F]FPEB. Interestingly, all three compounds inhibit radioligand binding to the prototypical MPEP/FPEB allosteric site in vitro. However, VU0092273 and VU0409106 bind to this site in a fully competitive manner, whereas the interaction of VU0360172 is noncompetitive. Thus, while close structural similarity between PET ligands and drug leads does not circumvent issues associated with differential binding to a given target, detailed molecular pharmacology analysis accurately predicts utility of ligand pairs for in vivo occupancy studies. [ABSTRACT FROM AUTHOR]

Winning the relay: The first total synthesis of stemaphylline N‐oxide has been completed utilizing a bistandem relay ring‐closing‐metathesis (RRCM) strategy, necessitated by the conformation of the requisite tetraene. This effort also gave unnatural 9a‐epi‐stemaphylline and 9a‐epi‐stemaphylline N‐oxide (see scheme). [ABSTRACT FROM AUTHOR]

Interferon (IFN)-stimulated gene 15 (ISG15), a ubiquitin-like protein, is covalently conjugated to host immune proteins such as MDA5 and IRF3 in a process called ISGylation, thereby promoting type I IFN induction to limit the replication of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). However, whether SARS-CoV-2 proteins can be directly targeted for ISGylation remains elusive. In this study, we identified the nucleocapsid (N) protein of SARS-CoV-2 as a major substrate of ISGylation catalyzed by the host E3 ligase HERC5; however, N ISGylation is readily removed through deISGylation by the papain-like protease (PLpro) activity of NSP3. Mass spectrometry analysis identified that the N protein undergoes ISGylation at four lysine residues (K266, K355, K387, and K388), and mutational analysis of these sites in the context of a SARS-CoV-2 replicon (N-4KR) abolished N ISGylation and alleviated ISGylation-mediated inhibition of viral RNA synthesis. Furthermore, our results indicated that HERC5 targets preferentially phosphorylated N protein for ISGylation to regulate its oligomeric assembly. These findings reveal a novel mechanism by which the host ISGylation machinery directly targets SARS-CoV-2 proteins to restrict viral replication and illuminate how an intricate interplay of host (HERC5) and viral (PLpro) enzymes coordinates viral protein ISGylation and thereby regulates virus replication. IMPORTANCE The role of protein ISGylation in regulating host cellular processes has been studied extensively; however, how ISG15 conjugation influences the activity of viral proteins, particularly coronaviral proteins, is largely unknown. Our study uncovered that the nucleocapsid (N) protein of SARS-CoV-2 is ISGylated by the HERC5 ISGylation machinery and that this modification impedes the functional assembly of N into oligomers ultimately inhibiting viral RNA synthesis. This antiviral restriction mechanism is antagonized by the PLpro deISGylation activity of SARS-CoV-2 NSP3. This study deepens our understanding of SARS-CoV-2 protein regulation by posttranslational modifications and may open new avenues for designing antiviral strategies for COVID-19. [ABSTRACT FROM AUTHOR]

The bat coronavirus HKU4 belongs to the same 2c lineage as that of the deadly Middle East Respiratory Syndrome coronavirus (MERS-CoV) and shows high sequence similarity, therefore potentiating a threat to the human population through a zoonotic shift or ‘spill over’ event. To date, there are no effective vaccines or antiviral treatments available that are capable of limiting the pathogenesis of any human coronaviral infection. An attractive target for the development of anti-coronaviral therapeutics is the 3C-like protease (3CL pro ), which is essential for the progression of the coronaviral life cycle. Herein, we report the screening results of a small, 230-member peptidomimetic library against HKU4-CoV 3CL pro and the identification of 43 peptidomimetic compounds showing good to excellent inhibitory potency of HKU4-CoV 3CL pro with IC 50 values ranging from low micromolar to sub-micromolar. We established structure–activity relationships (SARs) describing the important ligand-based features required for potent HKU4-CoV 3CL pro inhibition and identified a seemingly favored peptidic backbone for HKU4-CoV 3CL pro inhibition. To investigate this, a molecular sub-structural analysis of the most potent HKU4-CoV 3CL pro inhibitor was accomplished by the synthesis and testing of the lead peptidomimetic inhibitor’s sub-structural components, confirming the activity of the favored backbone ( 22A ) identified via SAR analysis. In order to elucidate the structural reasons for such potent HKU4-CoV 3CL pro inhibition by the peptidomimetics having the 22A backbone, we determined the X-ray structures of HKU4-CoV 3CL pro in complex with three peptidomimetic inhibitors. Sequence alignment of HKU4-CoV 3CL pro , and two other lineage C Betacoronaviruses 3CL pro ’s, HKU5-CoV and MERS-CoV 3CL pro , show that the active site residues of HKU4-CoV 3CL pro that participate in inhibitor binding are conserved in HKU5-CoV and MERS-CoV 3CL pro . Furthermore, we assayed our most potent HKU4-CoV 3CL pro inhibitor for inhibition of HKU5-CoV 3CL pro and found it to have sub-micromolar inhibitory activity (IC 50 = 0.54 ± 0.03 μM). The X-ray structures and SAR analysis reveal critical insights into the structure and inhibition of HKU4-CoV 3CL pro , providing fundamental knowledge that may be exploited in the development of anti-coronaviral therapeutics for coronaviruses emerging from zoonotic reservoirs. [ABSTRACT FROM AUTHOR]

Chronic elevation of plasma free fatty acid (FFA) levels is commonly associated with obesity, type 2 diabetes, cardiovascular disease and some cancers. Experimental evidence indicates FFA and their metabolites contribute to disease development through lipotoxicity. Previously, we identified a specific fatty acid transport inhibitor CB16.2, a.k.a. Lipofermata, using high throughput screening methods. In this study, efficacy of transport inhibition was measured in four cell lines that are models for myocytes (mmC2C12), pancreatic β-cells (rnINS-1E), intestinal epithelial cells (hsCaco-2), and hepatocytes (hsHepG2), as well as primary human adipocytes. The compound was effective in inhibiting uptake with IC 50 s between 3 and 6 μM for all cell lines except human adipocytes (39 μM). Inhibition was specific for long and very long chain fatty acids but had no effect on medium chain fatty acids (C6–C10), which are transported by passive diffusion. Derivatives of Lipofermata were evaluated to understand structural contributions to activity. Lipofermata prevented palmitate-mediated oxidative stress, induction of BiP and CHOP, and cell death in a dose-dependent manner in hsHepG2 and rnINS-1E cells, suggesting it will prevent induction of fatty acid-mediated cell death pathways and lipotoxic disease by channeling excess fatty acids to adipose tissue and away from liver and pancreas. Importantly, mice dosed orally with Lipofermata were not able to absorb 13 C-oleate demonstrating utility as an inhibitor of fatty acid absorption from the gut. [ABSTRACT FROM AUTHOR]

Summary Schizophrenia is associated with disruptions in N -methyl-D-aspartate glutamate receptor subtype (NMDAR)-mediated excitatory synaptic signaling. The metabotropic glutamate receptor subtype 5 (mGlu 5 ) is a closely associated signaling partner with NMDARs and regulates NMDAR function in forebrain regions implicated in the pathology of schizophrenia. Efficacy of mGlu 5 positive allosteric modulators (PAMs) in animal models of psychosis and cognition was previously attributed to potentiation of NMDAR function. To directly test this hypothesis, we identified VU0409551 as a novel mGlu 5 PAM that exhibits distinct stimulus bias and selectively potentiates mGlu 5 coupling to G αq -mediated signaling but not mGlu 5 modulation of NMDAR currents or NMDAR-dependent synaptic plasticity in the rat hippocampus. Interestingly, VU0409551 produced robust antipsychotic-like and cognition-enhancing activity in animal models. These data provide surprising new mechanistic insights into the actions of mGlu 5 PAMs and suggest that modulation of NMDAR currents is not critical for in vivo efficacy. Video Abstract [ABSTRACT FROM AUTHOR]

Background: Metabotropic glutamate receptor subtype 5 (mGlu5) activators have emerged as a novel approach to the treatment of schizophrenia. Positive allosteric modulators (PAMs) of mGlu5 have generated tremendous excitement and fueled major drug discovery efforts. Although mGlu5 PAMs have robust efficacy in preclinical models of schizophrenia, preliminary reports suggest that these compounds may induce seizure activity. Prototypical mGlu5 PAMs do not activate mGlu5 directly but selectively potentiate activation of mGlu5 by glutamate. This mechanism may be critical to maintaining normal activity-dependence of mGlu5 activation and achieving optimal in vivo effects. Methods: Using specially engineered mGlu5 cell lines incorporating point mutations within the allosteric and orthosteric binding sites, as well as brain slice electrophysiology and in vivo electroencephalography and behavioral pharmacology, we found that some mGlu5 PAMs have intrinsic allosteric agonist activity in the absence of glutamate. Results: Both in vitro mutagenesis and in vivo pharmacology studies demonstrate that VU0422465 is an agonist PAM that induces epileptiform activity and behavioral convulsions in rodents. In contrast, VU0361747, an mGlu5 PAMs optimized to eliminate allosteric agonist activity, has robust in vivo efficacy and does not induce adverse effects at doses that yield high brain concentrations. Conclusions: Loss of the absolute dependence of mGlu5 PAMs on glutamate release for their activity can lead to severe adverse effects. The finding that closely related mGlu5 PAMs can differ in their intrinsic agonist activity provides critical new insights that is essential for advancing these molecules through clinical development for treatment of schizophrenia. [ABSTRACT FROM AUTHOR]

Introduction: WDR5 is an epigenetic scaffolding protein that has attracted significant interest as an anti-cancer drug target, especially in MLL-rearranged leukemias. The most druggable 'WIN-site' on WDR5, which tethers WDR5 to chromatin, has been successfully targeted with multiple classes of exquisitely potent small-molecule protein-protein interaction inhibitors. Earlier progress has also been made on the development of WDR5 degraders and inhibitors at the 'WBM-site' on the opposite face of WDR5., Areas Covered: Based on an international survey of the patent literature using SciFinder from 2016-2024, herein we provide a comprehensive account of the chemical matter targeting WDR5, with a particular focus on proprietary compounds that are underreported in the existing academic literature. Our survey illuminates challenges for the field to overcome: a broad lack of chemical diversity, confusion about the molecular mechanism of WIN-site inhibitors, a paucity of brain-penetrant scaffolds despite emerging evidence of activity in brain cancers, sparse pharmacokinetic, metabolic, and disposition characterization, and the absence of safety or efficacy data in humans., Expert Opinion: It is our opinion that the best-in-class WIN-site inhibitors (from the imidazole class) merit advancement into clinical testing, likely against leukemia, which should provide much-needed clarity about the exciting but unproven potential of WDR5 as a next-generation therapeutic target.

Herein we detail the first disclosure of VU0467319 (VU319), an M 1 Positive Allosteric Modulator (PAM) clinical candidate that successfully completed a Phase I Single Ascending Dose (SAD) clinical trial. VU319 ( 16 ) is a moderately potent M 1 PAM (M 1 PAM EC 50 = 492 nM ± 2.9 nM, 71.3 ± 9.9% ACh Max), with minimal M 1 agonism (EC 50 > 30 μM), that displayed high CNS penetration ( K p s > 0.67 and K p,uu s > 0.9) and multispecies pharmacokinetics permissive of further development. Based on robust efficacy in multiple preclinical models of cognition, an ancillary pharmacology profile devoid of appreciable off-target activities, and a lack of cholinergic adverse effects (AEs) in rats, dogs and nonhuman primates, VU319 advanced into IND-enabling studies. After completing 4-week rat and dog GLP toxicology without AEs, including absence of cholinergic effects, the first in human Phase I SAD clinical trial of VU319 (NCT03220295) was performed at Vanderbilt, where a similar lack of adverse effects, including absence of cholinergic effects was noted. Moreover, signals of target engagement were seen at the highest dose tested. Thus, VU319 demonstrated the feasibility of achieving selective targeting of central M 1 muscarinic receptors without eliciting cholinergic AEs that have plagued other drugs targeting CNS cholinergic neurotransmission.

Purpose: There are no effective treatment options for patients with aggressive epithelioid hemangioendothelioma (EHE) driven by the TAZ-CAMTA1 (TC) fusion gene. Here, we aimed to understand the regulation of TC using pharmacologic tools and identify vulnerabilities that can potentially be exploited for the treatment of EHE., Experimental Design: TC is a transcriptional coregulator; we hypothesized that compounds that reduce TC nuclear levels, either through translocation of TC to the cytoplasm, or through degradation, would render TC less oncogenic. TC localization was monitored using immunofluorescence in an EHE tumor cell line. Two target-selective libraries were used to identify small molecules that reduce TC localization in the nucleus. The ability of the shortlisted hits to affect cell viability, apoptosis, and tumorigenesis was also evaluated., Results: Basal TC remained "immobile" in the nucleus; administration of cyclin-dependent kinase (CDK) inhibitors such as CGP60474 and dinaciclib (Dina) mobilized TC. "Mobile" TC shuttled between the nucleus and cytoplasm; however, it was eventually degraded through proteasomes. This dramatically suppressed the levels of TC-regulated transcripts and cell viability, promoted apoptosis, and reduced the area of metastatic lesions in the allograft model of EHE. We specifically identified that the inhibition of CDK9, a transcriptional CDK, destabilizes TC., Conclusions: The CDK inhibitor Dina exhibited antitumorigenic properties both in vitro and in vivo in EHE models. Dina has been rigorously tested in clinical trials and displayed an acceptable toxicity profile. Therefore, there is a potential therapeutic window for repurposing Dina for the treatment of EHE., (©2024 American Association for Cancer Research.)

Interferon (IFN)-stimulated gene 15 (ISG15), a ubiquitin-like protein, is covalently conjugated to host (immune) proteins such as MDA5 and IRF3 in a process called ISGylation, thereby limiting the replication of Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). However, whether SARS-CoV-2 proteins can be directly targeted for ISGylation remains elusive. In this study, we identified the nucleocapsid (N) protein of SARS-CoV-2 as a major substrate of ISGylation catalyzed by the host E3 ligase HERC5; however, N ISGylation is readily removed through de-ISGylation by the papain-like protease (PLpro) activity of NSP3. Mass spectrometry analysis identified that the N protein undergoes ISGylation at four lysine residues (K266, K355, K387 and K388), and mutational analysis of these sites in the context of a SARS-CoV-2 replicon (N-4KR) abolished N ISGylation and alleviated ISGylation-mediated inhibition of viral RNA synthesis. Furthermore, our results indicated that HERC5 targets preferentially phosphorylated N protein for ISGylation to regulate its oligomeric assembly. These findings reveal a novel mechanism by which the host ISGylation machinery directly targets SARS-CoV-2 proteins to restrict viral replication and illuminate how an intricate interplay of host (HERC5) and viral (PLpro) enzymes coordinates viral protein ISGylation and thereby regulates virus replication., Competing Interests: Competing interests The authors declare that they have no competing interests.

Citron kinase (CITK) is an AGC-family serine/threonine kinase that regulates cytokinesis. Despite knockdown experiments implicating CITK as an anticancer target, no selective CITK inhibitors exist. We transformed a previously reported kinase inhibitor with weak off-target CITK activity into a first-in-class CITK chemical probe, C3TD879 . C3TD879 is a Type I kinase inhibitor which potently inhibits CITK catalytic activity (biochemical IC 50 = 12 nM), binds directly to full-length human CITK in cells (NanoBRET K d < 10 nM), and demonstrates favorable DMPK properties for in vivo evaluation. We engineered exquisite selectivity for CITK (>17-fold versus 373 other human kinases), making C3TD879 the first chemical probe suitable for interrogating the complex biology of CITK. Our small-molecule CITK inhibitors could not phenocopy the effects of CITK knockdown in cell proliferation, cell cycle progression, or cytokinesis assays, providing preliminary evidence that the structural roles of CITK may be more important than its kinase activity.

Prostate cancer remains the second leading cause of cancer death in men in Western cultures. A deeper understanding of the mechanisms by which prostate cancer cells divide to support tumor growth could help devise strategies to overcome treatment resistance and improve survival. Here, we identified that the mitotic AGC family protein kinase citron kinase (CIT) is a pivotal regulator of prostate cancer growth that mediates prostate cancer cell interphase progression. Increased CIT expression correlated with prostate cancer growth induction and aggressive prostate cancer progression, and CIT was overexpressed in prostate cancer compared with benign prostate tissue. CIT overexpression was controlled by an E2F2-Skp2-p27 signaling axis and conferred resistance to androgen-targeted treatment strategies. The effects of CIT relied entirely on its kinase activity. Conversely, CIT silencing inhibited the growth of cell lines and xenografts representing different stages of prostate cancer progression and treatment resistance but did not affect benign epithelial prostate cells or nonprostatic normal cells, indicating a potential therapeutic window for CIT inhibition. CIT kinase activity was identified as druggable and was potently inhibited by the multikinase inhibitor OTS-167, which decreased the proliferation of treatment-resistant prostate cancer cells and patient-derived organoids. Isolation of the in vivo CIT substrates identified proteins involved in diverse cellular functions ranging from proliferation to alternative splicing events that are enriched in treatment-resistant prostate cancer. These findings provide insights into the regulation of aggressive prostate cancer cell behavior by CIT and identify CIT as a functionally diverse and druggable driver of prostate cancer progression., Significance: The poorly characterized protein kinase citron kinase is a therapeutic target in prostate cancer that drives tumor growth by regulating diverse substrates, which control several hallmarks of aggressive prostate cancer progression. See related commentary by Mishra et al., p. 4008., (©2023 American Association for Cancer Research.)

Cancer stem cells drive tumor growth and survival via self-renewal and therapeutic resistance, but the upstream mechanisms are not well defined. In this issue of PLOS Biology, a study in colon cancer reveals a new signalling network that links epigenetic regulation to these phenotypes., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Hubert et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Direct targeting of essential viral enzymes such as proteases, polymerases, and helicases has long been the major focus of antiviral drug design. Although successful for some viral enzymes, targeting viral helicases is notoriously difficult to achieve, demanding alternative strategies. Here, we show that the NS3 helicase of Zika virus (ZIKV) undergoes acetylation in its RNA-binding tunnel. Regulation of the acetylated state of K389 in ZIKV NS3 modulates RNA binding and unwinding and is required for efficient viral replication. NS3 acetylation is mediated by a specific isoform of the host acetyltransferase KAT5 (KAT5γ), which translocates from the nucleus to viral replication complexes upon infection. NS3 acetylation by KAT5γ and its proviral role are also conserved in West Nile virus (WNV), dengue virus (DENV), and yellow fever virus (YFV). Our study provides molecular insight into how a cellular acetyltransferase regulates viral helicase functions, unveiling a previously unknown target for antiviral drug development., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 Elsevier Inc. All rights reserved.)

α 1 -Adrenergic Receptors (ARs) regulate the sympathetic nervous system by the binding of norepinephrine (NE) and epinephrine (Epi) through different subtypes (α 1A , α 1B , α 1D ). α 1A -AR activation is hypothesized to be memory forming and cognitive enhancing but drug development has been stagnant due to unwanted side effects on blood pressure. We recently reported the pharmacological characterization of the first positive allosteric modulator (PAM) for the α 1A -AR with predictive pro-cognitive and memory properties. In this report, we now demonstrate the in vivo characteristics of Compound 3 (Cmpd-3) in two genetically-different Alzheimer's Disease (AD) mouse models. Drug metabolism and pharmacokinetic studies indicate sufficient brain penetrance and rapid uptake into the brain with low to moderate clearance, and a favorable inhibition profile against the major cytochrome p450 enzymes. Oral administration of Cmpd-3 (3-9 mg/kg QD) can fully rescue long-term potentiation defects and AD biomarker profile (amyloid β-40, 42) within 3 months of dosing to levels that were non-significant from WT controls and which outperformed donepezil (1 mg/kg QD). There were also significant effects on paired pulse facilitation and cognitive behavior. Long-term and high-dose in vivo studies with Cmpd-3 revealed no effects on blood pressure. Our results suggest that Cmpd-3 can maintain lasting therapeutic levels and efficacy with disease modifying effects with a once per day dosing regimen in AD mouse models with no observed side effects., Competing Interests: The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:The authors have filed the following patents: PCT/US2020/029583, filed 4/23/2020. European Patent (No. 20794530.4) filed 10/15/2021. US utility (17/605,801) filed 10/21/2021.Inventors: PEREZ, Dianne M.; STAUFFER, Shaun R.; MACDONALD, Jonathan. Allosteric Activators of the Alpha1A-Adrenergic receptor. European Patent (No. 20794530.4) filed June 10, 2022., (© 2023 The Authors.)

Glioblastomas (GBMs) are heterogeneous, treatment-resistant tumors driven by populations of cancer stem cells (CSCs). However, few molecular mechanisms critical for CSC population maintenance have been exploited for therapeutic development. We developed a spatially resolved loss-of-function screen in GBM patient-derived organoids to identify essential epigenetic regulators in the SOX2-enriched, therapy-resistant niche and identified WDR5 as indispensable for this population. WDR5 is a component of the WRAD complex, which promotes SET1 family-mediated Lys4 methylation of histone H3 (H3K4me), associated with positive regulation of transcription. In GBM CSCs, WDR5 inhibitors blocked WRAD complex assembly and reduced H3K4 trimethylation and expression of genes involved in CSC-relevant oncogenic pathways. H3K4me3 peaks lost with WDR5 inhibitor treatment occurred disproportionally on POU transcription factor motifs, including the POU5F1(OCT4)::SOX2 motif. Use of a SOX2/OCT4 reporter demonstrated that WDR5 inhibitor treatment diminished cells with high reporter activity. Furthermore, WDR5 inhibitor treatment and WDR5 knockdown altered the stem cell state, disrupting CSC in vitro growth and self-renewal, as well as in vivo tumor growth. These findings highlight the role of WDR5 and the WRAD complex in maintaining the CSC state and provide a rationale for therapeutic development of WDR5 inhibitors for GBM and other advanced cancers., (© 2023 Mitchell et al.; Published by Cold Spring Harbor Laboratory Press.)

α 1 -Adrenergic Receptors (ARs) are G-protein Coupled Receptors (GPCRs) that regulate the sympathetic nervous system via the binding and activation of norepinephrine (NE) and epinephrine (Epi). α 1 -ARs control various aspects of neurotransmission, cognition, cardiovascular functions as well as other organ systems. However, therapeutic drug development for these receptors, particularly agonists, has been stagnant due to unwanted effects on blood pressure regulation. We report the synthesis and characterization of the first positive allosteric modulator (PAM) for the α 1 -AR based upon the derivation of the α 1A -AR selective imidazoline agonist, cirazoline. Compound 3 (Cmpd-3) binds the α 1A -AR with high and low affinity sites (0.13pM; 54 ​nM) typical of GPCR agonists, and reverts to a single low affinity site of 100 ​nM upon the addition of GTP. Comparison of Cmpd-3 versus other orthosteric α 1A -AR-selective imidazoline ligands reveal unique properties that are consistent with a type I PAM. Cmpd-3 is both conformationally and ligand-selective for the α 1A -AR subtype. In competition binding studies, Cmpd-3 potentiates NE-binding at the α 1A -AR only on the high affinity state of NE with no effect on the Epi-bound α 1A -AR. Moreover, Cmpd-3 demonstrates signaling-bias and potentiates the NE-mediated cAMP response of the α 1A -AR at nM concentrations with no effects on the NE-mediated inositol phosphate response. There are no effects of Cmpd-3 on the signaling at the α 1B - or α 1D -AR subtypes. Cmpd-3 displays characteristics of a pure PAM with no intrinsic agonist properties. Specific derivation of Cmpd-3 at the R1 ortho -position recapitulated PAM characteristics. Our results characterize the first PAM for the α 1 -AR and holds promise for a first-in-class therapeutic to treat various diseases without the side effect of increasing blood pressure intrinsic to classical orthosteric agonists., Competing Interests: The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: The authors have filed the following patents: PCT/US2020/029583, filed 4/23/2020. European Patent (No. 20794530.4) filed 10/15/2021. US utility (17/605,801) filed 10/21/2021.Inventors: PEREZ, Dianne M.; STAUFFER, Shaun R.; MACDONALD, Jonathan. Allosteric Activators of the Alpha1A-Adrenergic receptor., (© 2022 The Authors.)

Metastatic outgrowth is supported by metabolic adaptations that may differ from the primary tumor of origin. However, it is unknown if such adaptations are therapeutically actionable. Here we report a novel aminopyridine compound that targets a unique Phosphogluconate Dehydrogenase (PGD)-dependent metabolic adaptation in distant metastases from pancreatic cancer patients. Compared to structurally similar analogs, 6-aminopicolamine (6AP) potently and selectively reversed PGD-dependent metastatic properties, including intrinsic tumorigenic capacity, excess glucose consumption, and global histone hyperacetylation. 6AP acted as a water-soluble prodrug that was converted into intracellular bioactive metabolites that inhibited PGD in vitro, and 6AP monotherapy demonstrated anti-metastatic efficacy with minimal toxicity in vivo. Collectively, these studies identify 6AP and possibly other 6-aminopyridines as well-tolerated prodrugs with selectivity for metastatic pancreatic cancers. If unique metabolic adaptations are a common feature of metastatic or otherwise aggressive human malignancies, then such dependencies could provide a largely untapped pool of druggable targets for patients with advanced cancers., (© 2022. The Author(s), under exclusive licence to Springer Nature Limited.)

The chromatin-associated protein WDR5 is a promising target for pharmacological inhibition in cancer. Drug discovery efforts center on the blockade of the "WIN site" of WDR5, a well-defined pocket that is amenable to small molecule inhibition. Various cancer contexts have been proposed to be targets for WIN site inhibitors, but a lack of understanding of WDR5 target genes and of the primary effects of WIN site inhibitors hampers their utility. Here, by the discovery of potent WIN site inhibitors, we demonstrate that the WIN site links WDR5 to chromatin at a small cohort of loci, including a specific subset of ribosome protein genes. WIN site inhibitors rapidly displace WDR5 from chromatin and decrease the expression of associated genes, causing translational inhibition, nucleolar stress, and p53 induction. Our studies define a mode by which WDR5 engages chromatin and forecast that WIN site blockade could have utility against multiple cancer types., (Copyright © 2019 The Author(s). Published by Elsevier Inc. All rights reserved.)

Selective activation of the M 1 subtype of muscarinic acetylcholine receptor, via positive allosteric modulation (PAM), is an exciting strategy to improve cognition in schizophrenia and Alzheimer's disease patients. However, highly potent M 1 ago-PAMs, such as MK-7622, PF-06764427, and PF-06827443, can engender excessive activation of M 1 , leading to agonist actions in the prefrontal cortex (PFC) that impair cognitive function, induce behavioral convulsions, and result in other classic cholinergic adverse events (AEs). Here, we report a fundamentally new and highly selective M 1 PAM, VU0486846. VU0486846 possesses only weak agonist activity in M 1 -expressing cell lines with high receptor reserve and is devoid of agonist actions in the PFC, unlike previously reported ago-PAMs MK-7622, PF-06764427, and PF-06827443. Moreover, VU0486846 shows no interaction with antagonist binding at the orthosteric acetylcholine (ACh) site (e.g., neither bitopic nor displaying negative cooperativity with [ 3 H]-NMS binding at the orthosteric site), no seizure liability at high brain exposures, and no cholinergic AEs. However, as opposed to ago-PAMs, VU0486846 produces robust efficacy in the novel object recognition model of cognitive function. Importantly, we show for the first time that an M 1 PAM can reverse the cognitive deficits induced by atypical antipsychotics, such as risperidone. These findings further strengthen the argument that compounds with modest in vitro M 1 PAM activity (EC 50 > 100 nM) and pure-PAM activity in native tissues display robust procognitive efficacy without AEs mediated by excessive activation of M 1 . Overall, the combination of compound assessment with recombinant in vitro assays (mindful of receptor reserve), native tissue systems (PFC), and phenotypic screens (behavioral convulsions) is essential to fully understand and evaluate lead compounds and enhance success in clinical development.

WDR5 is a chromatin-regulatory scaffold protein overexpressed in various cancers and a potential epigenetic drug target for the treatment of mixed-lineage leukemia. Here, we describe the discovery of potent and selective WDR5-WIN-site inhibitors using fragment-based methods and structure-based design. NMR-based screening of a large fragment library identified several chemically distinct hit series that bind to the WIN site within WDR5. Members of a 6,7-dihydro-5 H-pyrrolo[1,2- a]imidazole fragment class were expanded using a structure-based design approach to arrive at lead compounds with dissociation constants <10 nM and micromolar cellular activity against an AML-leukemia cell line. These compounds represent starting points for the discovery of clinically useful WDR5 inhibitors for the treatment of cancer.