Search

The glutamatergic system, located throughout the brain including the prefrontal cortex and nucleus accumbens, plays a critical role in reward and reinforcement processing, and mediates the psychotropic effects of addictive drugs such as cocaine. Glutamate transporters, including EAAT2/GLT-1, are responsible for removing glutamate from the synaptic cleft. Reduced expression of GLT-1 following chronic cocaine use and abstinence has been reported. Here, we demonstrate that targeting GLT-1 with a novel positive allosteric modulator (PAM), NA-014, results in reduction of cocaine-associated behaviors in rats. Pharmacokinetic analysis demonstrated that NA-014 is brain-penetrant and suitable for in vivo studies.We found that 15 and 30 mg/kg NA-014 significantly reduced cocaine-induced locomotion in males. Only the 15 mg/kg dose was effective in females and 60 mg/kg was ineffective in both sexes. Furthermore, 30 and 60 mg/kg NA-014 reduced expression of cocaine conditioned place preference (CPP) in males. 30 mg/kg NA-014 reduced expression of cocaine CPP in females and 15 mg/kg did not affect cocaine CPP in either sex, suggesting GLT-1 influences cocaine-associated behaviors in a sex-dependent manner. NA-014 did not elicit rewarding behavior, nor alter baseline locomotion. Twice daily/7-day administration of 100 mg/kg of NA-014 did not alter GLT-1 or GLAST expression in either sex in the prefrontal cortex (PFC). Collectively, these studies demonstrated that NA-014 reduced the locomotor stimulant and rewarding effects of cocaine in male and female rats. In the context of psychostimulant use disorders, our study suggests studying GLT-1 PAMs as alternatives to β-lactam compounds that increase GLT-1 protein levels., Competing Interests: Declaration of competing interest The authors declare the following competing financial interest(s): JMS owns equity in Alliance Discovery, Inc., Barer Institute, Context Therapeutics, and consults for Syndeavor Therapeutics, Inc. The other authors declare no competing interests. The other authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Cancer cells often use alternative nutrient sources to support their metabolism and proliferation. One important alternative nutrient source for many cancers is acetate. Acetate is metabolized into acetyl-coenzyme A (CoA) by acetyl-CoA synthetases 1 and 2 (ACSS1 and ACSS2), which are found in the mitochondria and cytosol, respectively. We show that ACSS1 and ACSS2 are differentially expressed in cancer. Melanoma, breast cancer, and acute myeloid leukemia cells expressing ACSS1 readily use acetate for acetyl-CoA biosynthesis and to fuel mitochondrial metabolism. ACSS1-dependent acetate metabolism decreases the relative contributions of glucose and glutamine to the tricarboxylic acid (TCA) cycle and alters the pentose phosphate pathway and redox state of cancer cells. ACSS1 knockdown decreases acute myeloid leukemia burden in vivo and inhibits melanoma tumor and metastatic growth. Our study highlights a key role for ACSS1-dependent acetate metabolism for cancer growth, raising the potential for ACSS1-targeting therapies in cancer., Competing Interests: Declaration of interests Z.T.S. and J.M.S. are scientific co-founders and consultants for Syndeavor Therapeutics., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Deubiquitylases (DUBs) are crucial in cell signalling and are often regulated by interactions within protein complexes. The BRCC36 isopeptidase complex (BRISC) regulates inflammatory signalling by cleaving K63-linked polyubiquitin chains on Type I interferon receptors (IFNAR1). As a Zn 2+ -dependent JAMM/MPN DUB, BRCC36 is challenging to target with selective inhibitors. We discovered first-in-class inhibitors, termed BRISC molecular glues (BLUEs), which stabilise a 16-subunit BRISC dimer in an autoinhibited conformation, blocking active sites and interactions with the targeting subunit SHMT2. This unique mode of action results in selective inhibition of BRISC over related complexes with the same catalytic subunit, splice variants and other JAMM/MPN DUBs. BLUE treatment reduced interferon-stimulated gene expression in cells containing wild type BRISC, and this effect was absent when using structure-guided, inhibitor-resistant BRISC mutants. Additionally, BLUEs increase IFNAR1 ubiquitylation and decrease IFNAR1 surface levels, offering a potential new strategy to mitigate Type I interferon-mediated diseases. Our approach also provides a template for designing selective inhibitors of large protein complexes by promoting, rather than blocking, protein-protein interactions., Competing Interests: Competing interests E.Z., R.G., J.M.S., and F.S. are named co-inventors in a patent application to use BRISC inhibitors as therapeutics (WO2024115713A1). J.M.S. owns equity in Alliance Discovery, Inc and the Barer Institute, Inc, and consults for Syndeavor Therapeutics, Inc.

We have synthesized a novel and highly selective severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) main protease peptide mimetic inhibitor mimicking the replicase 1ab recognition sequence -Val-Leu-Gln- and utilizing a cysteine selective acyloxymethyl ketone as the electrophilic warhead to target the active site Cys145. Utilizing a constrained cyclic peptide that locks the conformation between the P3 (Val) and P2 (Leu) residues, we identified a highly selective inhibitor that fills the P2 pocket occupied by the leucine residue sidechain of PF-00835231 and the dimethyl-3-azabicyclo-hexane motif in nirmatrelvir (PF-07321332). This strategy resulted in potent and highly selective Mpro inhibitors without inhibiting essential host cathepsin cysteine or serine proteases. The lead prototype compound 1 (MPro IC 50 = 230 ± 18 nM) also inhibits the replication of multiple SARS-CoV-2 variants in vitro , including SARS-CoV-2 variants of concern, and can synergize at lower concentrations with the viral RNA polymerase inhibitor, remdesivir, to inhibit replication. It also reduces SARS-CoV-2 replication in SARS-CoV-2 Omicron-infected Syrian golden hamsters without obvious toxicities, demonstrating in vivo efficacy. This novel lead structure provides the basis for optimization of improved agents targeting evolving SARS-CoV-2 drug resistance that can selectively act on Mpro versus host proteases and are less likely to have off-target effects due to non-specific targeting. Developing inhibitors against the active site of the main protease (Mpro), which is highly conserved across coronaviruses, is expected to impart a higher genetic barrier to evolving SARS-CoV-2 drug resistance. Drugs that selectively inhibit the viral Mpro are less likely to have off-target effects warranting efforts to improve this therapy., Competing Interests: J.M.S. receives funding from the NIH P30-CA010815-53; has patents pending to Wistar; owns equity in Alliance Discovery, Inc., Barer Institute, and Context Therapeutics; and consults for Syndeavor Therapeutics, Inc. All other authors declare no competing interests.

J-Lat cells are derivatives of the Jurkat CD4+ T cell line that contain a non-infectious, inducible HIV provirus with a GFP tag. While these cells have substantially advanced our understanding of HIV latency, their use by many laboratories in low and middle-income countries is restricted by limited access to flow cytometry. To overcome this barrier, we describe a modified J-Lat assay using a standard microplate reader that detects HIV-GFP expression following treatment with latency-reversing agents (LRAs). We show that HIV reactivation by control LRAs like prostratin and romidepsin is readily detected with dose dependence and with significant correlation and sensitivity to standard flow cytometry. For example, 10 µM prostratin induced a 20.1 ± 3.3-fold increase in GFP fluorescence in the microplate reader assay, which corresponded to 64.2 ± 5.0% GFP-positive cells detected by flow cytometery. Similarly, 0.3 µM prostratin induced a 1.7 ± 1.2-fold increase compared to 8.7 ± 5.7% GFP-positive cells detected. Using this method, we screen 79 epigenetic modifiers and identify molibresib, quisinostat, and CUDC-101 as novel LRAs. This microplate reader-based method offers accessibility to researchers in resource-limited regions to work with J-Lat cells and more actively participate in global HIV cure research efforts., Highlights: J-Lat T-cell lines are important to HIV cure research but require flow cytometryWe describe a method to work with J-Lat cells using a standard microplate readerThis assay can detect control LRAs similar to flow cytometry and discover new LRAsThis assay allows low-resourced laboratories to contribute to HIV cure research.

The regulation of dopamine (DA) removal from the synaptic cleft is a crucial process in neurotransmission and is facilitated by the sodium- and chloride-coupled dopamine transporter DAT. Psychostimulant drugs, cocaine, and amphetamine, both block the uptake of DA, while amphetamine also triggers the release of DA. As a result, they prolong or even amplify neurotransmitter signaling. Atypical inhibitors of DAT lack cocaine-like rewarding effects and offer a promising strategy for the treatment of drug use disorders. Here, we present the 3.2 Å resolution cryo-electron microscopy structure of the Drosophila melanogaster dopamine transporter (dDAT) in complex with the atypical non-competitive inhibitor AC-4-248. The inhibitor partially binds at the central binding site, extending into the extracellular vestibule, and locks the transporter in an outward open conformation. Our findings propose mechanisms for the non-competitive inhibition of DAT and attenuation of cocaine potency by AC-4-248 and provide a basis for the rational design of more efficacious atypical inhibitors., (© 2024 The Author(s). Journal of Neurochemistry published by John Wiley & Sons Ltd on behalf of International Society for Neurochemistry.)

The coronavirus disease 2019 (COVID-19) has spread worldwide with severe health, social, and economic repercussions. Although vaccines have significantly reduced the severity of symptoms and deaths, alternative medications derived from natural products (NPs) are vital to further decrease fatalities, especially in regions with low vaccine uptake. When paired with the latest computational developments, NPs, which have been used to cure illnesses and infections for thousands of years, constitute a renewed resource for drug discovery. In the present report, a combination of computational and in vitro methods reveals the repositioning of NPs and identifies salvinorin A and deacetylgedunin (DCG) as having potential anti-SARS-CoV-2 activities. Salvinorin A was found both in silico and in vitro to inhibit both SARS-CoV-2 spike/host ACE2 protein interactions, consistent with blocking viral cell entry, and well as live virus replication. Plant extracts from Azadirachta indica and Cedrela odorata , which contain high levels of DCG, inhibited viral cell replication by targeting the main protease (Mpro) and/or inhibited viral cell entry by blocking the interaction between spike RBD-ACE2 protein at concentrations lower than salvinorin A. Our findings suggest that salvinorin A represent promising chemical starting points where further optimization may result in effective natural product-derived and potent anti-SARS-CoV-2 inhibitors to supplement vaccine efforts., Competing Interests: There are no conflicts to declare., (This journal is © The Royal Society of Chemistry.)

Pancreatic ductal adenocarcinoma (PDAC), the most prevalent type of pancreatic cancer, is one of the deadliest forms of cancer with limited therapy options. Overexpression of the heat shock protein 70 (HSP70) is a hallmark of cancer that is strongly associated with aggressive disease and worse clinical outcomes. However, the underlying mechanisms by which HSP70 allows tumor cells to thrive under conditions of continuous stress have not been fully described. Here, we report that PDAC has the highest expression of HSP70 relative to normal tissue across all cancers analyzed. Furthermore, HSP70 expression is associated with tumor grade and is further enhanced in metastatic PDAC. We show that genetic or therapeutic ablation of HSP70 alters mitochondrial subcellular localization, impairs mitochondrial dynamics, and promotes mitochondrial swelling to induce apoptosis. Mechanistically, we find that targeting HSP70 suppresses the PTEN-induced kinase 1 (PINK1) mediated phosphorylation of dynamin-related protein 1 (DRP1). Treatment with the HSP70 inhibitor AP-4-139B was efficacious as a single agent in primary and metastatic mouse models of PDAC. In addition, we demonstrate that HSP70 inhibition promotes the AMP-activated protein kinase (AMPK) mediated phosphorylation of Beclin-1, a key regulator of autophagic flux. Accordingly, we find that the autophagy inhibitor hydroxychloroquine (HCQ) enhances the ability of AP-4-139B to mediate anti-tumor activity in vivo. Collectively, our results suggest that HSP70 is a multi-functional driver of tumorigenesis that orchestrates mitochondrial dynamics and autophagy. Moreover, these findings support the rationale for concurrent inhibition of HSP70 and autophagy as a novel therapeutic approach for HSP70-driven PDAC., (© 2024. The Author(s).)

Current small-molecule-based SARS-CoV-2 treatments have limited global accessibility and pose the risk of inducing viral resistance. Therefore, a marine algae and cyanobacteria extract library was screened for natural products that could inhibit two well-defined and validated COVID-19 drug targets, disruption of the spike protein/ACE-2 interaction and the main protease (M pro ) of SARS-CoV-2. Following initial screening of 86 extracts, we performed an untargeted metabolomic analysis of 16 cyanobacterial extracts. This approach led to the isolation of an unusual saturated fatty acid, jobosic acid (2,5-dimethyltetradecanoic acid, 1 ). We confirmed that 1 demonstrated selective inhibitory activity toward both viral targets while retaining some activity against the spike-RBD/ACE-2 interaction of the SARS-CoV-2 omicron variant. To initially explore its structure-activity relationship (SAR), the methyl and benzyl ester derivatives of 1 were semisynthetically accessed and demonstrated acute loss of bioactivity in both SARS-CoV-2 biochemical assays. Our efforts have provided copious amounts of a fatty acid natural product that warrants further investigation in terms of SAR, unambiguous determination of its absolute configuration, and understanding of its specific mechanisms of action and binding site toward new therapeutic avenues for SARS-CoV-2 drug development.

Quinazolines are an important class of heterocyclic compounds that have proven their significance, especially in the field of organic synthesis and medicinal chemistry because of their wide range of biological and pharmacological properties. Thus, numerous synthetic methods have been developed for the synthesis of quinazolines and their derivatives. This review article briefly outlines the new synthetic methods for compounds containing the quinazoline scaffold employing transition metal-catalyzed reactions.

Excitatory amino acid transporters (EAATs) are essential CNS proteins that regulate glutamate levels. Excess glutamate release and alteration in EAAT expression are associated with several CNS disorders. Previously, we identified positive allosteric modulators (PAM) of EAAT2, the main CNS transporter, and have demonstrated their neuroprotective properties in vitro . Herein, we report on the structure-activity relationships (SAR) for the analogs identified from virtual screening and from our medicinal chemistry campaign. This work identified several selective EAAT2 positive allosteric modulators (PAMs) such as compounds 4 (DA-023) and 40 (NA-014) from a library of analogs inspired by GT949, an early generation compound. This series also provides nonselective EAAT PAMs, EAAT inhibitors, and inactive compounds that may be useful for elucidating the mechanism of EAAT allosteric modulation.

Seven furanochromene-quinoline derivatives containing a hydrazone linker were synthesized by condensing a furanochromene hydrazide with quinoline 2-, 3-, 4-, 5-, 6-, and 8-carbaldehydes, including 8-hydroxyquinoline-2-carbaldehye. Structure-activity correlations were investigated to determine the influence of the location of the hydrazone linker on the quinoline unit on SARS-CoV-2 M pro enzyme inhibition. The 3-, 5-, 6- and 8-substituted derivatives showed moderate inhibition of SARS-CoV-2 M pro with IC 50 values ranging from 16 to 44 μM. Additionally, all of the derivatives showed strong interaction with the SARS-CoV-2 M pro substrate binding pocket, with docking energy scores ranging from -8.0 to -8.5 kcal/mol. These values are comparable to that of N3 peptide (-8.1 kcal/mol) and more favorable than GC-373 (-7.6 kcal/mol) and ML-188 (-7.5 kcal/mol), all of which are known SARS-CoV-2 M pro inhibitors. Furthermore, in silico absorption, distribution, metabolism, and excretion (ADME) profiles indicate that the derivatives have good drug-likeness properties. Overall, this study highlights the potential of the furanochromene-quinoline hydrazone scaffold as a SARS-CoV-2 M pro inhibitor., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

In the field of drug discovery, understanding how small molecule drugs interact with cellular components is crucial. Our study introduces a novel methodology to uncover primary drug targets using T andem A ffinity P urification for identification of D rug- B inding P roteins (TAP-DBP). Central to our approach is the generation of a FLAG-hemagglutinin (HA)-tagged chimeric protein featuring the FKBP12(F36V) adaptor protein and the TurboID enzyme. Conjugation of drug molecules with the FKBP12(F36V) ligand allows for the coordinated recruitment of drug-binding partners effectively enabling in-cell TurboID-mediated biotinylation. By employing a tandem affinity purification protocol based on FLAG-immunoprecipitation and streptavidin pulldown, alongside mass spectrometry analysis, TAP-DBP allows for the precise identification of drug-primary binding partners. Overall, this study introduces a systematic, unbiased method for identification of drug-protein interactions, contributing a clear understanding of target engagement and drug selectivity to advance the mode of action of a drug in cells.

Chemical prototypes with broad-spectrum antiviral activity are important toward developing new therapies that can act on both existing and emerging viruses. Binding of the SARS-CoV-2 spike protein to the host angiotensin-converting enzyme 2 (ACE2) receptor is required for cellular entry of SARS-CoV-2. Toward identifying new chemical leads that can disrupt this interaction, including in the presence of SARS-CoV-2 adaptive mutations found in variants like omicron that can circumvent vaccine, immune, and therapeutic antibody responses, we synthesized 5-chloro-3-(2-(2,4-dinitrophenyl)hydrazono)indolin-2-one (H 2 L) from the condensation reaction of 5-chloroisatin and 2,4-dinitrophenylhydrazine in good yield. H 2 L was characterised by elemental and spectral (IR, electronic, Mass) analyses. The NMR spectrum of H 2 L indicated a keto-enol tautomerism, with the keto form being more abundant in solution. H 2 L was found to selectively interfere with binding of the SARS-CoV-2 spike receptor-binding domain (RBD) to the host angiotensin-converting enzyme 2 receptor with a 50% inhibitory concentration (IC 50 ) of 0.26 μM, compared to an unrelated PD-1/PD-L1 ligand-receptor-binding pair with an IC 50 of 2.06 μM in vitro (Selectivity index = 7.9). Molecular docking studies revealed that the synthesized ligand preferentially binds within the ACE2 receptor-binding site in a region distinct from where spike mutations in SARS-CoV-2 variants occur. Consistent with these models, H 2 L was able to disrupt ACE2 interactions with the RBDs from beta, delta, lambda, and omicron variants with similar activities. These studies indicate that H 2 L-derived compounds are potential inhibitors of multiple SARS-CoV-2 variants, including those capable of circumventing vaccine and immune responses., Supplementary Information: The online version contains supplementary material available at 10.1007/s11696-023-03274-5., Competing Interests: Conflict of interestWe declare none., (© The Author(s) 2024.)

Isatin (indol-2,3-dione), a secondary metabolite of tryptophan, has been used as the core structure to design several compounds that have been tested and identified as potent inhibitors of apoptosis, potential antitumor agents, anticonvulsants, and antiviral agents. In this work, several analogs of isatin hybrids have been synthesized and characterized, and their activities were established as inhibitors of both Aurora A kinase and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike/host angiotensin-converting enzyme II (ACE2) interactions. Amongst the synthesized isatin hybrids, compounds 6a , 6f , 6g , and 6m exhibited Aurora A kinase inhibitory activities (with IC 50 values < 5 μ M), with GScore values of -7.9, -7.6, -8.2 and -7.7 kcal/mol, respectively. Compounds 6g and 6i showed activities in blocking SARS-CoV-2 spike/ACE2 binding (with IC 50 values in the range < 30  μ M), with GScore values of -6.4 and -6.6 kcal/mol, respectively. Compounds 6f , 6g , and 6i were both capable of inhibiting spike/ACE2 binding and blocking Aurora A kinase. Pharmacophore profiling indicated that compound 6g tightly fits Aurora A kinase and SARS-CoV-2 pharmacophores, while 6d fits SARS-CoV-2 and 6l fits Aurora A kinase pharmacophore. This work is a proof of concept that some existing cancer drugs may possess antiviral properties. Molecular modeling showed that the active compound for each protein adopted different binding modes, hence interacting with a different set of amino acid residues in the binding site. The weaker activities against spike/ACE2 could be explained by the small sizes of the ligands that fail to address the important interactions for binding to the ACE2 receptor site., Competing Interests: Conflict of interestThe authors declare no competing interests., (© This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply 2024.)