Design, synthesis, and biochemical and computational screening of novel oxindole derivatives as inhibitors of Aurora A kinase and SARS-CoV-2 spike/host ACE2 interaction.

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 IC50 values < 5 µM), 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 IC50 values in the range < 30 µM), 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.

5-chloro-3-(2-(2,4-dinitrophenyl) hydrazono)indolin-2-one: synthesis, characterization, biochemical and computational screening against SARS-CoV-2.

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 (H2L) from the condensation reaction of 5-chloroisatin and 2,4-dinitrophenylhydrazine in good yield. H2L was characterised by elemental and spectral (IR, electronic, Mass) analyses. The NMR spectrum of H2L indicated a keto-enol tautomerism, with the keto form being more abundant in solution. H2L 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 (IC50) of 0.26 µM, compared to an unrelated PD-1/PD-L1 ligand-receptor-binding pair with an IC50 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, H2L was able to disrupt ACE2 interactions with the RBDs from beta, delta, lambda, and omicron variants with similar activities. These studies indicate that H2L-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.

Synthesis and Structure-Activity Relationships for Glutamate Transporter Allosteric Modulators.

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.

Synthesis, SARS-CoV-2 main protease inhibition, molecular docking and in silico ADME studies of furanochromene-quinoline hydrazone derivatives.

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 Mpro enzyme inhibition. The 3-, 5-, 6- and 8-substituted derivatives showed moderate inhibition of SARS-CoV-2 Mpro with IC50 values ranging from 16 to 44 µM. Additionally, all of the derivatives showed strong interaction with the SARS-CoV-2 Mpro 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 Mpro 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 Mpro inhibitor.

A Tandem-Affinity Purification Method for Identification of Primary Intracellular Drug-Binding Proteins.

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 Tandem Affinity Purification for identification of Drug-Binding Proteins (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.

Synthesis and characterization of I-BET151 derivatives for use in identifying protein targets in the African trypanosome.

Trypanosoma brucei, the causative agent of Human African Trypanosomiasis (HAT) and animal trypanosomiases, cycles between a bloodstream form in mammals and a procyclic form in the gut of its insect vector. We previously discovered that the human bromodomain inhibitor I-BET151 causes transcriptome changes that resemble the transition from the bloodstream to the procyclic form. In particular, I-BET151 induces replacement of variant surface glycoprotein (VSG) with procyclin protein. While modest binding of I-BET151 to TbBdf2 and TbBdf3 has been demonstrated, it is unknown whether I-BET151 binds to other identified T. brucei bromodomain proteins and/or other targets. To identify target(s) in T. brucei, we have synthesized I-BET151 derivatives maintaining the key pharmacophoric elements with functionality useful for chemoproteomic approaches. We identified compounds that are potent in inducing expression of procyclin, delineating a strategy towards the design of drugs against HAT and other trypanosomiases. Furthermore, these derivatives represent useful chemical probes to elucidate the molecular mechanism underlying I-BET151-induced differentiation.

Acetate acts as a metabolic immunomodulator by bolstering T-cell effector function and potentiating antitumor immunity in breast cancer.

Acetate metabolism is an important metabolic pathway in many cancers and is controlled by acetyl-CoA synthetase 2 (ACSS2), an enzyme that catalyzes the conversion of acetate to acetyl-CoA. While the metabolic role of ACSS2 in cancer is well described, the consequences of blocking tumor acetate metabolism on the tumor microenvironment and antitumor immunity are unknown. We demonstrate that blocking ACSS2, switches cancer cells from acetate consumers to producers of acetate thereby freeing acetate for tumor-infiltrating lymphocytes to use as a fuel source. We show that acetate supplementation metabolically bolsters T-cell effector functions and proliferation. Targeting ACSS2 with CRISPR-Cas9 guides or a small-molecule inhibitor promotes an antitumor immune response and enhances the efficacy of chemotherapy in preclinical breast cancer models. We propose a paradigm for targeting acetate metabolism in cancer in which inhibition of ACSS2 dually acts to impair tumor cell metabolism and potentiate antitumor immunity.

Elevated pre-mRNA 3' end processing activity in cancer cells renders vulnerability to inhibition of cleavage and polyadenylation.

Cleavage and polyadenylation (CPA) is responsible for 3' end processing of eukaryotic poly(A)+ RNAs and preludes transcriptional termination. JTE-607, which targets CPSF-73, is the first known CPA inhibitor (CPAi) in mammalian cells. Here we show that JTE-607 perturbs gene expression through both transcriptional readthrough and alternative polyadenylation (APA). Sensitive genes are associated with features similar to those previously identified for PCF11 knockdown, underscoring a unified transcriptomic signature of CPAi. The degree of inhibition of an APA site by JTE-607 correlates with its usage level and, consistently, cells with elevated CPA activities, such as those with induced overexpression of FIP1, display greater transcriptomic disturbances when treated with JTE-607. Moreover, JTE-607 causes S phase crisis and is hence synergistic with inhibitors of DNA damage repair pathways. Together, our data reveal CPA activity and proliferation rate as determinants of CPAi-mediated cell death, raising the possibility of using CPAi as an adjunct therapy to suppress certain cancers.

A high-throughput screening assay for silencing established HIV-1 macrophage infection identifies nucleoside analogs that perturb H3K9me3 on proviral genomes.

HIV-infected macrophages are long-lived cells that represent a barrier to functional cure. Additionally, low-level viral expression by central nervous system (CNS) macrophages contributes to neurocognitive deficits that develop despite antiretroviral therapy (ART). We recently identified H3K9me3 as an atypical epigenetic mark associated with chronic HIV infection in macrophages. Thus, strategies are needed to suppress HIV-1 expression in macrophages, but the unique myeloid environment and the responsible macrophage/CNS-tropic strains require cell/strain-specific approaches. Here, we generated an HIV-1 reporter virus from a CNS-derived strain with intact auxiliary genes expressing destabilized luciferase. We employed this reporter virus in polyclonal infection of primary human monocyte-derived macrophages (MDM) for a high-throughput screen (HTS) to identify compounds that suppress virus expression from established macrophage infection. Screening ~6,000 known drugs and compounds yielded 214 hits. A secondary screen with 10-dose titration identified 24 meeting criteria for HIV-selective activity. Using three replication-competent CNS-derived macrophage-tropic HIV-1 isolates and viral gene expression readout in MDM, we confirmed the effect of three purine analogs, nelarabine, fludarabine, and entecavir, showing the suppression of HIV-1 expression from established macrophage infection. Nelarabine inhibited the formation of H3K9me3 on HIV genomes in macrophages. Thus, this novel HTS assay can identify suppressors of HIV-1 transcription in established macrophage infection, such as nucleoside analogs and HDAC inhibitors, which may be linked to H3K9me3 modification. This screen may be useful to identify new metabolic and epigenetic agents that ameliorate HIV-driven neuroinflammation in people on ART or prevent viral recrudescence from macrophage reservoirs in strategies to achieve ART-free remission. IMPORTANCE Macrophages infected by HIV-1 are a long-lived reservoir and a barrier in current efforts to achieve HIV cure and also contribute to neurocognitive complications in people despite antiretroviral therapy (ART). Silencing HIV expression in these cells would be of great value, but the regulation of HIV-1 in macrophages differs from T cells. We developed a novel high-throughput screen for compounds that can silence established infection of primary macrophages, and identified agents that downregulate virus expression and alter provirus epigenetic profiles. The significance of this assay is the potential to identify new drugs that act in the unique macrophage environment on relevant viral strains, which may contribute to adjunctive treatment for HIV-associated neurocognitive disorders and/or prevent viral rebound in efforts to achieve ART-free remission or cure.

Identification of a ß-arrestin-biased negative allosteric modulator for the ß2-adrenergic receptor.

Catecholamine-stimulated ß2-adrenergic receptor (ß2AR) signaling via the canonical Gs-adenylyl cyclase-cAMP-PKA pathway regulates numerous physiological functions, including the therapeutic effects of exogenous ß-agonists in the treatment of airway disease. ß2AR signaling is tightly regulated by GRKs and ß-arrestins, which together promote ß2AR desensitization and internalization as well as downstream signaling, often antithetical to the canonical pathway. Thus, the ability to bias ß2AR signaling toward the Gs pathway while avoiding ß-arrestin-mediated effects may provide a strategy to improve the functional consequences of ß2AR activation. Since attempts to develop Gs-biased agonists and allosteric modulators for the ß2AR have been largely unsuccessful, here we screened small molecule libraries for allosteric modulators that selectively inhibit ß-arrestin recruitment to the receptor. This screen identified several compounds that met this profile, and, of these, a difluorophenyl quinazoline (DFPQ) derivative was found to be a selective negative allosteric modulator of ß-arrestin recruitment to the ß2AR while having no effect on ß2AR coupling to Gs. DFPQ effectively inhibits agonist-promoted phosphorylation and internalization of the ß2AR and protects against the functional desensitization of ß-agonist mediated regulation in cell and tissue models. The effects of DFPQ were also specific to the ß2AR with minimal effects on the ß1AR. Modeling, mutagenesis, and medicinal chemistry studies support DFPQ derivatives binding to an intracellular membrane-facing region of the ß2AR, including residues within transmembrane domains 3 and 4 and intracellular loop 2. DFPQ thus represents a class of biased allosteric modulators that targets an allosteric site of the ß2AR.

Increased glucose availability sensitizes pancreatic cancer to chemotherapy.

Pancreatic Ductal Adenocarcinoma (PDAC) is highly resistant to chemotherapy. Effective alternative therapies have yet to emerge, as chemotherapy remains the best available systemic treatment. However, the discovery of safe and available adjuncts to enhance chemotherapeutic efficacy can still improve survival outcomes. We show that a hyperglycemic state substantially enhances the efficacy of conventional single- and multi-agent chemotherapy regimens against PDAC. Molecular analyses of tumors exposed to high glucose levels reveal that the expression of GCLC (glutamate-cysteine ligase catalytic subunit), a key component of glutathione biosynthesis, is diminished, which in turn augments oxidative anti-tumor damage by chemotherapy. Inhibition of GCLC phenocopies the suppressive effect of forced hyperglycemia in mouse models of PDAC, while rescuing this pathway mitigates anti-tumor effects observed with chemotherapy and high glucose.

The In-Cell Western immunofluorescence assay to monitor PROTAC mediated protein degradation.

The In-Cell Western plate-based immunofluorescence assay is a useful methodology for monitoring protein levels and provides a facile moderate through-put method for PROTAC and degrader optimization. The method is compared to other reported assays used for PROTAC development. The advantages of this method are the greater through-put compared to Western blots due to its plate-based method and the ease to transfer between cells lines. Adherent cell lines are preferred, although suspension cells can be used following recommended modifications and precautions to the protocol. This method requires a high-quality antibody that recognizes the protein epitope in its cellular context, and in general provides data similar to Western blots with higher assay through-put.

Targeted MDM2 Degradation Reveals a New Vulnerability for p53-Inactivated Triple-Negative Breast Cancer.

Triple-negative breast cancers (TNBC) frequently inactivate p53, increasing their aggressiveness and therapy resistance. We identified an unexpected protein vulnerability in p53-inactivated TNBC and designed a new PROteolysis TArgeting Chimera (PROTAC) to target it. Our PROTAC selectively targets MDM2 for proteasome-mediated degradation with high-affinity binding and VHL recruitment. MDM2 loss in p53 mutant/deleted TNBC cells in two-dimensional/three-dimensional culture and TNBC patient explants, including relapsed tumors, causes apoptosis while sparing normal cells. Our MDM2-PROTAC is stable in vivo, and treatment of TNBC xenograft-bearing mice demonstrates tumor on-target efficacy with no toxicity to normal cells, significantly extending survival. Transcriptomic analyses revealed upregulation of p53 family target genes. Investigations showed activation and a required role for TAp73 to mediate MDM2-PROTAC-induced apoptosis. Our data, challenging the current MDM2/p53 paradigm, show MDM2 is required for p53-inactivated TNBC cell survival, and PROTAC-targeted MDM2 degradation is an innovative potential therapeutic strategy for TNBC and superior to existing MDM2 inhibitors. SIGNIFICANCE: p53-inactivated TNBC is an aggressive, therapy-resistant, and lethal breast cancer subtype. We designed a new compound targeting an unexpected vulnerability we identified in TNBC. Our MDM2-targeted degrader kills p53-inactivated TNBC cells, highlighting the requirement for MDM2 in TNBC cell survival and as a new therapeutic target for this disease. See related commentary by Peuget and Selivanova, p. 1043. This article is highlighted in the In This Issue feature, p. 1027.

Thorectidiol A Isolated from the Marine Sponge Dactylospongia elegans Disrupts Interactions of the SARS-CoV-2 Spike Receptor Binding Domain with the Host ACE2 Receptor.

Thorectidiols isolated from the marine sponge Dactylospongia elegans (family Thorectidae, order Dictyoceratida) collected in Papua New Guinea are a family of symmetrical and unsymmetrical dimeric biphenyl meroterpenoid stereoisomers presumed to be products of oxidative phenol coupling of a co-occurring racemic monomer, thorectidol (3). One member of the family, thorectidiol A (1), has been isolated in its natural form, and its structure has been elucidated by analysis of NMR, MS, and ECD data. Acetylation of the sponge extract facilitated isolation of additional thorectidiol diacetate stereoisomers and the isolation of the racemic monomer thorectidol acetate (6). Racemic thorectidiol A (1) showed selective inhibition of the SARS-CoV-2 spike receptor binding domain (RBD) interaction with the host ACE2 receptor with an IC50 = 1.0 ± 0.7 µM.

Preclinical platforms to study therapeutic efficacy of human γδ T cells.

BACKGROUND: Gamma delta (γδ) T lymphocytes are promising candidate for adoptive T cell therapy, however, their treatment efficacy is not satisfactory. Vδ2 T cells are unique to primates and few suitable models are available to assay their anti-tumour function. METHODS: We tested human γδ T cell activation, tumour infiltration, and tumour-killing in four three-dimensional (3D) models, including unicellular, bicellular and multicellular melanoma spheroids, and patient-derived melanoma organoids. We studied the effects of checkpoint inhibitors on γδ T cells and performed a small molecule screen using these platforms. RESULTS: γδ T cells rapidly responded to melanoma cells and infiltrated melanoma spheroids better than αß T cells in PBMCs. Cancer-associated fibroblasts (CAFs) in bicellular spheroids, stroma cells in multicellular melanoma spheroids and inhibitory immune cells in organoids significantly inhibited immune cell infiltrates including γδ T cells and lessened their cytotoxicity to tumour cells. Tumour-infiltrating γδ T cells showed exhausted immunophenotypes with high checkpoints expression (CTLA-4, PD-1 and PD-L1). Immune checkpoint inhibitors increased γδ T cell infiltration of 3D models and killing of melanoma cells in all four 3D models. Our small molecule screen assay and subsequent mechanistic studies demonstrated that epigenetic modifiers enhanced the chemotaxis and cytotoxicity of γδ T cells through upregulating MICA/B, inhibiting HDAC6/7 pathway and downregulating the levels of PD-L1 and PD-L2 in CAFs and tumour cells. These compounds increased CXCR4 and CD107a expression, IFN-γ production and decreased PD-1 expression of γδ T cells. CONCLUSIONS: Tumour-infiltrating γδ T cells show exhausted immunophenotypes and limited anti-tumour capacity in melanoma 3D models. Checkpoint inhibitors and epigenetic modifiers enhance anti-tumour functions of γδ T cells. These four 3D models provided valuable preclinical platforms to test γδ T cell functions for immunotherapy.

Limited nutrient availability in the tumor microenvironment renders pancreatic tumors sensitive to allosteric IDH1 inhibitors.

Nutrient-deprived conditions in the tumor microenvironment (TME) restrain cancer cell viability due to increased free radicals and reduced energy production. In pancreatic cancer cells a cytosolic metabolic enzyme, wild-type isocitrate dehydrogenase 1 (wtIDH1), enables adaptation to these conditions. Under nutrient starvation, wtIDH1 oxidizes isocitrate to generate α-ketoglutarate (αKG) for anaplerosis and NADPH to support antioxidant defense. In this study, we show that allosteric inhibitors of mutant IDH1 (mIDH1) are potent wtIDH1 inhibitors under conditions present in the TME. We demonstrate that low magnesium levels facilitate allosteric inhibition of wtIDH1, which is lethal to cancer cells when nutrients are limited. Furthermore, the Food & Drug Administration (FDA)-approved mIDH1 inhibitor ivosidenib (AG-120) dramatically inhibited tumor growth in preclinical models of pancreatic cancer, highlighting this approach as a potential therapeutic strategy against wild-type IDH1 cancers.

Use of hyphenated analytical techniques to identify the bioactive constituents of Gunnera perpensa L., a South African medicinal plant, which potently inhibit SARS-CoV-2 spike glycoprotein-host ACE2 binding.

SARS-CoV-2, the causative agent of COVID-19, continues to cause global morbidity and mortality despite the increasing availability of vaccines. Alongside vaccines, antivirals are urgently needed to combat SARS-CoV-2 infection and spread, particularly in resource-limited regions which lack access to existing therapeutics. Small molecules isolated from medicinal plants may be able to block cellular entry by SARS-CoV-2 by antagonising the interaction of the viral spike glycoprotein receptor-binding domain (RBD) with the host angiotensin-converting enzyme II (ACE2) receptor. As the medicinal plant Gunnera perpensa L. is being used by some South African traditional healers for SARS-CoV-2/COVID-19 management, we hypothesised that it may contain chemical constituents that inhibit the RBD-ACE2 interaction. Using a previously described AlphaScreen-based protein interaction assay, we show here that the DCM:MeOH extract of G. perpensa readily disrupts RBD (USA-WA1/2020)-ACE2 interactions with a half-maximal inhibition concentration (IC50) of < 0.001 µg/mL, compared to an IC50 of 0.025 µg/mL for the control neutralising antibody REGN10987. Employing hyphenated analytical techniques like UPLC-IMS-HRMS (method developed and validated as per the International Conference on Harmonization guidelines), we identified two ellagitannins, punicalin (2.12% w/w) and punicalagin (1.51% w/w), as plant constituents in the DCM:MeOH extract of G. perpensa which antagonised RBD-ACE2 binding with respective IC50s of 9 and 29 nM. This good potency makes both compounds promising leads for development of future entry-based SARS-CoV-2 antivirals. The results also highlight the advantages of combining reverse pharmacology (based on medicinal plant use) with hyphenated analytical techniques to expedite identification of urgently needed antivirals.

Ghost mitochondria drive metastasis through adaptive GCN2/Akt therapeutic vulnerability.

Cancer metabolism, including in mitochondria, is a disease hallmark and therapeutic target, but its regulation is poorly understood. Here, we show that many human tumors have heterogeneous and often reduced levels of Mic60, or Mitofilin, an essential scaffold of mitochondrial structure. Despite a catastrophic collapse of mitochondrial integrity, loss of bioenergetics, and oxidative damage, tumors with Mic60 depletion slow down cell proliferation, evade cell death, and activate a nuclear gene expression program of innate immunity and cytokine/chemokine signaling. In turn, this induces epithelial-mesenchymal transition (EMT), activates tumor cell movements through exaggerated mitochondrial dynamics, and promotes metastatic dissemination in vivo. In a small-molecule drug screen, compensatory activation of stress response (GCN2) and survival (Akt) signaling maintains the viability of Mic60-low tumors and provides a selective therapeutic vulnerability. These data demonstrate that acutely damaged, "ghost" mitochondria drive tumor progression and expose an actionable therapeutic target in metastasis-prone cancers.

Allosteric Modulator KM822 Attenuates Behavioral Actions of Amphetamine in Caenorhabditis elegans through Interactions with the Dopamine Transporter DAT-1.

Aberrant dopamine (DA) signaling is associated with several psychiatric disorders, such as autism, bipolar disorder, addiction, and Parkinson's disease, and several medications that target the DA transporter (DAT) can induce or treat these disorders. In addition, psychostimulants, such as cocaine and D-amphetamine (AMPH), rely on the competitive interactions with the transporter's substrate binding site to produce their rewarding effects. Agents that exhibit noncompetitive, allosteric modulation of DAT remain an important topic of investigation due to their potential therapeutic applications. We previously identified a novel allosteric modulator of human DAT, KM822, that can decrease the affinity of cocaine for DAT and attenuate cocaine-elicited behaviors; however, whether DAT is the sole mediator of KM822 actions in vivo is unproven given the large number of potential off-target sites. Here, we provide in silico and in vitro evidence that the allosteric site engaged by KM822 is conserved between human DAT and Caenorhabditis elegans DAT-1. KM822 binds to a similar pocket in DAT-1 as previously identified in human DAT. In functional dopamine uptake assays, KM822 affects the interaction between AMPH and DAT-1 by reducing the affinity of AMPH for DAT-1. Finally, through a combination of genetic and pharmacological in vivo approaches we provide evidence that KM822 diminishes the behavioral actions of AMPH on swimming-induced paralysis through a direct allosteric modulation of DAT-1. More broadly, our findings demonstrate allosteric modulation of DAT as a behavior modifying strategy and suggests that Caenorhabditis elegans can be operationalized to identify and investigate the interactions of DAT allosteric modulators. SIGNIFICANCE STATEMENT: We previously demonstrated that the dopamine transporter (DAT) allosteric modulator KM822 decreases cocaine affinity for human DAT. Here, using in silico and in vivo genetic approaches, we extend this finding to interactions with amphetamine, demonstrating evolutionary conservation of the DAT allosteric site. In Caenorhabditis elegans, we report that KM822 suppresses amphetamine behavioral effects via specific interactions with DAT-1. Our findings reveal Caenorhabditis elegans as a new tool to study allosteric modulation of DAT and its behavioral consequences.

Targeting cancer metabolism in the era of precision oncology.

One hundred years have passed since Warburg discovered alterations in cancer metabolism, more than 70 years since Sidney Farber introduced anti-folates that transformed the treatment of childhood leukaemia, and 20 years since metabolism was linked to oncogenes. However, progress in targeting cancer metabolism therapeutically in the past decade has been limited. Only a few metabolism-based drugs for cancer have been successfully developed, some of which are in - or en route to - clinical trials. Strategies for targeting the intrinsic metabolism of cancer cells often did not account for the metabolism of non-cancer stromal and immune cells, which have pivotal roles in tumour progression and maintenance. By considering immune cell metabolism and the clinical manifestations of inborn errors of metabolism, it may be possible to isolate undesirable off-tumour, on-target effects of metabolic drugs during their development. Hence, the conceptual framework for drug design must consider the metabolic vulnerabilities of non-cancer cells in the tumour immune microenvironment, as well as those of cancer cells. In this Review, we cover the recent developments, notable milestones and setbacks in targeting cancer metabolism, and discuss the way forward for the field.