IspH inhibitors kill Gram-negative bacteria and mobilize immune clearance.

Isoprenoids are vital for all organisms, in which they maintain membrane stability and support core functions such as respiration1. IspH, an enzyme in the methyl erythritol phosphate pathway of isoprenoid synthesis, is essential for Gram-negative bacteria, mycobacteria and apicomplexans2,3. Its substrate, (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMBPP), is not produced in metazoans, and in humans and other primates it activates cytotoxic Vγ9Vδ2 T cells at extremely low concentrations4-6. Here we describe a class of IspH inhibitors and refine their potency to nanomolar levels through structure-guided analogue design. After modification of these compounds into prodrugs for delivery into bacteria, we show that they kill clinical isolates of several multidrug-resistant bacteria-including those from the genera Acinetobacter, Pseudomonas, Klebsiella, Enterobacter, Vibrio, Shigella, Salmonella, Yersinia, Mycobacterium and Bacillus-yet are relatively non-toxic to mammalian cells. Proteomic analysis reveals that bacteria treated with these prodrugs resemble those after conditional IspH knockdown. Notably, these prodrugs also induce the expansion and activation of human Vγ9Vδ2 T cells in a humanized mouse model of bacterial infection. The prodrugs we describe here synergize the direct killing of bacteria with a simultaneous rapid immune response by cytotoxic γδ T cells, which may limit the increase of antibiotic-resistant bacterial populations.

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.

Metabolic control of BRISC-SHMT2 assembly regulates immune signalling.

Serine hydroxymethyltransferase 2 (SHMT2) regulates one-carbon transfer reactions that are essential for amino acid and nucleotide metabolism, and uses pyridoxal-5'-phosphate (PLP) as a cofactor. Apo SHMT2 exists as a dimer with unknown functions, whereas PLP binding stabilizes the active tetrameric state. SHMT2 also promotes inflammatory cytokine signalling by interacting with the deubiquitylating BRCC36 isopeptidase complex (BRISC), although it is unclear whether this function relates to metabolism. Here we present the cryo-electron microscopy structure of the human BRISC-SHMT2 complex at a resolution of 3.8 Å. BRISC is a U-shaped dimer of four subunits, and SHMT2 sterically blocks the BRCC36 active site and inhibits deubiquitylase activity. Only the inactive SHMT2 dimer-and not the active PLP-bound tetramer-binds and inhibits BRISC. Mutations in BRISC that disrupt SHMT2 binding impair type I interferon signalling in response to inflammatory stimuli. Intracellular levels of PLP regulate the interaction between BRISC and SHMT2, as well as inflammatory cytokine responses. These data reveal a mechanism in which metabolites regulate deubiquitylase activity and inflammatory signalling.

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.

Cryo-EM structure of the dopamine transporter with a novel atypical non-competitive inhibitor bound to the orthosteric site.

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.

Design of novel and highly selective SARS-CoV-2 main protease inhibitors.

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 IC50 = 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.

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.

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.

Metabolomic Analysis and Antiviral Screening of a Marine Algae Library Yield Jobosic Acid (2,5-Dimethyltetradecanoic Acid) as a Selective Inhibitor of SARS-CoV-2.

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 (Mpro) 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.

Recent Advances in Metal-Catalyzed Approaches for the Synthesis of Quinazoline Derivatives.

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.

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.

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.

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.

BCL6 BTB-specific inhibitor reversely represses T-cell activation, Tfh cells differentiation, and germinal center reaction in vivo.

Inhibition of the BCL6 BTB domain results in killing Diffuse Large B-cell Lymphoma (DLBL) cells, reducing the T-cell dependent germinal center (GC) reaction in mice, and reversing GC hyperplasia in nonhuman primates. The available BCL6 BTB-specific inhibitors are poorly water soluble, thus, limiting their absorption in vivo and our understanding of therapeutic strategy targeting GC. We synthesized a prodrug (AP-4-287) from a potent BCL6 BTB inhibitor (FX1) with improved aqueous solubility and pharmacokinetics (PK) in mice. We also evaluated its in vivo biological activity on humoral immune responses using the sheep red blood cells (SRBC)-vaccination mouse model. AP-4-287 had a significant higher aqueous solubility and was readily converted to FX1 in vivo after intraperitoneally (i.p.) administration, but a shorter half-life in vivo. Importantly, AP-4-287 treatment led to a reversible effect on (1) the reduction in the frequency of splenic Ki67+ CD4+ T cells, Tfh cells, and GC B cells; (2) lower GC formation following vaccination; and (3) a decrease in the titers of antigen-specific IgG and IgM antibodies. Our study advances the preclinical development of drug targeting BCL6 BTB domain for the treatment of diseases that are associated with abnormal BCL6 elevation.

Selective inhibition of Ph-positive ALL cell growth through kinase-dependent and -independent effects by CDK6-specific PROTACs.

Expression of the cell cycle regulatory gene CDK6 is required for Philadelphia-positive (Ph+) acute lymphoblastic leukemia (ALL) cell growth, whereas expression of the closely related CDK4 protein is dispensable. Moreover, CDK6 silencing is more effective than treatment with the dual CDK4/6 inhibitor palbociclib in suppressing Ph+ ALL in mice, suggesting that the growth-promoting effects of CDK6 are, in part, kinase-independent in Ph+ ALL. Accordingly, we developed CDK4/6-targeted proteolysis-targeting chimeras (PROTACs) that inhibit CDK6 enzymatic activity in vitro, promote the rapid and preferential degradation of CDK6 over CDK4 in Ph+ ALL cells, and markedly suppress S-phase cells concomitant with inhibition of CDK6-regulated phospho-RB and FOXM1 expression. No such effects were observed in CD34+ normal hematopoietic progenitors, although CDK6 was efficiently degraded. Treatment with the CDK6-degrading PROTAC YX-2-107 markedly suppressed leukemia burden in mice injected with de novo or tyrosine kinase inhibitor-resistant primary Ph+ ALL cells, and this effect was comparable or superior to that of the CDK4/6 enzymatic inhibitor palbociclib. These studies provide "proof of principle" that targeting CDK6 with PROTACs that inhibit its enzymatic activity and promote its degradation represents an effective strategy to exploit the "CDK6 dependence" of Ph+ ALL and, perhaps, of other hematologic malignancies. Moreover, they suggest that treatment of Ph+ ALL with CDK6-selective PROTACs would spare a high proportion of normal hematopoietic progenitors, preventing the neutropenia induced by treatment with dual CDK4/6 inhibitors.

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.

The Natural Stilbenoid (-)-Hopeaphenol Inhibits Cellular Entry of SARS-CoV-2 USA-WA1/2020, B.1.1.7, and B.1.351 Variants.

Antivirals are urgently needed to combat the global SARS-CoV-2/COVID-19 pandemic, supplement existing vaccine efforts, and target emerging SARS-CoV-2 variants of concern. Small molecules that interfere with binding of the viral spike receptor binding domain (RBD) to the host angiotensin-converting enzyme II (ACE2) receptor may be effective inhibitors of SARS-CoV-2 cell entry. Here, we screened 512 pure compounds derived from natural products using a high-throughput RBD/ACE2 binding assay and identified (-)-hopeaphenol, a resveratrol tetramer, in addition to vatalbinoside A and vaticanol B, as potent and selective inhibitors of RBD/ACE2 binding and viral entry. For example, (-)-hopeaphenol disrupted RBD/ACE2 binding with a 50% inhibitory concentration (IC50) of 0.11 µM, in contrast to an IC50 of 28.3 µM against the unrelated host ligand/receptor binding pair PD-1/PD-L1 (selectivity index, 257.3). When assessed against the USA-WA1/2020 variant, (-)-hopeaphenol also inhibited entry of a VSVΔG-GFP reporter pseudovirus expressing SARS-CoV-2 spike into ACE2-expressing Vero-E6 cells and in vitro replication of infectious virus in cytopathic effect and yield reduction assays (50% effective concentrations [EC50s], 10.2 to 23.4 µM) without cytotoxicity and approaching the activities of the control antiviral remdesivir (EC50s, 1.0 to 7.3 µM). Notably, (-)-hopeaphenol also inhibited two emerging variants of concern, B.1.1.7/Alpha and B.1.351/Beta in both viral and spike-containing pseudovirus assays with similar or improved activities over the USA-WA1/2020 variant. These results identify (-)-hopeaphenol and related stilbenoid analogues as potent and selective inhibitors of viral entry across multiple SARS-CoV-2 variants of concern.

BCL6 BTB-specific inhibition via FX1 treatment reduces Tfh cells and reverses lymphoid follicle hyperplasia in Indian rhesus macaque (Macaca mulatta).

BACKGROUND: The BTB domain of B-cell lymphoma 6 (BCL6) protein was identified as a therapeutic target for B-cell lymphoma. This study compared the pharmacokinetics (PK) of the BCL6 BTB inhibitor (FX1) between mice and macaques, as well as evaluating its lymphoid suppressive effect in uninfected macaques with lymphoid hyperplasia. MATERIALS AND METHODS: Eight uninfected adult Indian rhesus macaques (Macaca mulatta) were used in the study, four animals carrying lymphoid tissue hyperplasia. Plasma FX1 levels were measured by HPLC-MS/MS. Lymph node biopsies were used for H&E and immunohistochemistry staining, as well as mononuclear cell isolation for flow cytometry analysis. RESULTS: Inhibition of the BCL6 BTB domain with FX1 led to a reduction in the frequency of GC, Tfh CD4+ , and Tfh precursor cells, as well as resolving lymphoid hyperplasia, in rhesus macaques. CONCLUSIONS: B-cell lymphoma 6 inhibition may represent a novel strategy to reduce hyperplastic lymphoid B-cell follicles and decrease Tfh cells.