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.

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.

Fragile X protein mitigates TDP-43 toxicity by remodeling RNA granules and restoring translation.

RNA dysregulation is a newly recognized disease mechanism in amyotrophic lateral sclerosis (ALS). Here we identify Drosophila fragile X mental retardation protein (dFMRP) as a robust genetic modifier of TDP-43-dependent toxicity in a Drosophila model of ALS. We find that dFMRP overexpression (dFMRP OE) mitigates TDP-43 dependent locomotor defects and reduced lifespan in Drosophila. TDP-43 and FMRP form a complex in flies and human cells. In motor neurons, TDP-43 expression increases the association of dFMRP with stress granules and colocalizes with polyA binding protein in a variant-dependent manner. Furthermore, dFMRP dosage modulates TDP-43 solubility and molecular mobility with overexpression of dFMRP resulting in a significant reduction of TDP-43 in the aggregate fraction. Polysome fractionation experiments indicate that dFMRP OE also relieves the translation inhibition of futsch mRNA, a TDP-43 target mRNA, which regulates neuromuscular synapse architecture. Restoration of futsch translation by dFMRP OE mitigates Futsch-dependent morphological phenotypes at the neuromuscular junction including synaptic size and presence of satellite boutons. Our data suggest a model whereby dFMRP is neuroprotective by remodeling TDP-43 containing RNA granules, reducing aggregation and restoring the translation of specific mRNAs in motor neurons.

Ubiquilin-2 (UBQLN2) binds with high affinity to the C-terminal region of TDP-43 and modulates TDP-43 levels in H4 cells: characterization of inhibition by nucleic acids and 4-aminoquinolines.

Recently, it was reported that mutations in the ubiquitin-like protein ubiquilin-2 (UBQLN2) are associated with X-linked amyotrophic lateral sclerosis (ALS), and that both wild-type and mutant UBQLN2 can co-localize with aggregates of C-terminal fragments of TAR DNA binding protein (TDP-43). Here, we describe a high affinity interaction between UBQLN2 and TDP-43 and demonstrate that overexpression of both UBQLN2 and TDP-43 reduces levels of both exogenous and endogenous TDP-43 in human H4 cells. UBQLN2 bound with high affinity to both full length TDP-43 and a C-terminal TDP-43 fragment (261-414 aa) with KD values of 6.2nM and 8.7nM, respectively. Both DNA oligonucleotides and 4-aminoquinolines, which bind to TDP-43, also inhibited UBQLN2 binding to TDP-43 with similar rank order affinities compared to inhibition of oligonucleotide binding to TDP-43. Inhibitor characterization experiments demonstrated that the DNA oligonucleotides noncompetitively inhibited UBQLN2 binding to TDP-43, which is consistent with UBQLN2 binding to the C-terminal region of TDP-43. Interestingly, the 4-aminoquinolines were competitive inhibitors of UBQLN2 binding to TDP-43, suggesting that these compounds also bind to the C-terminal region of TDP-43. In support of the biochemical data, co-immunoprecipitation experiments demonstrated that both TDP-43 and UBQLN2 interact in human neuroglioma H4 cells. Finally, overexpression of UBQLN2 in the presence of overexpressed full length TDP-43 or C-terminal TDP-43 (170-414) dramatically lowered levels of both full length TDP-43 and C-terminal TDP-43 fragments (CTFs). Consequently, these data suggest that UBQLN2 enhances the clearance of TDP-43 and TDP-43 CTFs and therefore may play a role in the development of TDP-43 associated neurotoxicity.

Molecular glues that inhibit specific Zn2+-dependent DUB activity and inflammation.

Deubiquitylases (DUBs) play a pivotal role in cell signalling and are often regulated by homo- or hetero-interactions within protein complexes. The BRCC36 isopeptidase complex (BRISC) regulates inflammatory signalling by selectively cleaving K63-linked polyubiquitin chains on Type I interferon receptors (IFNAR1). BRCC36 is a Zn2+-dependent JAMM/MPN DUB, a challenging ubiquitin protease class for the design of selective inhibitors. We identified first-in-class DUB inhibitors that act as BRISC molecular glues (BLUEs). BLUEs inhibit DUB activity by stabilising a BRISC dimer consisting of 16 subunits. The BLUE-stabilised BRISC dimer is an autoinhibited conformation, whereby the active sites and interactions with the recruiting subunit SHMT2 are blocked. This unique mode of action leads to highly selective inhibitors for BRISC over related complexes with the same catalytic subunit, splice variants and other JAMM/MPN DUBs. Structure-guided inhibitor resistant mutants confirm BLUEs on-target activity in cells, and BLUE treatment results in reduced interferon-stimulated gene (ISG) expression in human peripheral blood mononuclear cells from Scleroderma patients, a disease linked with aberrant IFNAR1 activation. BLUEs represent a new class of molecules with potential utility in Type I interferon-mediated diseases and a template for designing selective inhibitors of large protein complexes by promoting protein-protein interactions instead of blocking them.

Novel inhibitors of heat shock protein Hsp70-mediated luciferase refolding that bind to DnaJ.

Inhibitors of both heat shock proteins Hsp90 and Hsp70 have been identified in assays measuring luciferase refolding containing rabbit reticulocyte lysate or purified chaperone components. Here, we report the discovery of a series of phenoxy-N-arylacetamides that disrupt Hsp70-mediated luciferase refolding by binding to DnaJ, the bacterial homolog of human Hsp40. Inhibitor characterization experiments demonstrated negative cooperativity with respect to DnaJ and luciferase concentration, but varying the concentration of ATP had no effect on potency. Thermal shift analysis suggested a direct interaction with DnaJ, but not with Hsp70. These compounds may be useful tools for studying DnaJ/Hsp40 in various cellular processes.

Novel sulfamoyl benzamides as selective CB(2) agonists with improved in vitro metabolic stability.

A lead optimization campaign in our previously reported sulfamoyl benzamide class of CB(2) agonists was conducted to improve the in vitro metabolic stability profile in this series while retaining high potency and selectivity for the CB(2) receptor. From this study, compound 14, N-(3,4-dimethyl-5-(morpholinosulfonyl)phenyl)-2,2-dimethylbutanamide, was identified as a potent and selective CB(2) agonist exhibiting moderate in vitro metabolic stability and oral bioavailability. Compound 14 demonstrated in vivo efficacy in a rat model of post-surgical pain.

Novel pyridine derivatives as potent and selective CB2 cannabinoid receptor agonists.

Replacement of the phenyl ring in our previous (morpholinomethyl)aniline carboxamide cannabinoid receptor ligands with a pyridine ring led to the discovery of a novel chemical series of CB2 ligands. Compound 3, that is, 2,2-dimethyl-N-(5-methyl-4-(morpholinomethyl)pyridin-2-yl)butanamide was identified as a potent and selective CB2 agonist exhibiting in vivo efficacy after oral administration in a rat model of neuropathic pain.

Discovery of N-(3-(morpholinomethyl)-phenyl)-amides as potent and selective CB2 agonists.

Recently sulfamoyl benzamides were identified as a novel series of cannabinoid receptor ligands. Replacing the sulfonamide functionality and reversing the original carboxamide bond led to the discovery of N-(3-(morpholinomethyl)-phenyl)-amides as potent and selective CB(2) agonists. Selective CB(2) agonist 31 (K(i)=2.7; CB(1)/CB(2)=190) displayed robust activity in a rodent model of postoperative pain.

CB2 selective sulfamoyl benzamides: optimization of the amide functionality.

Previous research within our laboratories identified sulfamoyl benzamides as novel cannabinoid receptor ligands. Optimization of the amide linkage led to the reverse amide 40. The compound exhibited robust antiallodynic activity in a rodent pain model when administered intraperitoneally. Efficacy after oral administration was observed only when ABT, a cytochrome P450 suicide inhibitor, was coadministered.

Structure-based design of small-molecule inhibitors of EBNA1 DNA binding blocks Epstein-Barr virus latent infection and tumor growth.

Epstein-Barr virus (EBV) is a DNA tumor virus responsible for 1 to 2% of human cancers including subtypes of Burkitt's lymphoma, Hodgkin's lymphoma, gastric carcinoma, and nasopharyngeal carcinoma (NPC). Persistent latent infection drives EBV-associated tumorigenesis. Epstein-Barr nuclear antigen 1 (EBNA1) is the only viral protein consistently expressed in all EBV-associated tumors and is therefore an attractive target for therapeutic intervention. It is a multifunctional DNA binding protein critical for viral replication, genome maintenance, viral gene expression, and host cell survival. Using a fragment-based approach and x-ray crystallography, we identify a 2,3-disubstituted benzoic acid series that selectively inhibits the DNA binding activity of EBNA1. We characterize these inhibitors biochemically and in cell-based assays, including chromatin immunoprecipitation and DNA replication assays. In addition, we demonstrate the potency of EBNA1 inhibitors to suppress tumor growth in several EBV-dependent xenograft models, including patient-derived xenografts for NPC. These inhibitors selectively block EBV gene transcription and alter the cellular transforming growth factor-ß (TGF-ß) signaling pathway in NPC tumor xenografts. These EBNA1-specific inhibitors show favorable pharmacological properties and have the potential to be further developed for the treatment of EBV-associated malignancies.

Use of receptor chimeras to identify small molecules with high affinity for the dynorphin A binding domain of the kappa opioid receptor.

A series of 2-substituted sulfamoyl arylacetamides of general structure 2 were prepared as potent kappa opioid receptor agonists and the affinities of these compounds for opioid and chimeric receptors were compared with those of dynorphin A. Compounds 2e and 2i were identified as non-peptide small molecules that bound to chimeras 3 and 4 with high affinities similar to dynorphin A, resulting in K(i) values of 1.5 and 1.2 nM and 1.3 and 2.2 nM, respectively.

Novel trans-3,4-dimethyl-4-(3-hydroxyphenyl)piperidines as mu opioid receptor antagonists with improved opioid receptor selectivity profiles.

A series of N-substituted trans-3,4-dimethyl-4-(3-hydroxyphenyl)piperidines, mu opioid receptor antagonists, analogs of alvimopan, were prepared using solid phase methodology. This study led to the identification of a highly selective mu opioid receptor antagonist, which interacts selectively with mu peripheral receptors.

Discovery of a series of aminopiperidines as novel iNOS inhibitors.

Nitric oxide (NO), a mediator of various physiological and pathophysiological processes, is synthesized by three isozymes of nitric oxide synthase (NOS). Potential candidate clinical drugs should be devoid of inhibitory activity against endothelial NOS (eNOS), since eNOS plays an important role in maintaining normal blood pressure and flow. A new series of aminopiperidines as potent inhibitors of iNOS were identified from a HTS lead. From this study, we identified compound 33 as a potent iNOS inhibitor, with >25-fold selectivity over eNOS and 16-fold selectivity over nNOS.

Sulfamoyl benzamides as novel CB2 cannabinoid receptor ligands.

Sulfamoyl benzamides were identified as a novel series of cannabinoid receptor ligands. Starting from a screening hit 8 that had modest affinity for the cannabinoid CB(2) receptor, a parallel synthesis approach and initial SAR are described, leading to compound 27 with 120-fold functional selectivity for the CB(2) receptor. This compound produced robust antiallodynic activity in rodent models of postoperative pain and neuropathic pain without traditional cannabinergic side effects.

Pharmacological characterization of human kappa/mu opioid receptor chimeras that retain high affinity for dynorphin A.

Arylacetamide analgesics that stimulate kappa opioid receptors in the central nervous system mediate dysphoria and psychosis as well as analgesia. However, the naturally occurring peptide agonist, dynorphin A, is analgesic in the absence of dysphoria and psychosis, indicating that the therapeutic effects of kappa opioid agonists may be separated from their side effects. As part of our effort to discover kappa opioid receptor analgesics lacking side effects, we designed and constructed two mu/kappa chimeric receptors, composed primarily of amino acid residues derived from the mu opioid receptor, that were expected to bind dynorphin A with high affinity. In one, extracellular loop 2 and transmembrane domain 4 were derived from the kappa opioid receptor and in the other, only extracellular loop 2 was derived from the kappa opioid receptor. Most competitors of [(3)H]diprenorphine binding from a variety of structural classes bound to the chimeras with affinities similar to those with which they bound to the mu opioid receptor. In contrast, dynorphin A analogs bound to the chimeras with affinities similar to those with which they bound to the kappa opioid receptor. Pharmacological characterization of [(35)S]GTPgammaS binding mediated by the chimera with extracellular loop 2 derived from the kappa opioid receptor showed that it behaved as if it were mu opioid receptor with high affinity for dynorphin A analogs. These chimeras may be useful in identifying novel kappa receptor agonists that bind to the second extracellular loop of the receptor and share the desirable therapeutic profile of dynorphin A.

[(3)H]Alvimopan binding to the micro opioid receptor: comparative binding kinetics of opioid antagonists.

Alvimopan is a novel peripheral micro opioid antagonist in clinical development for the management of post-operative ileus and opioid-induced bowel dysfunction. We hypothesized that the long duration of action of alvimopan might be related to a slower dissociation rate from the micro opioid receptor compared to other shorter acting antagonists. The dissociation rate of alvimopan from the micro opioid receptor (t(1/2)=30--44 min) was comparable to that of the long acting partial agonist buprenorphine (t(1/2)=44 min), but was slower than those of the antagonists naloxone (t(1/2)=0.82 min) and N-methylnaltrexone (t(1/2)=0.46 min). Also, increases in the apparent affinities and potencies of buprenorphine and alvimopan, but not of naloxone and methylnaltrexone, were observed upon preincubation with the micro opioid receptor. Consistent with its long duration of action, alvimopan has a slow dissociation rate from the micro opioid receptor compared to other shorter acting antagonists and may be more potent if administered prior to dosing with exogenous opioids.

Structure-activity relationships of dynorphin a analogues modified in the address sequence.

The peptide [Pro3]Dyn A(1-11)-NH2 2 exhibits high affinity (K(i) = 2.4 nM) and over 2000-fold selectivity for the opioid receptor. Stepwise removal of the C-terminal residues from this ligand demonstrated that its positively charged Arg residues, particularly Arg6 and Arg7, were crucial for binding to the kappa receptor. Analogues shorter than seven amino acids lacked significant affinity for opioid receptors. Comparison with a series of truncated analogues of Dyn A showed that the relative losses in binding potency differed only slightly between the two series. The neutral residues Ile8 and Pro10 could be removed without significant loss in affinity for the kappa receptor. Their replacement, in the Pro3 analogue, with additional Arg residues led to analogues with improved kappa affinity (e.g., [Pro3,Arg8]Dyn A(1-11)-NH2 20: K(i)(kappa) = 0.44 nM). This type of modification did not compromise the high kappa selectivity of the Pro3 analogues. These findings support the view that a negatively charged domain in the putative second extracellular loop of the kappa receptor selectively recognizes residues 6-11 of dynorphin through electrostatic interactions. As with parent compound 2, analogue 20 and related compounds displayed kappa antagonist properties.

Synthesis and biological activities of cyclic lanthionine enkephalin analogues: delta-opioid receptor selective ligands.

The synthesis and biological test results of a series of enkephalin analogues incorporating the lanthionine modification are presented. The syntheses of four monosulfide-bridged analogues of enkephalins, Tyr-c[D-Ala(L)-Gly-Phe-D-Ala(L)]-OH (1a), Tyr-c[D-Val(L)-Gly-Phe-D-Ala(L)]-OH (1b), Tyr-c[D-Ala(L)-Gly-Phe-Ala(L)]-OH (1c), and Tyr-c[D-Val(L)-Gly-Phe-Ala(L)]-OH (1d), where Ala(L) and Val(L) denote the lanthionine amino acid ends linked by a monosulfide bridge to form the lanthionine structure, were successfully carried out via preparation of the linear peptide on solid support and cyclization in solution. In vitro binding assays against mu-, delta-, and kappa-opioid receptors and in vitro tests using GPI and MVD assays revealed that the dimethyl lanthionine analogues 1b and 1d, denoted as D-Val(L) in position 2, show substantial selectivity toward the delta-opioid receptor, while the unsubstituted analogues 1a and 1c, denoted as D-Ala(L) in position 2, bind to both mu- and delta-opioid receptors. The in vivo thermal escape assay by intrathecal administration showed that the analogues 1b and 1d are among the most potent ligands at producing antinociception through the delta-opioid receptor. The picomolar potencies of analogues 1a and 1c in the intrathecal (it.) assay strongly indicate that mu- and delta-opioid receptors interact synergistically to modulate the antinociceptive responses.

Antipruritic and antihyperalgesic actions of loperamide and analogs.

Loperamide and three of its analogs were evaluated for their ability to inhibit binding to cloned human opioid receptor subtypes and to produce antipruritus and antinociception following local s.c. administration to rodents. All four compounds were fully efficacious agonists with affinities of 2 to 4 nM for the cloned human mu opioid receptor. Local s.c. injection of loperamide, ADL 01-0001 or ADL 01-0002 at the same site as the introduction of the pruritogenic compound 48/80 resulted in antipruritic activity in a mouse model of itch. Similarly, i.paw or i.pl. administration of compounds ADL 01-0001, ADL 01-0002 and ADL 01-0003 to inflamed paws caused potent antinociception, inhibiting late phase formalin-induced flinching, Freund's adjuvant-induced mechanical hyperalgesia and tape stripping-induced mechanical hyperalgesia. Loperamide and its analogs were efficacious in animal models of itch and inflammatory pain, and may have potential therapeutic utility as antipruritic and antihyperalgesic agents.