Biology subjects

Reed, J. C.

Publications and source records attributed to Reed, J. C..

7 recordsLinked to original sources

bioRxiv2023

Protein Assembly Modulation: A New Approach to ALS Therapeutics

Amyotrophic Lateral Sclerosis (ALS) is a neurodegenerative disease with a complex, multifactorial pathophysiology, most commonly manifest as loss of motor neurons. We introduce a new mechanism of ALS pathogenesis via a novel drug-like small molecule series that targets protein disulfide isomerase (PDI) within a previously unappreciated transient and energy-dependent multi-protein complex. This novel drug was found to have activity in cellular models for both familial and sporadic ALS, as well as in transgenic worms, flies, and mice bearing a diversity of human genes with ALS-associated mutations. These compounds were initially identified as modulators of human immunodeficiency virus (HIV) capsid assembly in cell-free protein synthesis and assembly (CFPSA) systems, with demonstrated antiviral activity in cell culture. Their advancement as ALS-therapeutics, and the subsequent separation of activity against HIV and ALS in chemical subseries through structure-activity-relationship optimization, may provide insights into the molecular mechanisms governing pathophysiology of disordered homeostasis relevant to ALS.

neuroscience↗

bioRxiv2022

Small Molecule Assembly Modulators with Pan-Cancer Therapeutic Efficacy

Two structurally-unrelated small molecule chemotypes, represented by compounds PAV-617 and PAV-951 with antiviral activity in cell culture against monkeypox virus (MPXV) and human immunodeficiency virus (HIV) respectively, were studied for anti-cancer efficacy. Each exhibited apparent pan-cancer cytotoxicity, reasonable pharmacokinetics, and non-toxicity in mice at active concentrations. Anti-tumor properties of both chemotypes, were validated in mouse xenografts against A549 human lung cancer and, for one of the chemotypes, against HT-29 colorectal cancer. The targets of these compounds are unconventional: each binds to a different transient, energy-dependent multi-protein complex containing the protein TRIM28/KAP1, an allosteric modulator known to regulate mechanisms underlying viral and nonviral disease states including cancer. Treatment with these compounds alters the target multi-protein complexes in a manner consistent with allosteric modulation as their mechanism of action. These compounds appear to remove a block, crucial for cancer survival and progression, on the homeostatic linkage of uncontrolled cellular proliferation to apoptosis. These compounds provide starting points for development of next-generation non-toxic, pan-cancer therapeutics.

cancer biology↗

bioRxiv2021

ATM phosphorylates PP2A subunit A resulting in nuclearexport and spatiotemporal regulation of the DNA damageresponse

Ataxia telangiectasia mutated (ATM) is a serine-threonine protein kinase and important regulator of the DNA damage response (DDR). One critical ATM target is the structural subunit A (PR65) of protein phosphatase 2A (PP2A), known to regulate diverse cellular processes such as mitosis and cell growth as well as dephosphorylating many proteins during the recovery from the DDR. We generated mouse embryonic fibroblasts expressing PR65-WT, -S401A (cannot be phosphorylated), and -S401D (phosphomimetic) transgenes. Significantly, S401 mutants exhibited extensive chromosomal aberrations, impaired DNA double-strand break (DSB) repair and underwent increased mitotic catastrophe after radiation. Our study demonstrates that the phosphorylation of a single, critical PR65 amino acid (S401) by ATM fundamentally controls the DDR, and balances DSB repair quality, cell survival and growth by spatiotemporal PR65 nuclear-cytoplasmic shuttling mediated by the nuclear export receptor CRM1.

cancer biology↗

bioRxiv2021

From COVID-19 to the Common Cold: Novel Host-Targeted, Pan-Respiratory Antiviral Small Molecule Therapeutics

We present a novel small molecule antiviral chemotype that was identified by an unconventional cell-free protein synthesis and assembly-based phenotypic screen for modulation of viral capsid assembly. Activity of PAV-431, a representative compound from the series, has been validated against infectious virus in multiple cell culture models for all six families of viruses causing most respiratory disease in humans. In animals this chemotype has been demonstrated efficacious for Porcine Epidemic Diarrhea Virus (a coronavirus) and Respiratory Syncytial Virus (a paramyxovirus). PAV-431 is shown to bind to the protein 14-3-3, a known allosteric modulator. However, it only appears to target the small subset of 14-3-3 which is present in a dynamic multi-protein complex whose components include proteins implicated in viral lifecycles and in innate immunity. The composition of this target multi-protein complex appears to be modified upon viral infection and largely restored by PAV-431 treatment. Our findings suggest a new paradigm for understanding, and drugging, the host-virus interface, which leads to a new clinical therapeutic strategy for treatment of respiratory viral disease.

biochemistry↗

bioRxiv2020

Identification of an antiretroviral small molecule that appears to be a host-targeting inhibitor of HIV-1 assembly

Given the projected increase in multidrug resistant HIV-1, there is an urgent need for development of antiretrovirals that act on virus life-cycle stages that are not targeted by antiretrovirals currently in use. Host-targeting drugs are of particular interest because they can offer a high barrier to resistance. Here we report identification of two related small molecules that inhibit HIV-1 late events, a stage of the HIV-1 life cycle for which potent and specific inhibitors are lacking. This chemotype was discovered using cell-free protein synthesis and assembly systems that recapitulate intracellular host-catalyzed viral capsid assembly pathways. These compounds inhibit replication of HIV-1 in human T cell lines and PBMCs and are effective against a primary isolate. They reduce virus production, likely by inhibiting a post-translational step in HIV-1 Gag assembly. Notably, the compound colocalizes with HIV-1 Gag in situ; however, unexpectedly, selection experiments failed to identify compound-specific resistance mutations in gag or pol, even though known resistance mutations developed in a parallel nelfinavir selection. Thus, we hypothesized that instead of binding to Gag directly, these compounds might localize to assembly intermediates, the intracellular multiprotein complexes containing Gag and host factors that are formed during immature HIV-1 capsid assembly. Indeed, imaging of infected cells showed colocalization of the compound with two host enzymes found in assembly intermediates, ABCE1 and DDX6. While the exact target and mechanism of action of this chemotype remain to be determined, these findings suggest that these compounds represent first-in-class, host-targeting inhibitors of intracellular events in HIV-1 assembly. IMPORTANCEThe success of antiretroviral treatment for HIV-1 is at risk of being undermined by the growing problem of drug resistance. Thus, there is a need to identify antiretrovirals that act on viral life cycle stages not targeted by drugs in use, such as the events of HIV-1 Gag assembly. To address this gap, we developed a compound screen that recapitulates the intracellular events of HIV-1 assembly, including viral-host interactions that promote assembly. This effort led to identification of a new chemotype that inhibits HIV-1 replication at nanomolar concentrations by inhibiting virus production. This compound colocalized with Gag and two host enzymes that facilitate capsid assembly but resistance selection did not result in compound-specific mutations in gag, suggesting that the chemotype does not directly target Gag. We hypothesize that this chemotype may represent a first-in-class inhibitor of virus production that acts by targeting a viral-host complex important for HIV-1 Gag assembly.

microbiology↗

bioRxiv2017

The ABCE1 capsid assembly pathway is conserved between primate lentiviruses and the non-primate lentivirus feline immunodeficiency virus

During immature capsid assembly in cells, the Gag protein of HIV-1 and other primate lentiviruses co-opts a host RNA granule, forming a pathway of assembly intermediates that contains host components, including two cellular enzymes shown to facilitate assembly, ABCE1 and DDX6. Here we asked whether a non-primate lentivirus, feline immunodeficiency virus (FIV), also forms such RNA-granule-derived intracellular capsid assembly intermediates. First, we found that, unlike for HIV-1, the FIV completed immature capsid and the largest putative assembly intermediate are unstable during analysis. Next, we identified in situ cross-linking conditions that overcame this problem and revealed the presence of FIV Gag complexes that correspond in size to early and late HIV-1 assembly intermediates. Because assembly-defective HIV-1 Gag mutants are arrested at specific intracellular assembly intermediates, we asked if a similar arrest is also observed for FIV. We analyzed four FIV Gag mutants, including three not previously studied that we identified based on sequence and structural similarity to HIV-1 Gag, and found that each is assembly-defective and arrested at the same intermediate as the corresponding HIV-1 mutant. Further evidence that these FIV Gag-containing complexes correspond to assembly intermediates came from co-immunoprecipitation studies demonstrating that FIV Gag is associated with ABCE1 and DDX6, as shown previously for HIV-1. Finally, we validated these co-immunoprecipitations with a proximity ligation assay that revealed co-localization between assembly-competent FIV Gag and ABCE1 in situ. Together, these data offer novel structure-function insights and indicate that primate and non-primate lentiviruses form intracellular capsid assembly intermediates derived from ABCE1-containing RNA granules.\n\nImportanceLike HIV-1, FIV Gag assembles into immature capsids; however, it is not known whether FIV Gag progresses through a pathway of immature capsid assembly intermediates derived from host RNA granules, as shown for HIV-1 Gag. Here we asked whether FIV Gag forms complexes similar in size to HIV-1 assembly intermediates and if FIV Gag is associated with ABCE1 and DDX6, two host enzymes that facilitate HIV-1 immature capsid assembly that are found in HIV-1 assembly intermediates. Our studies identified FIV Gag-containing complexes that closely resemble HIV-1 capsid assembly intermediates, showed that known and novel assembly-defective FIV Gag mutants fail to progress past these putative intermediates, and utilized biochemical and imaging approaches to demonstrate association of FIV Gag with ABCE1 and DDX6. Thus, we conclude that viral-host interactions important for immature capsid assembly are conserved between primate and non-primate lentiviruses, and could yield important targets for future antiviral strategies.

microbiology↗

bioRxiv2017

HIV-1 initiates genomic RNA packaging in a unique subset of host RNA granules

How HIV-1 genomic RNA (gRNA) is packaged into assembling virus remains unclear. Here, we use biochemical and in situ approaches to identify the complex in which the capsid protein Gag first associates with gRNA, termed the packaging initiation complex. First, we show that in the absence of assembling Gag, non-nuclear non-translating gRNA is nearly absent from the soluble fraction of provirus-expressing cells, and is found instead primarily in complexes >30S. When we express a Gag mutant known to be arrested at packaging initiation, we find only one complex containing Gag and gRNA; thus, this complex corresponds to the packaging initiation complex. This [~]80S complex also contains two cellular facilitators of assembly, ABCE1 and the RNA granule protein DDX6, and therefore corresponds to a co-opted host RNA granule and a previously described capsid assembly intermediate. Additionally, we find this granule-derived packaging initiation complex in HIV-1-infected H9 T cells, and demonstrate that wild-type Gag forms both the packaging initiation complex and a larger granule-derived complex corresponding to a late packaging/assembly intermediate. We also demonstrate that packaging initiation complexes are far more numerous than P bodies in situ. Finally, we show that Gag enters the [~]80S granule to form the packaging initiation complex via a two-step mechanism. In a step that is independent of a gRNA-binding domain, Gag enters a broad class of RNA granules, most of which lack gRNA. In a second step that is dependent on the gRNA-binding nucleocapsid domain of Gag or a heterologous gRNA-binding domain, Gag enters a gRNA-containing subset of these granules. Thus, we conclude that packaging in cells does not result from random encounters between Gag and gRNA; instead our data support a fundamentally different model in which Gag is directed to gRNA within a unique host RNA granule to initiate this critical event in HIV-1 replication.\n\nNontechnical SummaryTo form infectious virus, the HIV-1 capsid protein Gag must associate with and package the viral genomic RNA (gRNA) during the virus assembly process. HIV-1 Gag first associates with gRNA in the cytoplasm, forming a complex termed the packaging initiation complex; this complex subsequently targets to the plasma membrane where Gag completes the assembly and packaging process before releasing the virus from the cell. Although the packaging initiation complex is critical for infectious virus formation, its identity and composition, and the mechanism by which it is formed, remain unknown. Here we identify the packaging initiation complex, and demonstrate that it corresponds to a host RNA granule that is co-opted by the virus. RNA granules are diverse complexes utilized by host cells for all aspects of RNA storage and metabolism besides translation. Our study also defines the mechanism by which HIV-1 Gag enters this host RNA granule to form the packaging initiation complex, and reveal that it involves two steps that depend on different regions of Gag. Our finding that Gag co-opts a poorly studied host complex to first associate with gRNA during packaging provides a new paradigm for understanding this critical event in the viral life cycle.

microbiology↗