Biology subjects

Smithgall, T. E.

Publications and source records attributed to Smithgall, T. E..

5 recordsLinked to original sources

bioRxiv2026

HIV-1 Nef Homodimerization as a Structural Mechanism for Kinase Activation and Small Molecule Inhibitor Action

The HIV-1 accessory protein Nef plays a central role in viral pathogenesis by enhancing viral replication, modulating cellular signaling, and evading immune recognition, making it a compelling target for therapeutic intervention. Nef lacks enzymatic activity and instead functions through diverse interactions with host cell proteins including the Src-family tyrosine kinase, Hck. Kinase activation may requires Nef homodimerization, as mutations disrupting the Nef dimer interface impair kinase activation as well as many other Nef functions. In the present study, we investigated the structural consequences of dimer interface mutations and their impact on Nef interactions with Hck regulatory domains. Using size-exclusion chromatography, multi-angle light scattering and crystallography, we found that mutations at dimer interface residues Leu112 and Phe121 abolish recombinant Nef protein dimerization while preserving the overall Nef fold, resulting in monomeric 1:1 complexes with Hck SH3 or SH3-SH2 domain proteins. These findings demonstrate that the broad phenotypic effects of interface mutations arise from loss of Nef dimerization rather than global misfolding or perturbation of SH3 binding. We also investigated the effects of small molecule Nef inhibitors on homodimer formation. These compounds, like the dimerization-defective mutations, suppress kinase activation, viral replication and restore immune recognition of HIV-infected cells. Using a SplitFAST fluorescence complementation assay, we provide direct evidence that these inhibitors disrupt Nef homodimer formation in solution. Co-crystallization of a wild-type Nef:SH3 complex with an inhibitor also prevented homodimer formation. Computational docking identified a shared pocket for six active Nef inhibitors formed by the Nef dimer interface but lost in the monomer. Together, our findings support homodimerization as a structural feature essential for many Nef functions and validate disruption of this interface as a promising therapeutic strategy against HIV-1.

microbiology↗

bioRxiv2025

Small molecule intervention of actin-binding protein profilin1 reduces tumor angiogenesis in renal cell carcinoma

Angiogenesis plays a key role in the development and progression of renal cell carcinoma (RCC). Actin-binding protein profilin-1 (Pfn1) is overexpressed in clear cell RCC predominantly in tumor-associated vascular endothelial cells (ECs). We previously demonstrated that that EC-selective (over)expression of Pfn1 accelerates RCC progression, and conversely, genetic loss of EC-Pfn1 dramatically inhibits tumor angiogenesis impeding tumor initiation and/or progression in RCC, suggesting that Pfn1 could be an actionable therapeutic target in RCC. In this study, we demonstrate that 4,4-((4-bromophenyl)methylene)bis(3,5-dimethyl-1H-pyrazole), a small molecule that we had previously identified as an inhibitor of Pfn1-actin interaction, directly binds to Pfn1 and attenuates tumor angiogenesis when directly administered into subcutaneous RCC tumors. Next, we undertook a chemical optimization approach to design and synthesize 4,4-((4-(trifluoromethyl)phenyl)methylene)bis(3,5-dimethyl-1H-pyrazole), a structural analog of our originally identified inhibitor, that exhibits improved anti-angiogenic efficacy in vitro and in vivo. Finally, we demonstrate that Pfn1 inhibitor is amenable to lipid microbubble encapsulation and release in the tumor microenvironment (TME) by ultrasound-mediated disruption of circulating microbubbles to achieve anti-angiogenic and anti-tumor benefit. In summary, our findings suggest that tumor-localized release of Pfn1 inhibitor could be a potential therapeutic strategy in RCC.

cell biology↗

bioRxiv2024

An SH3-binding allosteric modulator stabilizes the global conformation of the AML-associated Src-family kinase, Hck

While ATP-site inhibitors for protein-tyrosine kinases are often effective drugs, their clinical utility can be limited by off-target activity and acquired resistance mutations due to the conserved nature of the ATP-binding site. However, combining ATP-site and allosteric kinase inhibitors can overcome these shortcomings in a double-drugging framework. Here we explored the allosteric effects of two pyrimidine diamines, PDA1 and PDA2, on the conformational dynamics and activity of the Src-family tyrosine kinase Hck, a promising drug target for acute myeloid leukemia. Using 1H-15N HSQC NMR, we mapped the binding site for both analogs to the SH3 domain. Despite the shared binding site, PDA1 and PDA2 had opposing effects on near-full-length Hck dynamics by hydrogen-deuterium exchange mass spectrometry, with PDA1 stabilizing and PDA2 disrupting the overall kinase conformation. Kinase activity assays were consistent with these observations, with PDA2 enhancing kinase activity while PDA1 was without effect. Molecular dynamics simulations predicted selective bridging of the kinase domain N-lobe and SH3 domain by PDA1, a mechanism of allosteric stabilization supported by site-directed mutagenesis of N-lobe contact sites. Cellular thermal shift assays confirmed SH3 domaindependent interaction of PDA1 with wild-type Hck in myeloid leukemia cells and with a kinase domain gatekeeper mutant (T338M). These results identify PDA1 as a starting point for Src-family kinase allosteric inhibitor development that may work in concert with ATP-site inhibitors to suppress the evolution of resistance.

pharmacology and toxicology↗

bioRxiv2023

PROTAC-mediated Degradation of HIV-1 Nef Efficiently Restores Cell-surface CD4 and MHC-I Expression and Blocks HIV-1 Replication

The HIV-1 Nef accessory factor is critical to the viral life cycle in vivo where it promotes immune escape of HIV-infected cells and viral persistence. While these features identify Nef as an attractive antiretroviral drug target, Nef lacks enzymatic activity and an active site, complicating development of occupancy-based drugs. Here we describe the development of proteolysis targeting chimeras (PROTACs) for the targeted degradation of Nef. Nef-binding compounds, based on a previously reported hydroxypyrazole core, were coupled to ligands for ubiquitin E3 ligases via flexible linkers. The resulting bivalent PROTACs induced formation of a ternary complex between Nef and the Cereblon E3 ubiquitin ligase, resulting in ubiquitylation of Nef and proteolytic degradation. Nef-directed PROTACs efficiently rescued Nef-mediated MHC-I and CD4 downregulation in T cells and suppressed HIV-1 replication in donor PBMCs. Targeted degradation of Nef is anticipated to reverse all HIV-1 Nef functions and may help restore adaptive immune responses against HIV-1 reservoir cells in vivo.

pharmacology and toxicology↗

bioRxiv2021

Nef dimerization defect abrogates HIV viremia and associated immune dysregulation in the Bone Marrow-Liver-Thymus-Spleen (BLTS) humanized mouse model

Loss of function mutations in the human immunodeficiency virus (HIV) negative factor (Nef) gene are associated with reduced viremia, robust T cell immune responses, and delayed acquired immunodeficiency syndrome (AIDS) progression in humans. In vitro studies have shown that mutations in the Nef dimerization interface significantly attenuate viral replication and impair host defense. However, in vivo, mechanistic studies on the role of Nef dimerization in HIV infection are lacking. Humanized rodents with human immune cells are robust platforms for investigating the interactions between HIV and the human immune system. The bone marrow-liver-thymus-spleen (BLTS) humanized mouse model carries human immune cells and lymphoid tissues that facilitate anti-viral immune responses. Here, we employed the BLTS-humanized mouse model to demonstrate that preventing Nef dimerization abrogates HIV viremia and the associated immune dysregulation. This suggests that Nef dimerization may be a therapeutic target for future HIV cure strategies.

immunology↗