Global changes in the RNA binding specificity of HIV-1 gag regulate virion genesis.

The HIV-1 Gag protein orchestrates all steps of virion genesis, including membrane targeting and RNA recruitment into virions. Using crosslinking-immunoprecipitation (CLIP) sequencing, we uncover several dramatic changes in the RNA-binding properties of Gag that occur during virion genesis, coincident with membrane binding, multimerization, and proteolytic maturation. Prior to assembly, and after virion assembly and maturation, the nucleocapsid domain of Gag preferentially binds to psi and Rev Response elements in the viral genome, and GU-rich mRNA sequences. However, during virion genesis, this specificity transiently changes in a manner that facilitates genome packaging; nucleocapsid binds to many sites on the HIV-1 genome and to mRNA sequences with a HIV-1-like, A-rich nucleotide composition. Additionally, we find that the matrix domain of Gag binds almost exclusively to specific tRNAs in the cytosol, and this association regulates Gag binding to cellular membranes.

Perioperative outcomes of robotic versus laparoscopic paraesophageal hernia repair: a NSQIP analysis.

BACKGROUND: Studies comparing outcomes between robotic and laparoscopic paraesophageal hernia repairs have yielded conflicting results. We sought to analyze early postoperative complications between these approaches using a newly available NSQIP variable indicating robot use. METHODS: We queried the 2022 ACS NSQIP database for adult patients undergoing elective minimally invasive hiatal hernia repair. Chi-squared and Kruskal-Wallis tests were used to compare cohort characteristics. Logistic, linear, and Cox proportional hazards regression analyses were used to compare perioperative outcomes between the laparoscopic and robotic groups. RESULTS: We identified 4345 patients who underwent repair using a laparoscopic (2778 patients; 63.9%) or robotic (1567 patients; 36.1%) approach. Most (73.1%) were female, and the median age was 65 (IQR 55, 73). Patients who underwent robotic repair were younger (median age 64 vs 66), had a slightly higher body mass index (BMI; median 30.2 vs 29.9), and were more likely to have hypertension (53.0% vs 48.5%), all p < 0.01. On unadjusted analysis the robotic approach was associated with decreased 30-day mortality (0.0% vs 0.4%, p < 0.01). After adjusting for age, gender, race, BMI, and hypertension, the robotic approach was not associated with increased major complications (5.6% vs 5.1%, AOR 1.13, 95% CI 0.86, 1.49), minor complications (0.9% vs 1.5%, AOR 1.20, 95% CI 0.74, 1.93), or unplanned readmission (6.5% vs 5.5%, AHR 1.17, 95% CI 0.89, 1.54), all p ≥ 0.26. After adjusting for age and hypertension, the robotic cohort had an increased risk of myocardial infarction (AOR 2.53, 95% CI 1.01, 6.33, p = 0.048) and pulmonary embolism (AOR 2.76, 95% CI 1.17, 6.49, p = 0.02), although none resulted in 30-day mortality. CONCLUSIONS: Robotic and laparoscopic paraesophageal hernia repairs had similar overall complication and readmission rates. The robotic cohort had an increased risk of myocardial infarction and pulmonary embolism but no 30-day mortality. Current data support the use of both robotic and laparoscopic approaches for paraesophageal hernia repair.

Genome-Wide Analysis of Heterogeneous Nuclear Ribonucleoprotein (hnRNP) Binding to HIV-1 RNA Reveals a Key Role for hnRNP H1 in Alternative Viral mRNA Splicing.

Alternative splicing of HIV-1 mRNAs increases viral coding potential and controls the levels and timing of gene expression. HIV-1 splicing is regulated in part by heterogeneous nuclear ribonucleoproteins (hnRNPs) and their viral target sequences, which typically repress splicing when studied outside their native viral context. Here, we determined the location and extent of hnRNP binding to HIV-1 mRNAs and their impact on splicing in a native viral context. Notably, hnRNP A1, hnRNP A2, and hnRNP B1 bound to many dispersed sites across viral mRNAs. Conversely, hnRNP H1 bound to a few discrete purine-rich sequences, a finding that was mirrored in vitro hnRNP H1 depletion and mutation of a prominent viral RNA hnRNP H1 binding site decreased the use of splice acceptor A1, causing a deficit in Vif expression and replicative fitness. This quantitative framework for determining the regulatory inputs governing alternative HIV-1 splicing revealed an unexpected splicing enhancer role for hnRNP H1 through binding to its target element.IMPORTANCE Alternative splicing of HIV-1 mRNAs is an essential yet quite poorly understood step of virus replication that enhances the coding potential of the viral genome and allows the temporal regulation of viral gene expression. Although HIV-1 constitutes an important model system for general studies of the regulation of alternative splicing, the inputs that determine the efficiency with which splice sites are utilized remain poorly defined. Our studies provide an experimental framework to study an essential step of HIV-1 replication more comprehensively and in much greater detail than was previously possible and reveal novel cis-acting elements regulating HIV-1 splicing.

Benzopyrimidodiazepinone inhibitors of TNK2.

The SAR of a series of benzopyrimidodiazepinone inhibitors of TNK2 was developed, starting from the potent and selective compound XMD8-87. A diverse set of anilines was introduced in an effort to improve the in vivo PK profile and minimize the risk of quinone diimine formation.

Discovery of a series of benzopyrimidodiazepinone TNK2 inhibitors via scaffold morphing.

The protein kinase TNK2 (ACK1) is an emerging drug target for a variety of indications, in particular for cancer where it plays a key role transmitting cell survival, growth and proliferative signals via modification of multiple downstream effectors by unique tyrosine phosphorylation events. Scaffold morphing based on our previous TNK2 inhibitor XMD8-87 identified urea 17 from which we developed the potent and selective compound 32. A co-crystal structure was obtained showing 32 interacting primarily with the main chain atoms of an alanine residue of the hinge region. Additional H-bonds exist between the urea NHs and the Thr205 and Asp270 residues.

Tetherin Inhibits Cell-Free Virus Dissemination and Retards Murine Leukemia Virus Pathogenesis.

The relative contributions of cell-free virion circulation and direct cell-to-cell transmission to retroviral dissemination and pathogenesis are unknown. Tetherin/Bst2 is an antiviral protein that blocks enveloped virion release into the extracellular milieu but may not inhibit cell-to-cell virus transmission. We developed live-cell imaging assays which show that tetherin does not affect Moloney murine leukemia virus (MoMLV) spread, and only minimally affects vesicular stomatitis virus (VSV) spread, to adjacent cells in a monolayer. Conversely, cell-free MLV and VSV virion yields and VSV spread to distal cells were dramatically reduced by tetherin. To elucidate the roles of tetherin and cell-free virions during in vivo viral dissemination and pathogenesis, we developed mice carrying an inducible human tetherin (hTetherin) transgene. While ubiquitous hTetherin expression was detrimental to the growth and survival of mice, restriction of hTetherin expression to hematopoietic cells gave apparently healthy mice. The expression of hTetherin in hematopoietic cells had little or no effect on the number of MoMLV-infected splenocytes and thymocytes. However, hTetherin expression significantly reduced cell-free plasma viremia and also delayed MoMLV-induced disease. Overall, these results suggest that MoMLV spread within hematopoietic tissues and cell monolayers involves cell-to-cell transmission that is resistant to tetherin but that virion dissemination via plasma is inhibited by tetherin and is required for full MoMLV pathogenesis.IMPORTANCE Retroviruses are thought to spread primarily via direct cell-to-cell transmission, yet many have evolved to counteract an antiviral protein called tetherin, which may selectively inhibit cell-free virus release. We generated a mouse model with an inducible tetherin transgene in order to study how tetherin affects retroviral dissemination and on which cell types its expression is required to do so. We first developed a novel in vitro live-cell imaging assay to demonstrate that while tetherin does indeed dramatically reduce cell-free virus spreading, it has little to no effect on direct cell-to-cell transmission of either vesicular stomatitis virus (VSV) or the retrovirus MoMLV. Using our transgenic mouse model, we found that tetherin expression on hematopoietic cells resulted in the specific reduction of MoMLV cell-free plasma viremia but not the number of infected hematopoietic cells. The delay in disease associated with this scenario suggests a role for cell-free virus in retroviral disease progression.

Progress in genome-inspired treatment decisions for multifocal lung adenocarcinoma.

INTRODUCTION: Multifocal lung adenocarcinoma (MFLA) is becoming increasingly recognized as a distinct subset of lung cancer, with unique biology, disease course, and treatment outcomes. While definitions remain controversial, MFLA is characterized by the development and concurrent presence of multiple independent (non-metastatic) lesions on the lung adenocarcinoma spectrum. Disease progression typically follows an indolent course measured in years, with a lower propensity for nodal and distant metastases than other more common forms of non-small cell lung cancer. AREAS COVERED: Traditional imaging and histopathological analyses of tumor biopsies are frequently unable to fully characterize the disease, prompting interest in molecular diagnosis. We highlight some of the key questions in the field, including accurate definitions to identify and stage MLFA, molecular tests to stratify patients and treatment decisions, and the lack of clinical trial data to delineate best management for this poorly understood subset of lung cancer patients. We review the existing literature and progress toward a genomic diagnosis for this unique disease entity. EXPERT OPINION: Multifocal lung adenocarcinoma behaves differently than other forms of non-small cell lung cancer. Progress in molecular diagnosis may enhance potential for accurate definition, diagnosis, and optimizing treatment approach.

Selective Macrocyclic Inhibitors of DYRK1A/B.

Dual-specificity tyrosine-(Y)-phosphorylation regulated kinase 1A (DYRK1A) is a therapeutic target of interest due to the roles it plays in both neurological diseases and cancer. We present the development of the first macrocyclic inhibitors of DYRK1A. Initial lead inhibitor JH-XIV-68-3 (3) displayed selectivity for DYRK1A and close family member DYRK1B in biochemical and cellular assays, and demonstrated antitumor efficacy in head and neck squamous cell carcinoma (HNSCC) cell lines. However, we noted that it suffered from rapid aldehyde oxidase (AO)-mediated metabolism. To overcome this liability, we generated a derivative (JH-XVII-10 (10)), where fluorine was introduced to block the 2-position of the azaindole and render the molecule resistant to AO activity. We showed that 10 maintains remarkable potency and selectivity in biochemical and cellular assays as well as antitumor efficacy in HNSCC cell lines and improved metabolic stability. Therefore, 10 represents a promising new scaffold for developing DYRK1A-targeting chemical probes and therapeutics.

Serial evaluation of segmental esophageal reconstruction using a polyurethane scaffold in a pig model.

Background: Many esophageal pathologies are clinically treated by resection and reconstruction of the esophagus. Surgical esophagectomy remains a morbid procedure and despite minimally invasive advances, has changed little in decades. Novel approaches to esophageal segmental resection and reconstruction are an unmet need. Methods: Circumferential thoracic esophageal transection was performed in both male and female pigs and the defects reconstructed using 5 or 10 cm polyurethane (PU) tubular grafts and stented. A subset were treated with stent only. Animals were survived to 14, 30, 60, and 399 days. Tissues were evaluated histologically, and via non-invasive serial endoscopy and contrast swallowing studies in long-term animals. Results: Luminal patency was achieved in all animals with no clinical evidence of leak. In short-term animals, there was healing noted in all cases with a variably sized region of ulceration remaining at the most central part of the repaired tube (between the proximal and distal anastomosis). In four long-term animals following stent removal, two resumed normal diet and thrived, while two animals were euthanized prior to the proposed endpoint because of stricture formation and inability to tolerate a normal diet. Re-epithelialization was observed in all groups, and more complete over time. Conclusions: The PU scaffold provides a matrix across which formation of new tissue can occur. The mechanisms through which this happens remain unclear, but likely a combination of fibrosis and tissue contraction, in conjunction with new tissue formation.

Selective degradation-inducing probes for studying cereblon (CRBN) biology.

Targeted protein degradation represents a rapidly growing area in drug discovery and development. Moreover, small molecules that induce the targeted degradation of a given protein also represent an important addition to the chemical probes toolbox as these compounds can achieve selective protein knockdown, thus providing an approach that is orthogonal to genetic knockdowns. In order to develop degradation-inducing chemical probes for studying cereblon (CRBN) biology, we generated six CRBN-CRBN (homo-PROTAC) degraders and six CRBN-VHL (hetero-PROTAC) degraders. From these compounds we identified two potent and selective CRBN degraders (ZXH-4-130 and ZXH-4-137), both of which are CRBN-VHL compounds. We characterized these lead degraders by quantitative proteomics in five cell lines (MM1.S, Kelly, SK-N-DZ, HEK293T, and MOLT-4) and observed high selectivity for CRBN in all cell lines. Furthermore, we directly compared our compounds to current lead CRBN degraders and demonstrated how these probes can be used as chemical knockdown reagents for studying CRBN-dependent processes. Overall, our work provides a roadmap for thorough degrader characterization by combination western and proteomic analysis, as illustrated by the identification of ZXH-4-130 and ZXH-4-137 as CRBN-knockdown tool compounds suitable for cell-based studies.

A Gut-Restricted Lithocholic Acid Analog as an Inhibitor of Gut Bacterial Bile Salt Hydrolases.

Bile acids play crucial roles in host physiology by acting both as detergents that aid in digestion and as signaling molecules that bind to host receptors. Gut bacterial bile salt hydrolase (BSH) enzymes perform the gateway reaction leading to the conversion of host-produced primary bile acids into bacterially modified secondary bile acids. Small molecule probes that target BSHs will help elucidate the causal roles of these metabolites in host physiology. We previously reported the development of a covalent BSH inhibitor with low gut permeability. Here, we build on our previous findings and describe the development of a second-generation gut-restricted BSH inhibitor with enhanced potency, reduced off-target effects, and durable in vivo efficacy. Structure-activity relationship (SAR) studies focused on the bile acid core identified a compound, AAA-10, containing a C3-sulfonated lithocholic acid scaffold and an alpha-fluoromethyl ketone warhead as a potent pan-BSH inhibitor. This compound inhibits BSH activity in mouse and human fecal slurry, bacterial cultures, and purified BSH proteins and displays reduced toxicity against mammalian cells compared to first generation compounds. Oral administration of AAA-10 to wild-type mice for 5 days resulted in a decrease in the abundance of the secondary bile acids deoxycholic acid (DCA) and lithocholic acid (LCA) in the mouse GI tract with low systemic exposure of AAA-10, demonstrating that AAA-10 is an effective tool for inhibiting BSH activity and modulating bile acid pool composition in vivo.

Selective Degradation of GSPT1 by Cereblon Modulators Identified via a Focused Combinatorial Library.

Cereblon (CRBN) is an E3 ligase adapter protein that can be reprogrammed by imide-class compounds such as thalidomide, lenalidomide, and pomalidomide to induce the degradation of neo-substrate proteins. In order to identify additional small molecule CRBN modulators, we implemented a focused combinatorial library approach where we fused an imide-based CRBN-binding pharmacophore to a heterocyclic scaffold, which could be further elaborated. We screened the library for CRBN-dependent antiproliferative activity in the multiple myeloma cell line MM1.S and identified five hit compounds. Quantitative chemical proteomics of hit compounds revealed that they induced selective degradation of GSPT1, a translation termination factor that is currently being explored as a therapeutic target for the treatment of acute myeloid leukemia. Molecular docking studies with CRBN and GSPT1 followed by analogue synthesis identified a possible hydrogen bond interaction with the central pyrimidine ring as a molecular determinant of hit compounds' selectivity. This study demonstrates that a focused combinatorial library design, phenotypic screening, and chemical proteomics can provide a suitable workflow to efficiently identify novel CRBN modulators.

Chemically Induced Degradation of Anaplastic Lymphoma Kinase (ALK).

We present the development of the first small molecule degraders that can induce anaplastic lymphoma kinase (ALK) degradation, including in non-small-cell lung cancer (NSCLC), anaplastic large-cell lymphoma (ALCL), and neuroblastoma (NB) cell lines. These degraders were developed through conjugation of known pyrimidine-based ALK inhibitors, TAE684 or LDK378, and the cereblon ligand pomalidomide. We demonstrate that in some cell types degrader potency is compromised by expression of drug transporter ABCB1. In addition, proteomic profiling demonstrated that these compounds also promote the degradation of additional kinases including PTK2 (FAK), Aurora A, FER, and RPS6KA1 (RSK1).

SRPKIN-1: A Covalent SRPK1/2 Inhibitor that Potently Converts VEGF from Pro-angiogenic to Anti-angiogenic Isoform.

The SRPK family of kinases regulates pre-mRNA splicing by phosphorylating serine/arginine (SR)-rich splicing factors, signals splicing control in response to extracellular stimuli, and contributes to tumorigenesis, suggesting that these splicing kinases are potential therapeutic targets. Here, we report the development of the first irreversible SRPK inhibitor, SRPKIN-1, which is also the first kinase inhibitor that forms a covalent bond with a tyrosine phenol group in the ATP-binding pocket. Kinome-wide profiling demonstrates its selectivity for SRPK1/2, and SRPKIN-1 attenuates SR protein phosphorylation at submicromolar concentrations. Vascular endothelial growth factor (VEGF) is a known target for SRPK-regulated splicing and, relative to the first-generation SRPK inhibitor SRPIN340 or small interfering RNA-mediated SRPK knockdown, SRPKIN-1 is more potent in converting the pro-angiogenic VEGF-A165a to the anti-angiogenic VEGF-A165b isoform and in blocking laser-induced neovascularization in a murine retinal model. These findings encourage further development of SRPK inhibitors for treatment of age-related macular degeneration.