Association of Nicotine Cessation Time on the Incidence of Recurrent Incisional Hernia Repair and Postoperative Surgical Site Occurrences.

BACKGROUND: Nicotine products are believed to be associated with a higher incidence of incisional hernia (IH) recurrence and postoperative complications after IH repair. METHODS: A retrospective analysis of the Abdominal Core Health Quality Collaborative (ACHQC) database was performed. Outcomes included risk of IH recurrence, 30-day surgical site infection (SSI), and 30-day surgical site occurrence (SSO). RESULTS: We included 14,663 patients. Nicotine users who quit within 1 year of surgery had a 26% higher risk of IH recurrence compared to patients who quit more than a year before surgery or never users. Patients who quit using nicotine within 1 year of surgery had a 54% higher odds of SSI compared to former nicotine users who quit more than a year before surgery. CONCLUSION: Former nicotine users with less than 1 year of nicotine use cessation before surgery exhibited worse outcomes than those with more than a year of cessation or no prior use.

Identification of 2-Sulfonyl/Sulfonamide Pyrimidines as Covalent Inhibitors of WRN Using a Multiplexed High-Throughput Screening Assay.

Werner syndrome protein (WRN) is a multifunctional enzyme with helicase, ATPase, and exonuclease activities that are necessary for numerous DNA-related transactions in the human cell. Recent studies identified WRN as a synthetic lethal target in cancers characterized by genomic microsatellite instability resulting from defects in DNA mismatch repair pathways. WRN's helicase activity is essential for the viability of these high microsatellite instability (MSI-H) cancers and thus presents a therapeutic opportunity. To this end, we developed a multiplexed high-throughput screening assay that monitors exonuclease, ATPase, and helicase activities of full-length WRN. This screening campaign led to the discovery of 2-sulfonyl/sulfonamide pyrimidine derivatives as novel covalent inhibitors of WRN helicase activity. The compounds are specific for WRN versus other human RecQ family members and show competitive behavior with ATP. Examination of these novel chemical probes established the sulfonamide NH group as a key driver of compound potency. One of the leading compounds, H3B-960, showed consistent activities in a range of assays (IC50 = 22 nM, KD = 40 nM, KI = 32 nM), and the most potent compound identified, H3B-968, has inhibitory activity IC50 ∼ 10 nM. These kinetic properties trend toward other known covalent druglike molecules. Our work provides a new avenue for screening WRN for inhibitors that may be adaptable to different therapeutic modalities such as targeted protein degradation, as well as a proof of concept for the inhibition of WRN helicase activity by covalent molecules.

Characterization of autonomic symptom burden in long COVID: A global survey of 2,314 adults.

Background: Autonomic dysfunction is a known complication of post-acute sequelae of SARS-CoV-2 (PASC)/long COVID, however prevalence and severity are unknown. Objective: To assess the frequency, severity, and risk factors of autonomic dysfunction in PASC, and to determine whether severity of acute SARS-CoV-2 infection is associated with severity of autonomic dysfunction. Design: Cross-sectional online survey of adults with PASC recruited through long COVID support groups between October 2020 and August 2021. Participants: 2,413 adults ages 18-64 years with PASC including patients who had a confirmed positive test for COVID-19 (test-confirmed) and participants who were diagnosed with COVID-19 based on clinical symptoms alone. Main measures: The main outcome measure was the Composite Autonomic Symptom 31 (COMPASS-31) total score, used to assess global autonomic dysfunction. Test-confirmed hospitalized vs. test-confirmed non-hospitalized participants were compared to determine if the severity of acute SARS-CoV-2 infection was associated with the severity autonomic dysfunction. Key results: Sixty-six percent of PASC patients had a COMPASS-31 score >20, suggestive of moderate to severe autonomic dysfunction. COMPASS-31 scores did not differ between test-confirmed hospitalized and test-confirmed non-hospitalized participants [28.95 (15.62, 46.60) vs. 26.4 (13.75, 42.10); p = 0.06]. Conclusions: Evidence of moderate to severe autonomic dysfunction was seen in 66% of PASC patients in our study, independent of hospitalization status, suggesting that autonomic dysfunction is highly prevalent in the PASC population and independent of the severity of acute COVID-19 illness.

Biochemical and structural basis for the pharmacological inhibition of nuclear hormone receptor PPARγ by inverse agonists.

Recent studies have reported that the peroxisome proliferator-activated receptor gamma (PPARγ) pathway is activated in approximately 40% of patients with muscle-invasive bladder cancer. This led us to investigate pharmacological repression of PPARγ as a possible intervention strategy. Here, we characterize PPARγ antagonists and inverse agonists and find that the former behave as silent ligands, whereas inverse agonists (T0070907 and SR10221) repress downstream PPARγ target genes leading to growth inhibition in bladder cancer cell lines. To understand the mechanism, we determined the ternary crystal structure of PPARγ bound to T0070907 and the corepressor (co-R) peptide NCOR1. The structure shows that the AF-2 helix 12 (H12) rearranges to bind inside the ligand-binding domain, where it forms stabilizing interactions with the compound. This dramatic movement in H12 unveils a large interface for co-R binding. In contrast, the crystal structure of PPARγ bound to a SR10221 analog shows more subtle structural differences, where the compound binds and pushes H12 away from the ligand-binding domain to allow co-R binding. Interestingly, we found that both classes of compound promote recruitment of co-R proteins in biochemical assays but with distinct conformational changes in H12. We validate our structural models using both site-directed mutagenesis and chemical probes. Our findings offer new mechanistic insights into pharmacological modulation of PPARγ signaling.

Covalent ERα Antagonist H3B-6545 Demonstrates Encouraging Preclinical Activity in Therapy-Resistant Breast Cancer.

Nearly 30% of patients with relapsed breast cancer present activating mutations in estrogen receptor alpha (ERα) that confer partial resistance to existing endocrine-based therapies. We previously reported the development of H3B-5942, a covalent ERα antagonist that engages cysteine-530 (C530) to achieve potency against both wild-type (ERαWT) and mutant ERα (ERαMUT). Anticipating that the emergence of C530 mutations could promote resistance to H3B-5942, we applied structure-based drug design to improve the potency of the core scaffold to further enhance the antagonistic activity in addition to covalent engagement. This effort led to the development of the clinical candidate H3B-6545, a covalent antagonist that is potent against both  ERαWT/MUT, and maintains potency even in the context of ERα C530 mutations. H3B-6545 demonstrates significant activity and superiority over standard-of-care fulvestrant across a panel of ERαWT and ERαMUT palbociclib sensitive and resistant models. In summary, the compelling preclinical activity of H3B-6545 supports its further development for the potential treatment of endocrine therapy-resistant ERα+ breast cancer harboring wild-type or mutant ESR1, as demonstrated by the ongoing clinical trials (NCT03250676, NCT04568902, NCT04288089). SUMMARY: H3B-6545 is an ERα covalent antagonist that exhibits encouraging preclinical activity against CDK4/6i naïve and resistant ERαWT and ERαMUT tumors.

Preparing for the long-haul: Autonomic complications of COVID-19.

As global numbers of COVID-19 grow, chronic neurological symptoms, including those of autonomic dysfunction, are being reported with increasing frequency. Mounting evidence suggests that many patients experience chronic and sometimes debilitating symptoms long after their acute infectious period, leading to the new diagnostic category of post-acute COVID syndrome. Many symptoms of post-acute COVID syndrome appear autonomic in nature, suggesting that autonomic impairment may play a central role in the underlying pathophysiology. In this review, we discuss the autonomic symptoms and manifestations of post-acute COVID syndrome, potential mechanisms involved, and future directions for a better understanding of this novel condition.

Structural basis of intron selection by U2 snRNP in the presence of covalent inhibitors.

Intron selection during the formation of prespliceosomes is a critical event in pre-mRNA splicing. Chemical modulation of intron selection has emerged as a route for cancer therapy. Splicing modulators alter the splicing patterns in cells by binding to the U2 snRNP (small nuclear ribonucleoprotein)-a complex chaperoning the selection of branch and 3' splice sites. Here we report crystal structures of the SF3B module of the U2 snRNP in complex with spliceostatin and sudemycin FR901464 analogs, and the cryo-electron microscopy structure of a cross-exon prespliceosome-like complex arrested with spliceostatin A. The structures reveal how modulators inactivate the branch site in a sequence-dependent manner and stall an E-to-A prespliceosome intermediate by covalent coupling to a nucleophilic zinc finger belonging to the SF3B subunit PHF5A. These findings support a mechanism of intron recognition by the U2 snRNP as a toehold-mediated strand invasion and advance an unanticipated drug targeting concept.

Biomarkers and heterogeneous fibroblast phenotype associated with incisional hernia.

Development of incisional hernia (IH) is multifactorial but inflammation and abdominal wall ECM (extracellular matrix) disorganization are key pathological events. We investigated if the differential expression of fibroblast biomarkers reflects the cellular milieu and the dysregulated ECM in IH tissues. Expression of fibroblast biomarkers, including connective tissue growth factor, alpha-smooth muscle actin (α-SMA), CD34 (cluster of differentiation 34), cadherin-11 and fibroblast specific protein 1 (FSP1), was examined by histology and immunofluorescence in the hernial-fascial ring/neck tissue (HRT) and hernia sack tissue (HST) harvested from the patients undergoing hernia surgery and compared with normal fascia (FT) and peritoneum (PT) harvested from brain-dead healthy subjects undergoing organ procurement for transplantation. The H&E staining revealed alterations in tissue architecture, fibroblast morphology, and ECM organization in the IH tissues compared to control. The biomarker for undifferentiated fibroblasts, CD34, was significantly higher in HST and decreased in HRT than the respective FT and PT controls. Also, the findings revealed an increased level of CTGF (connective tissue growth factor) with decrease in α-SMA in both HRT and HST compared to the controls. In addition, an increased level of FSP1 (fibroblast specific protein 1) and cadherin-11 in HRT with decreased level in HST were observed relative to the respective controls (FT and PT). Hence, these findings support the heterogeneity of fibroblast population at the laparotomy site that could contribute to the development of IH. Understanding the mechanisms causing the phenotype switch of these fibroblasts would open novel strategies to prevent the development of IH following laparotomy.

Discovery of Aminopyrazole Derivatives as Potent Inhibitors of Wild-Type and Gatekeeper Mutant FGFR2 and 3.

Fibroblast growth factor receptors (FGFR) 2 and 3 have been established as drivers of numerous types of cancer with multiple drugs approved or entering late stage clinical trials. A limitation of current inhibitors is vulnerability to gatekeeper resistance mutations. Using a combination of targeted high-throughput screening and structure-based drug design, we have developed a series of aminopyrazole based FGFR inhibitors that covalently target a cysteine residue on the P-loop of the kinase. The inhibitors show excellent activity against the wild-type and gatekeeper mutant versions of the enzymes. Further optimization using SAR analysis and structure-based drug design led to analogues with improved potency and drug metabolism and pharmacokinetics properties.

The SF3b Complex is an Integral Component of the Spliceosome and Targeted by Natural Product-Based Inhibitors.

In this chapter, the essential role of the SF3b multi-protein complex will be discussed in the context of the overall spliceosome. SF3b is critical during spliceosome assembly for recognition of the branch point (BP) adenosine and, by de facto, selection of the 3' splice site. This complex is highly dynamic, undergoing significant conformational changes upon loading of the branch duplex RNA and in its relative positioning during spliceosomal remodeling from the A, pre-B, B, Bact and B* complexes. Ultimately, during the spliceosome activation phase, SF3b must be displaced to unmask the branch point adenosine for the first splicing reaction to occur. In certain cancers, such as the hematological malignancies CML, CLL and MDS, the SF3b subunit SF3B1 is frequently mutated. Recent studies suggest these mutations lead to inappropriate branch point selection and mis-splicing events that appear to be drivers of disease. Finally, the SF3b complex is the target for at least three different classes of natural product-based inhibitors. These inhibitors bind in the BP adenosine-binding pocket and demonstrate a pre-mRNA competitive mechanism of action resulting in either intron retention or exon skipping. These compounds are extremely useful as chemical probes to isolate and characterize early stages of spliceosome assembly. They are also being explored preclinically and clinically as possible agents for hematological cancers.

Regulatory role of cardiomyocyte metabolism via AMPK activation in modulating atrial structural, contractile, and electrical properties following atrial fibrillation.

The number of patients diagnosed with atrial fibrillation (AF) has been rising due to increased incidence, enhanced detection methods, and greater survival rates following diagnosis. Due to this increase, AF is now the most commonly diagnosed arrhythmia in clinical practice. AF is characterized by irregular, high-frequency contractions of atrial myocytes that lead to turbulent blood flow and the potential for thrombus formation, stroke, or heart failure. These high-frequency contractions of the atrial myocytes cause an imbalance between metabolic supply and demand. Although advances have been made in understanding the pathophysiology of AF, the etiology and underlying pathogenic mechanism remain unknown. However, recent evidence suggests that cardiomyocyte metabolism involving 5' AMP-activated protein kinase (AMPK) activation is altered in patients with AF. Here, we critically reviewed the current understanding of AMPK activation in AF and how it could affect structural, contractile, and electrophysiological cellular properties in the pathogenesis of AF.

Selected conditions associated with an increased incidence of incisional hernia: A review of molecular biology.

BACKGROUND: Incisional hernias (IH) following a laparotomy, on average, occur in 10-20% of patients, however, little is known about its molecular basis. Thus, a better understanding of the molecular mechanisms could lead to the identification of key target(s) to intervene pre-and post-operatively. METHODS: We examined the current literature describing the molecular mechanisms of IH and overlap these factors with smoking, abdominal aortic aneurysm, obesity, diabetes mellitus, and diverticulitis. RESULTS: The expression levels of collagen I and III, matrix metalloproteinases, and tissue inhibitors of metalloproteases are abnormal in the extracellular matrix (ECM) of IH patients and ECM disorganization has an overlap with these comorbid conditions. CONCLUSION: Understanding the pathophysiology of IH development and associated risk factors will allow physicians to identify patients that may be at increased risk for IH and to possibly act preemptively to decrease the incidence of IH.