Biophysical and Biochemical Characterization of Structurally Diverse Small Molecule Hits for KRAS Inhibition.

We describe six compounds as early hits for the development of direct inhibitors of KRAS, an important anticancer drug target. We show that these compounds bind to KRAS with affinities in the low micromolar range and exert different effects on its interactions with binding partners. Some of the compounds exhibit selective binding to the activated form of KRAS and inhibit signal transduction through both the MAPK or the phosphatidylinositide 3-kinase PI3K-protein kinase B (AKT) pathway in cells expressing mutant KRAS. Most inhibit intrinsic and/or SOS-mediated KRAS activation while others inhibit RAS-effector interaction. We propose these compounds as starting points for the development of non-covalent allosteric KRAS inhibitors.

The Resurgence of Lymphogranuloma Venereum: Changing Presentation of Lymphogranuloma Venereum in the Era of HIV Preexposure Prophylaxis, 2004 to 2022.

BACKGROUND: Before the early 2000s, the sexually transmitted infection lymphogranuloma venereum (LGV) was rare in high-income countries. Initially, most cases in these countries were among symptomatic men who have sex with men (MSM) living with HIV. In the context of widespread HIV preexposure prophylaxis (PrEP), LGV's epidemiology may be changing. We aimed to characterize the epidemiology and clinical presentation of LGV in the PrEP era. METHODS: A retrospective chart review was performed on all LGV cases occurring between November 2004 to October 2022 in British Columbia (BC), Canada. Cases were stratified by having occurred before (2004-2017) or after widespread PrEP availability in BC (2018-2022). Annual rates and test positivity percentages were calculated. Bivariate logistic regression was performed to identify drivers of asymptomatic infection in the PrEP era. RESULTS: Among 545 cases identified, 205 (37.6%) occurred pre-PrEP and 340 (62.4%) occurred during the PrEP era. Most cases were among MSM (97.2%). The estimated rate of LGV has doubled from 2018 to 2022, reaching 1535.2 cases per 100,000 PrEP users. Most PrEP-era cases were among HIV-negative individuals (65.3%), particularly those on PrEP (72.6%). Cases in the PrEP era were often asymptomatic compared with pre-PrEP (38.6% vs. 19.3%; P < 0.001). Users of PrEP were more likely to experience asymptomatic infection compared with HIV-negative PrEP nonusers (odds ratio, 2.07; 95% confidence interval, 1.07-3.99). CONCLUSIONS: In the context of increased asymptomatic testing, LGV may be increasing in BC. Most infections now occur among HIV-negative MSM. A high proportion of infections are asymptomatic.

Small-Molecule Inhibition of KRAS through Conformational Selection.

Mutations in KRAS account for about 20% of human cancers. Despite the major progress in recent years toward the development of KRAS inhibitors, including the discovery of covalent inhibitors of the G12C KRAS variant for the treatment of non-small-cell lung cancer, much work remains to be done to discover broad-acting inhibitors to treat many other KRAS-driven cancers. In a previous report, we showed that a 308.4 Da small-molecule ligand [(2R)-2-(N'-(1H-indole-3-carbonyl)hydrazino)-2-phenyl-acetamide] binds to KRAS with low micro-molar affinity [Chem. Biol. Drug Des.2019; 94(2):1441-1456]. Binding of this ligand, which we call ACA22, to the p1 pocket of KRAS and its interactions with residues at beta-strand 1 and the switch loops have been supported by data from nuclear magnetic resonance spectroscopy and microscale thermophoresis experiments. However, the inhibitory potential of the compound was not demonstrated. Here, we show that ACA22 inhibits KRAS-mediated signal transduction in cells expressing wild type (WT) and G12D mutant KRAS and reduces levels of guanosine triphosphate-loaded WT KRAS more effectively than G12D KRAS. We ruled out the direct effect on nucleotide exchange or effector binding as possible mechanisms of inhibition using a variety of biophysical assays. Combining these observations with binding data that show comparable affinities of the compound for the active and inactive forms of the mutant but not the WT, we propose conformational selection as a possible mechanism of action of ACA22.

A Prospective Comparative Study of Laparoscopic Totally Extraperitoneal (TEP) and Laparoscopic Transabdominal Preperitoneal (TAPP) Inguinal Hernial Repair.

Introduction Inguinal hernia is a common surgical problem throughout the world. Currently, the management options available are open mesh hernioplasty and laparoscopic mesh repair. Laparoscopic mesh repair can be performed by either transabdominal preperitoneal (TAPP) repair or totally extraperitoneal (TEP) repair. Many studies comparing the two procedures have been unable to establish the superiority of one procedure over the other and have yielded conflicting results. Thus, we performed this study to compare TAPP and TEP. Aim The aim of this study is to compare the clinical outcomes and safety of laparoscopic TEP and laparoscopic TAPP for inguinal hernia repair. Materials and methods Patients were randomly divided into two groups on the basis of surgical procedures. The first group of patients underwent laparoscopic TAPP mesh repair, and the second group of patients underwent laparoscopic TEP mesh repair. Their intraoperative and postoperative findings were noted. Patients were followed up at regular intervals for up to six months. Results The mean age and mean weight distribution between the two groups were not significant. The duration of surgery needed (in minutes) for TAPP was found to be significantly less compared to TEP. In the TEP group, conversion to open occurred for three subjects (6.7%) while there was no conversion in the TAPP group. Postoperative pain at 24 hrs was found to be higher in TAPP subjects compared to that in TEP subjects, but the difference was statistically insignificant. Tolerance to a liquid diet started few hours after surgery was found to be the same in both groups. Association of the duration of hospital stays with the type of surgery was not significant. Six subjects (13.2%) showed hematoma in the TEP group while five subjects (11%) in the TAPP group showed hematoma after one week of surgery. Eight subjects (17.6%) showed seroma in the TEP group while three subjects (15.4%) in the TAPP group showed seroma after one week of surgery. Two subjects (4.4%) showed superficial wound infection in both the TEP group and TAPP group after one week of surgery. Four subjects each (8.9%) showed scrotal edema in the TEP group as well as the TAPP group after one week of surgery. No subject showed port site hernia without closure of the sheath at one-week, one-month, and six-month follow-up visits. Two subjects (4.4%) each showed groin pain in the TEP group as well as the TAPP group after one week of surgery. There were no instances of bowel obstruction or mesh infection. Conclusion TEP is a more skill-demanding procedure as compared to TAPP and thus takes more time to perform. However, it is superior on account of not breaching the peritoneum. TAPP is favorable for larger hernias. The choice of procedure should be individualized according to the patient's characteristics and surgeon's preference.

Pelvic Synovial Sarcoma Clinically Masquerading as an Ovarian Malignancy.

Background: Synovial sarcoma, a malignant mesenchymal tumor, commonly involves the extremities and is rarely found in the pelvis. Cytology with a biphasic pattern can suggest the diagnosis of synovial sarcoma. Case Report: A 32-year-old female presented with complaints of abdominal distension. She was initially evaluated in another hospital where she underwent ultrasound-guided fine needle aspiration cytology (FNAC) of the abdominal mass. The mass was diagnosed as ovarian adenocarcinoma, and the patient underwent 3 cycles of chemotherapy. After 3 months, she was referred to our institute for surgical excision of the mass. Contrast-enhanced computed tomography of the abdomen suggested a well-circumscribed, heterogeneous, solid-cystic mass in the left adnexal region measuring 13.9 × 10 × 9.1 cm and compressing the adjacent structures. No previous radiologic investigations were available. We reviewed the FNAC slide from the outside hospital and made a preliminary diagnosis of biphasic synovial sarcoma. The patient underwent debulking surgery consisting of panhysterectomy with excision of the pelvic mass. Microscopic examination of the pelvic mass showed a biphasic tumor composed of epithelial and mesenchymal components, suggestive of synovial sarcoma. The immunohistochemistry profile supported the morphologic diagnosis. Bilateral ovaries were unremarkable. The patient received 4 cycles of adjuvant chemotherapy and is presently asymptomatic. Conclusion: Although primary pelvic synovial sarcoma is a rare occurrence, this case illustrates that synovial sarcoma can be diagnosed or at least suspected on FNAC. Because of the importance of adjuvant chemotherapy, synovial sarcoma must be high on the list of differential diagnoses of high-grade intra-abdominal masses.

As the World Struggles With the COVID-19 Pandemic, Another Emergency Threat Arrives on the Horizon, the Monkeypox: A Systematic Review.

The whole world got threatened by COVID-19, which made a significant loss in various sectors and pushed the world into a deep valley. Now a new threat, the emerging outbreak of monkeypox is rapidly spreading across the globe and is currently being observed in more than 110 countries with 79,473 confirmed cases and 50 deaths. Data were collected from PubMed, EMBASE, MEDLINE, Cochrane, Scopus database, African Journals OnLine, internet library sub-Saharan Africa, and Google Scholar. Most data were taken from the democratic Republic of Congo, the Central African Republic, Cameroon, the Republic of Congo, Liberia, Nigeria, the US, and the UK. Case reports, outbreak investigations, epidemiological studies, and surveillance studies were reviewed to find epidemiological details about the outbreak. A total of 50 peer-reviewed articles and 20 grey literature articles, including 9050 cases, were identified for data extraction. Our systematic review revealed that the group most affected is male (95.5%), with a median age of 33.8 years. A total of 55% of the transmission was sexually transmitted. The most commonly reported symptoms such as vesicular-pustular rashes (97.54%), fever (55.25%), inguinal lymphadenopathy (53.6%), exanthema (40.21%), fatigue, headache, asthenia (26.32%), myalgia (16.33%), vesicles and ulcers (30.61%) in the anogenital regions were some of the significant findings. The case fatality rate was observed to be up to 8.65%. The most affected country was the USA, which has the most fatalities in younger ages involved in homosexuality, suffering from HIV or sexually transmitted diseases (STDs).

Sleep testing during the pandemic.

BACKGROUND: The COVID-19 pandemic disrupted the U.S. healthcare system, reducing the capacity available for unrelated conditions, such as sleep disordered breathing, and increasing concerns about the safety of in-lab testing. This study characterizes how the pandemic impacted the assessment of sleep disordered breathing and use of associated services. METHODS: Sleep testing claims occurring between January 2019 and June 2021 were extracted from the database of a national healthcare organization. Utilization was trended. Logistic regressions were run to assess the association between quarter of initial testing, whether testing was followed by treatment, and whether testing was followed by a clinical visit with a diagnosis related to sleep apnea, after controlling for patient-related factors. A Cox proportional hazards model assessed factors influencing time to treatment. Finally, a logistic regression assessed factors influencing the finality of home-based testing. RESULTS: In Q2 2021, home-based testing utilization was 134% of its initial level, while in-lab and split night testing were both at 61% of initial levels. Patients receiving initial home-based testing did not significantly differ in their likelihood of treatment, but were significantly less likely to have a clinical visit for sleep apnea (P < 0.01). Patients initially tested in 2021 were treated significantly more quickly than those initially tested in Q1 2019. Home-based testing occurring in Q4 2019 or later was significantly more likely to be definitive than home-based testing occurring Q1 2019. CONCLUSIONS: Home-based sleep testing increased significantly and durably in 2020, and was associated with faster time to treatment than initial in-lab testing.

Utility of Antibodies in the Diagnoses of Thyroid Diseases: A Review Article.

Thyroid problems are among the most widespread endocrine illnesses, affecting individuals in India and the global population. A thyroid function test is used to diagnose, screen, and monitor patients. Hyperthyroidism is a clinical condition due to excessive circulation of thyroid hormone; in contrast, hypothyroidism is due to a deficiency of thyroid hormone. Graves' disease (GD) is a form of hyperthyroidism due to thyroid-stimulating hormone receptor autoantibodies (TRAb), and anti-thyroid peroxidase antibodies (anti-TPO antibodies). The most common reason for hypothyroidism is Hashimoto's thyroiditis (HT), in which patients have thyroid receptor antibodies (TRAb), antibodies to thyroid peroxidase (TPO), and thyroglobulin antibodies. Many essential genes, including the thyroid-specific genes thyroglobulin (TSGT), TSH-receptor gene, human leukocyte antigen (HLA) genes, cytotoxic T lymphocyte-associated antigen (CTLA) genes, thyroglobulin gene, vitamin D receptor gene, and many immune-regulatory genes were associated with autoimmune thyroid diseases' (AITDs') etiology. This review paper aims to determine if antibodies are beneficial in detecting autoimmune thyroid disease or not. We have also discussed the etiology of autoimmune thyroid illness, serum antibodies in autoimmune thyroid disease, pathophysiology, and TSH receptor features.

Prescription Drug Insurance and Cost-Related Medication Nonadherence Among Lesbian, Gay, and Bisexual Individuals in Canada.

Purpose: This study estimates the frequency of uninsurance for prescription drugs and cost-related medication nonadherence (CRNA) among lesbian, gay, and bisexual (LGB) persons in Canada, compared with the heterosexual population. Methods: Logistic regression was used to quantify associations between sexual orientation, insurance status, and CRNA within the national probability-based Canadian Community Health Survey, 2015-2016. This sample included 98,413 individuals aged 15-80 years, including 2803 LGB individuals. Results: From our sample of Canadians, 22.2% of LGB respondents reported being uninsured for prescription drugs, compared with 20.0% of heterosexual persons (unadjusted odds ratio [UOR] 1.14, 95% confidence interval [CI] 0.97-1.35). LGB individuals had more than twice the odds of reporting CRNA compared with heterosexual individuals (UOR 2.48, 95% CI 1.99-3.10). This disparity was most pronounced among bisexual respondents, who had over three times the odds of reporting CRNA in comparison to heterosexual respondents (UOR 3.45, 95% CI 2.65-4.51). The odds ratio (OR) for CRNA comparing bisexual with heterosexual individuals remained statistically significant after adjustment for race/ethnicity, gender/sex, and age (OR 2.67, 95% CI 1.97-3.61) and was further attenuated with adjustment for partnership status, employment status, income, educational attainment, prescription drug insurance status, general health status, and immigration status (OR 2.09, 95% CI 1.51-2.89). Conclusion: LGB Canadians reported more CRNA but comparable prescription drug insurance frequencies to heterosexual persons. Factors pertaining to medication access (e.g., income, partnership status) and health needs appear to be the most important contributors to disparities.

KRAS Inhibitor that Simultaneously Inhibits Nucleotide Exchange Activity and Effector Engagement.

We describe a small molecule ligand ACA-14 (2-hydroxy-5-{[(2-phenylcyclopropyl) carbonyl] amino} benzoic acid) as an initial lead for the development of direct inhibitors of KRAS, a notoriously difficult anticancer drug target. We show that the compound binds to KRAS near the switch regions with affinities in the low micromolar range and exerts different effects on KRAS interactions with binding partners. Specifically, ACA-14 impedes the interaction of KRAS with its effector Raf and reduces both intrinsic and SOS-mediated nucleotide exchange rates. Likely as a result of these effects, ACA-14 inhibits signal transduction through the MAPK pathway in cells expressing mutant KRAS and inhibits the growth of pancreatic and colon cancer cells harboring mutant KRAS. We thus propose compound ACA-14 as a useful initial lead for the development of broad-acting inhibitors that target multiple KRAS mutants and simultaneously deplete the fraction of GTP-loaded KRAS while abrogating the effector-binding ability of the already GTP-loaded fraction.

Design and development of a portable resistive sensor based on α-MnO2 /GQD nanocomposites for trace quantification of Pb(II) in water.

The occurrence of heavy metal ions in food chain is appearing to be a major problem for mankind. The traces of heavy metals, especially Pb(II) ions present in water bodies remains undetected, untreated, and it remains in the food cycle causing serious health hazards for human and livestock. The consumption of Pb(II) ions may lead to serious medical complications including multiple organ failure which can be fatal. The conventional methods of heavy metal detection are costly, time-consuming and require laboratory space. There is an immediate need to develop a cost-effective and portable sensing system which can easily be used by the common man without any technical knowhow. A portable resistive device with miniaturized electronics is developed with microfluidic well and α-MnO2 /GQD nanocomposites as a sensing material for the sensitive detection of Pb(II). α-MnO2 /GQD nanocomposites which can be easily integrated with the miniaturized electronics for real-time on-field applications. The proposed sensor exhibited a tremendous potential to be integrated with conventional water purification appliances (household and commercial) to give an indication of safety index for the drinking water. The developed portable sensor required low sample volume (200 µL) and was assessed within the Pb(II) concentration range of 0.001 nM to 1 uM. The Limit of Detection (LoD) and sensitivity was calculated to be 0.81 nM and 1.05 kΩ/nM/mm2 , and was validated with the commercial impedance analyser. The shelf-life of the portable sensor was found to be ∼45 days.

Overexpression and repression of key rate-limiting enzymes (acetyl CoA carboxylase and HMG reductase) to enhance fatty acid production from Rhodotorula mucilaginosa.

Implementing two-way strategies to enhance the lipid production in Rhodotorula mucilaginosa with the help of metabolic engineering was focused on the overexpression of acetyl coenzyme A carboxylase (ACC1 carboxylase) gene and repression of 3-hydroxy 3-methylglutaryl reductase (HMG-CoA reductase). Using an inducer (sodium citrate) and inhibitor (rosuvastatin), the amounts of biomass, lipid, and carotenoid were estimated. In the presence of inhibitor (200 mM), 62% higher lipid concentration was observed, while 44% enhancement was recorded when inducer (3 mM) was used. A combination of both inhibitor and inducer resulted in a 57% increase in lipid concentration by the oleaginous yeast. These results were again confirmed by real-time polymerase chain reaction by targeting the expression of the genes coding for ACC1 carboxylase and 13-fold increase was recorded in the presence of inducer as compared with control. This combined strategy (inducer and inhibitor use) has been reported for the first time as far as the best of our knowledge. The metabolic engineering strategies reported here will be a powerful approach for the enhanced commercial production of lipids.

Multi-target, ensemble-based virtual screening yields novel allosteric KRAS inhibitors at high success rate.

RAS mutations account for >15% of all human tumors, and of these ~85% are due to mutations in a particular RAS gene: KRAS. Recent studies revealed that KRAS harbors four druggable allosteric sites. Here, we have (a) used molecular simulations to generate ensembles of wild type and four major oncogenic KRAS mutants (G12V, G12D, G13D, and Q61H); (b) characterized the druggability of each allosteric pocket in each protein; (c) conducted extensive ensemble-based virtual screening using pocket-tailored ligand libraries; (d) prioritized hits through hierarchical postdocking analysis; and (e) validated predicted hits with NMR. Of the 785 diverse potential hits identified by our in silico analysis, we tested 90 for their ability to bind KRAS using NMR and found that nine cause backbone amide chemical shift perturbations of residues near the functionally responsive switch loops, suggesting potential binding. We conducted detailed biophysical analyses on a novel indole-based compound to demonstrate the potential of our workflow to yield lead compounds. We believe the detailed information documented in this work regarding the druggability profile of each allosteric site and the chemical fingerprints of compounds that target them will serve as vital resources for future structure-based drug design efforts against KRAS, a high-value target for cancer therapy.

Synthesis, SAR and docking studies of substituted aryl phenylthiazolyl phenylcarboxamide as potential protein tyrosine phosphatase 1B (PTP1B) inhibitors.

In our continued effort to discover novel PTP1B inhibitor with improved in vivo activity, we attempted to optimize our previously discovered lead compound by replacing the sulfonyl group with benzoyl group to yield compound II. Additional structural modifications were performed on compound II to yield a series of 24 aryl phenylthiazolyl phenylcarboxamides as potential PTP1B inhibitors. Of the 24 tested, 6 compounds showed good PTP1B inhibitory activity while compound 38 as the most promising one. The plausible PTP1B-binding site interaction of compound 38 showed favourable binding similar to known PTP1B binders and suggests its selectivity towards PTP1B. Compound 38 also showed promising antihyperglycaemic, antidyslipidaemic and insulin resistant reversal activities in vivo in STZ model and db/db mice model. Altogether, the compound 38 presents an excellent candidate for future PTP1B targeted drug discovery.

Discovery of High-Affinity Noncovalent Allosteric KRAS Inhibitors That Disrupt Effector Binding.

Approximately 15% of all human tumors harbor mutant KRAS, a membrane-associated small GTPase and notorious oncogene. Mutations that render KRAS constitutively active will lead to uncontrolled cell growth and cancer. However, despite aggressive efforts in recent years, there are no drugs on the market that directly target KRAS and inhibit its aberrant functions. In the current work, we combined structure-based design with a battery of cell and biophysical assays to discover a novel pyrazolopyrimidine-based allosteric KRAS inhibitor that binds to activated KRAS with sub-micromolar affinity and disrupts effector binding, thereby inhibiting KRAS signaling and cancer cell growth. These results show that pyrazolopyrimidine-based compounds may represent a first-in-class allosteric noncovalent inhibitors of KRAS. Moreover, by studying two of its analogues, we identified key chemical features of the compound that interact with a set of specific residues at the switch regions of KRAS and play critical roles for its high-affinity binding and unique mode of action, thus providing a blueprint for future optimization efforts.

Evaluation of a Program for Improving Advanced Imaging Interpretation.

OBJECTIVES: Self-referred imaging has grown rapidly, raising concerns about increased costs and compromised quality of care. A quality improvement program using imaging interpretation criteria was designed by a national payer to ensure that noninvasive diagnostic images are interpreted by appropriately trained physicians. The objective of this program evaluation was to compare self-referral rates before and after institution of the imaging interpretation criteria program. METHODS: The imaging interpretation criteria program allocated privileges to bill for advanced imaging interpretation according to physician specialty. Nonradiologist physicians could obtain exemptions by appeal. Some physicians were not restricted in their billing because of successful appeals of the restrictions or the timing of their contract renewals. Self-referral rates were compared between the period 12 months before and 25 months after the program was initiated using t tests. The preprogram and postprogram self-referral rate for computed tomography and magnetic resonance imaging in aggregate was calculated both for the physicians that came into contact with the program and nationally, and then was stratified based on physician appeal status and reimbursement restrictions. RESULTS: The program was associated with significantly less frequent self-referrals by physicians whose appeals were denied (17.4%-8.2%; P = 0.0011) and by physicians notified of the program but not subject to it (24.8%-18.5%; P = 0.026). Self-referrals in the program states declined from 19.9% to 13.7% (P < 0.01). CONCLUSIONS: A significant reduction in image interpretations billed by physicians working outside of the scope of their training occurred after the implementation of the imaging interpretation criteria program.

Lipid Lowering Oxopropanylindole Hydrazone Derivatives with Antioxidant and Anti-hyperglycemic Activity.

A series of substituted oxopropanylindole hydrazone derivatives was synthesized and evaluated for anti-oxidant and anti-dyslipidemic activity. Of the 12 tested, 3 compounds (6c, 7b and 7d) showed good anti-oxidant activity, compound 6c attenuated LDL oxidation by 32%. The compounds 6c and 7d also showed good anti-dyslipidemic activity by reducing serum levels of total cholesterol (TC), phospholipids (PL) and triglycerides (TG). These two compounds were further evaluated for antiadipogenic and anti-hyperglycemic activity, where 6c showed 44% reduction in lipid accumulation and 20.5% and 24.3% reduction in blood glucose at 5h and 24h respectively, as compared to standard drug metformin. Thus, compounds 6c and 7d with balanced anti-oxidant and anti-dyslipidimic activities may be excellent candidates for lead optimization and drug development for the treatment of metabolic disorders.

Integrated Ligand and Structure-Based Investigation of Structural Requirements for Silent Information Regulator 1 (SIRT1) Activation.

A series of imidazothiazole and oxazolopyridine derivatives as human Silent Information Regulator 1 (SIRT1) activators were subjected to the integrated 2D and 3D QSAR approaches. The derived 3D QSAR models yielded high cross-validated q2 values of 0.682 and 0.628 for CoMFA and CoMSIA, respectively. The non-cross validated values of r2 training = 0.89; predictive r2 test = 0.69 for CoMFA and r2=0.87; predictive r2 test =0.67 for CoMSIA reflected the statistical significance of the developed model. The steric, electrostatic, hydrophobic and hydrogen bond acceptor interactions have been found important in describing the variation in human SIRT1 activation. Further, 2D QSAR model for the same dataset yielded high statistical significance and derived 2D model's parameters corroborated with the 3D model in terms of features. Derived model was also validated by the crystal structure of active conformation of SIRT1. Developed models may be useful for the identification of potential novel human SIRT1 activators as a therapeutic agent.