Acquired Resistance to KRASG12C Inhibition in Cancer.

BACKGROUND: Clinical trials of the KRAS inhibitors adagrasib and sotorasib have shown promising activity in cancers harboring KRAS glycine-to-cysteine amino acid substitutions at codon 12 (KRASG12C). The mechanisms of acquired resistance to these therapies are currently unknown. METHODS: Among patients with KRASG12C -mutant cancers treated with adagrasib monotherapy, we performed genomic and histologic analyses that compared pretreatment samples with those obtained after the development of resistance. Cell-based experiments were conducted to study mutations that confer resistance to KRASG12C inhibitors. RESULTS: A total of 38 patients were included in this study: 27 with non-small-cell lung cancer, 10 with colorectal cancer, and 1 with appendiceal cancer. Putative mechanisms of resistance to adagrasib were detected in 17 patients (45% of the cohort), of whom 7 (18% of the cohort) had multiple coincident mechanisms. Acquired KRAS alterations included G12D/R/V/W, G13D, Q61H, R68S, H95D/Q/R, Y96C, and high-level amplification of the KRASG12C allele. Acquired bypass mechanisms of resistance included MET amplification; activating mutations in NRAS, BRAF, MAP2K1, and RET; oncogenic fusions involving ALK, RET, BRAF, RAF1, and FGFR3; and loss-of-function mutations in NF1 and PTEN. In two of nine patients with lung adenocarcinoma for whom paired tissue-biopsy samples were available, histologic transformation to squamous-cell carcinoma was observed without identification of any other resistance mechanisms. Using an in vitro deep mutational scanning screen, we systematically defined the landscape of KRAS mutations that confer resistance to KRASG12C inhibitors. CONCLUSIONS: Diverse genomic and histologic mechanisms impart resistance to covalent KRASG12C inhibitors, and new therapeutic strategies are required to delay and overcome this drug resistance in patients with cancer. (Funded by Mirati Therapeutics and others; ClinicalTrials.gov number, NCT03785249.).

Selected Approaches to Disrupting Protein-Protein Interactions within the MAPK/RAS Pathway.

Within the MAPK/RAS pathway, there exists a plethora of protein-protein interactions (PPIs). For many years, scientists have focused efforts on drugging KRAS and its effectors in hopes to provide much needed therapies for patients with KRAS-mutant driven cancers. In this review, we focus on recent strategies to inhibit RAS-signaling via disrupting PPIs associated with SOS1, RAF, PDEδ, Grb2, and RAS.

Design and evaluation of achiral, non-atropisomeric 4-(aminomethyl)phthalazin-1(2H)-one derivatives as novel PRMT5/MTA inhibitors.

MRTX1719 is an inhibitor of the PRMT5/MTA complex and recently entered clinical trials for the treatment of MTAP-deleted cancers. MRTX1719 is a class 3 atropisomeric compound that requires a chiral synthesis or a chiral separation step in its preparation. Here, we report the SAR and medicinal chemistry design strategy, supported by structural insights from X-ray crystallography, to discover a class 1 atropisomeric compound from the same series that does not require a chiral synthesis or a chiral separation step in its preparation.

A scoping review of the roles, training, and impact of community health workers in oral health.

OBJECTIVE: To synthesize English or Spanish-language literature on community health workers' (CHWs') roles, training, and impact in oral health. BASIC RESEARCH DESIGN: A scoping review conducted in accordance with the Arksey and O'Malley (2005) methodological framework. METHOD: Electronic literature searches were conducted in Medline (Ovid), Embase (Ovid), DOSS, CINAHL, Web of Science, and Global Health CAB from inception of the databases to April 2020. Three reviewers independently conducted the title and abstract and full-text reviews. This was followed by data charting by three reviewers and data summarizing by two reviewers. RESULTS: Out of the 36 articles that met the inclusion criteria, most took place in the United States (n=15) with most published between 2012 and 2019 (12). CHWs were incorporated in programs that focused on access to dental care (n=10), oral health promotion only (9), early childhood caries (8), oral health promotion and services (5), and oral cancer screening (4). Common roles included providing oral health education and behavior change motivation to community members, facilitating utilization of dental services, and the delivery of diagnostic and dental services to community members. Training and outcomes were not consistently described across studies. CONCLUSION: CHWs have been used in oral health programs and interventions across a wide range of locations and contexts. The implementation and scaling-up of oral health CHW programs requires appropriate provision of training as well as community embedded monitoring and evaluation structures based on rigorous methods with clearly defined outcomes.

Time-Resolved Fuel Density Profiles of the Stagnation Phase of Indirect-Drive Inertial Confinement Implosions.

The implosion efficiency in inertial confinement fusion depends on the degree of stagnated fuel compression, density uniformity, sphericity, and minimum residual kinetic energy achieved. Compton scattering-mediated 50-200 keV x-ray radiographs of indirect-drive cryogenic implosions at the National Ignition Facility capture the dynamic evolution of the fuel as it goes through peak compression, revealing low-mode 3D nonuniformities and thicker fuel with lower peak density than simulated. By differencing two radiographs taken at different times during the same implosion, we also measure the residual kinetic energy not transferred to the hot spot and quantify its impact on the implosion performance.

Chemical proteomics reveals new targets of cysteine sulfinic acid reductase.

Cysteine sulfinic acid or S-sulfinylation is an oxidative post-translational modification (OxiPTM) that is known to be involved in redox-dependent regulation of protein function but has been historically difficult to analyze biochemically. To facilitate the detection of S-sulfinylated proteins, we demonstrate that a clickable, electrophilic diazene probe (DiaAlk) enables capture and site-centric proteomic analysis of this OxiPTM. Using this workflow, we revealed a striking difference between sulfenic acid modification (S-sulfenylation) and the S-sulfinylation dynamic response to oxidative stress, which is indicative of different roles for these OxiPTMs in redox regulation. We also identified >55 heretofore-unknown protein substrates of the cysteine sulfinic acid reductase sulfiredoxin, extending its function well beyond those of 2-cysteine peroxiredoxins (2-Cys PRDX1-4) and offering new insights into the role of this unique oxidoreductase as a central mediator of reactive oxygen species-associated diseases, particularly cancer. DiaAlk therefore provides a novel tool to profile S-sulfinylated proteins and study their regulatory mechanisms in cells.

Divergent trends in the prevalence of asthma-like symptoms and asthma in a developing country: three repeated surveys between 2002 and 2016.

INTRODUCTION AND OBJECTIVES: There have been differences in temporal trends of asthma prevalence by geographic region and economic prosperity. The aim of this study was to assess temporal trends in asthma prevalence among young adolescents in Skopje, Republic of North Macedonia as a developing country with a low asthma prevalence. SUBJECTS AND METHODS: Data were obtained from three cross-sectional surveys (2002, 2006, and 2016) of adolescents (12-15 years) from randomly selected schools in Skopje. Trends in the prevalence of asthma and asthma-like symptoms were investigated descriptively and using multiple logistic regression to adjust for potential confounding factors. RESULTS: The prevalence of asthma increased, although the changes were not statistically significant (2002: 1.7%; 2006: 2.0%; 2016: 2.8%; p=0.075). Statistically significant (p<0.05) reductions in wheeze prevalence over time (2002, 2006, 2016) were observed for current wheeze (8.8%, 7.2%, 5.5%), exercise-induced wheeze (14.2%, 7.9%, 1.9%), and night dry cough (16.5%, 13.5%, 9.6%). After adjustment for potential confounding factors, there was an increase in asthma likelihood by year compared to 2002 (2006: OR=1.22, 95%CI=0.67-2.22; 2016: OR=2.45, 95%CI=1.24-4.84). In the adjusted analyses, associations between year and the asthma-like symptoms confirmed the descriptive results, except for current wheeze, where statistical significance disappeared. CONCLUSIONS: Divergent trends in prevalence with a decrease in asthma-like symptoms and an increase in physician-diagnosed asthma in Skopje during a period of 14 years were established. Improved asthma labelling and effective preventative treatment of symptoms may explain some of these changes, although changes in environment and lifestyle could not be ruled out.

Prevalence of and factors associated with underdiagnosis of pediatric asthma in Batumi, Georgia.

BACKGROUND AND OBJECTIVE: A recent survey in Batumi, Georgia showed a low prevalence of asthma in children (1.8%). A potential explanation is underdiagnosis of asthma. To investigate this, we conducted a follow up to the survey with the objective of estimating the level of childhood asthma underdiagnosis and to describe factors related to it. METHODS: Subjects included 437 survey participants who had a history of asthma-like symptoms and no diagnosis of asthma. All children underwent clinical examination (spirometry, skin prick tests, FeNO measurement) to identify new cases of asthma. The distribution of host and environmental factors was compared between the group with newly identified asthma and a group of 59 children with previously known asthma (diagnosed asthma). RESULTS: Clinical investigation identified 107 cases of undiagnosed asthma. The corrected asthma prevalence estimate was 5.1% (95%CI: 4.4%-5.9%) suggesting that 65% of asthma cases were undiagnosed. Compared to children with diagnosed asthma, children with undiagnosed asthma were younger (8.2±1.6 vs. 9.3±2.1; p=0.0005), had less frequent history of allergic disorders (38.3% vs. 64.4%; p=0.001), and a lower prevalence of parental asthma (1.8% vs. 8.4%; p=0.04). The groups did not differ in terms of environmental characteristics except for more exposure to passive smoking in the undiagnosed asthma group (p=0.01). Multivariate analysis confirmed results of simple analyses. CONCLUSION: In Batumi, 65% of children with asthma remain undiagnosed. Older age of a child, coexisting allergic disorders, and parental asthma seem to facilitate diagnosis. Implementation of current diagnostic guidelines should improve diagnostic accuracy of pediatric asthma in Batumi.

Eight-year follow-up of a randomized clinical trial comparing ultrasound-guided foam sclerotherapy with surgical stripping of the great saphenous vein.

BACKGROUND: This was an 8-year follow-up of an RCT comparing ultrasound-guided foam sclerotherapy (UGFS) with high ligation and surgical stripping (HL/S) of the great saphenous vein (GSV). METHODS: Patients were randomized to UGFS or HL/S of the GSV. The primary outcome was the recurrence of symptomatic GSV reflux. Secondary outcomes were patterns of reflux according to recurrent varices after surgery, Clinical Etiologic Anatomic Pathophysiologic (CEAP) classification, Venous Clinical Severity Score (VCSS) and EuroQol Five Dimensions (EQ-5D™) quality-of-life scores. RESULTS: Of 430 patients originally randomized (230 UGFS, 200 HL/S), 227 (52·8 per cent; 123 UGFS, 103 HL/S) were available for analysis after 8 years. The proportion of patients free from symptomatic GSV reflux at 8 years was lower after UGFS than HL/S (55·1 versus 72·1 per cent; P = 0·024). The rate of absence of GSV reflux, irrespective of venous symptoms, at 8 years was 33·1 and 49·7 per cent respectively (P = 0·009). More saphenofemoral junction (SFJ) failure (65·8 versus 41·7 per cent; P = 0·001) and recurrent reflux in the above-knee GSV (72·5 versus 20·4 per cent; P = 0·001) was evident in the UGFS group. The VCSS was worse than preoperative scores in both groups after 8 years; CEAP classification and EQ-5D® scores were similar in the two groups. CONCLUSION: Surgical stripping had a technically better outcome in terms of recurrence of GSV and SFJ reflux than UGFS in the long term. Long-term follow-up suggests significant clinical progression of venous disease measured by VCSS in both groups, but less after surgery. Registration number: NCT02304146 (http://www.clinicaltrials.gov).

Characterisation of feline renal cortical fibroblast cultures and their transcriptional response to transforming growth factor ß1.

BACKGROUND: Chronic kidney disease (CKD) is common in geriatric cats, and the most prevalent pathology is chronic tubulointerstitial inflammation and fibrosis. The cell type predominantly responsible for the production of extra-cellular matrix in renal fibrosis is the myofibroblast, and fibroblast to myofibroblast differentiation is probably a crucial event. The cytokine TGF-ß1 is reportedly the most important regulator of myofibroblastic differentiation in other species. The aim of this study was to isolate and characterise renal fibroblasts from cadaverous kidney tissue of cats with and without CKD, and to investigate the transcriptional response to TGF-ß1. RESULTS: Cortical fibroblast cultures were successfully established from the kidney tissue of cats with normal kidney function (FCF) and cats with chronic kidney disease (CKD-FCF). Both cell types expressed the mesenchymal markers vimentin, CD44 and CD29, and were negative for the epithelial marker cytokeratin, mesangial cell marker desmin and endothelial cell marker vWF. Only CKD-FCF expressed VCAM-1, a cell marker associated with inflammation. Incubation with TGF-ß1 (0-10 ng/ml) induced a concentration dependent change in cell morphology, and upregulation of myofibroblast marker gene α-SMA expression alongside collagen 1α1, fibronectin, TGF-ß1 and CTGF mRNA. These changes were blocked by the TGF-ß1 receptor 1 antagonist SB431542 (5 µM). CONCLUSIONS: FCF and CKD-FCF can be cultured via a simple method and represent a model for the investigation of the progression of fibrosis in feline CKD. The findings of this study suggest TGF-ß1 may be involved in fibroblast-myofibroblast transition in feline CKD, as in other species.

An international comparison of risk factors between two regions with distinct differences in asthma prevalence.

BACKGROUND AND PURPOSE: Investigation of the geographic variation in asthma prevalence can improve our understanding of asthma etiology and management. The purpose of our investigation was to compare the prevalence of asthma and wheeze among adolescents living in two distinct international regions and to investigate reasons for observed differences. METHODS: A cross-sectional survey of 13-14 year olds was completed in Saskatoon, Canada (n=1200) and Skopje, Republic of Macedonia (n=3026), as part of the International Study of Asthma and Allergies in Childhood (ISAAC) Phase 3 study. Surveys were self-completed by students following the ISAAC protocol. Multiple logistic regression models were used to investigate associations with reports of asthma and current wheeze. A mediation analysis was then completed. RESULTS: Asthma prevalence was much higher in Saskatoon than Skopje (21.3% vs. 1.7%) as was the prevalence of current wheeze (28.2% vs. 8.8%). Higher paracetamol (acetaminophen) use was a consistent risk factor for asthma and wheeze in both locations and showed dose-response relationships. In both countries, paracetamol use and physical activity mediated some of the association for both asthma and wheeze. In Saskatoon, among those with current wheeze, 42.6% reported ever having a diagnosis of asthma compared to 10.2% among Skopje adolescents. CONCLUSIONS: The results suggest that the variation in risk factors between the two locations may explain some of the differences in the prevalence of asthma and wheeze between these two study sites. However, diagnostic labeling patterns should not be ruled out as another potential explanatory factor.

Identification of AHCY inhibitors using novel high-throughput mass spectrometry.

S-adenosylhomocysteine hydrolase (AHCY) catalyzes the reversible hydrolysis of S-adenosylhomocysteine (SAH) to adenosine and l-homocysteine. This enzyme is frequently overexpressed in many tumor types and is considered to be a validated anti-tumor target. In order to enable the development of small molecule AHCY inhibitors as targeted cancer therapeutics we developed an assay based on a RapidFire high-throughput mass spectrometry detection system, which allows the direct measurement of AHCY enzymatic activity. This technique avoids many of the problems associate with the previously reported method of using a thiol-reactive fluorescence probes to measure AHCY activity. Screening of a ∼500,000 compound library using this technique identified multiple SAH competitive hits. Co-crystal structures of the hit compounds complexed with AHCY were obtained showing that the compounds indeed bind in the SAH site of the enzyme. In addition, some hit compounds increased the SAH levels in HCT116 cells and showed growth inhibition. These compounds could be promising starting points for the optimization of cancer treatments.

Arteriovenous Fistulae for Haemodialysis: A Systematic Review and Meta-analysis of Efficacy and Safety Outcomes.

BACKGROUND: Arteriovenous fistulae are the currently recommended gold standard vascular access modality for haemodialysis because of their prolonged patency, improved durability, and low risk of infection for those that mature. However, notable disadvantages are observed in terms of protracted maturation time, associated high rates of catheter use, and substantial abandonment rates. The aim of this study was to quantitatively summarize the outcomes of fistula patency, infection, maturation, and abandonment published in the scientific literature. METHODS: This was a systematic review and meta-analyses of studies evaluating fistula outcomes. Literature searches were conducted in multiple databases to identify observational and interventional studies of mean fistula patency rates at 1 year, infection risk, maturation time, and abandonment. Digitisation software was used to simulate individual patient level data from Kaplan-Meier survival plots. RESULTS: Over 8000 studies were reviewed, and from these, 318 studies were included comprising 62,712 accesses. For fistulas the primary unassisted, primary assisted, and secondary patency rates at one year were 64%, 73% and 79% respectively, however not all fistulas reported as patent could be confirmed as being clinically useful for dialysis (i.e. functional patency). For fistulas that were reported as mature, mean time to maturation was 3.5 months, however only 26% of created fistulas were reported as mature at 6 months and 21% of fistulas were abandoned without use. Overall risk of infection in fistula patients was 4.1% and the overall rate per 100 access days was 0.018. CONCLUSIONS: Reported fistula patency rates may overstate their potential clinical utility when time to maturation, maturation rate, abandonment and infection are considered. Protracted maturation times, abandonment and infection all have a significant impact on evaluating the clinical utility of fistula creation. A rigorous and consistent set of outcomes definitions for hemodialysis access are necessary to clarify factors contributing to fistula success and the clinical consequence of fistula failure.

Design, synthesis and optimization of 7-substituted-pyrazolo[4,3-b]pyridine ALK5 (activin receptor-like kinase 5) inhibitors.

A series of potent ALK5 inhibitors were designed using a SBDD approach and subsequently optimized to improve drug likeness. Starting with a 4-substituted quinoline screening hit, SAR was conducted using a ALK5 binding model to understand the binding site and optimize activity. The resulting inhibitors displayed excellent potency but were limited by high in vitro clearance in rat and human microsomes. Using a scaffold morphing strategy, these analogs were transformed into a related pyrazolo[4,3-b]pyridine series with improved ADME properties.

Structure-based optimization of 1H-imidazole-2-carboxamides as Axl kinase inhibitors utilizing a Mer mutant surrogate.

Axl has been a target of interest in the oncology field for several years based on its role in various oncogenic processes. To date, no wild-type Axl crystal structure has been reported. Herein, we describe the structure-based optimization of a novel chemotype of Axl inhibitors, 1H-imidazole-2-carboxamide, using a mutated kinase homolog, Mer(I650M), as a crystallographic surrogate. Iterative optimization of the initial lead compound (1) led to compound (21), a selective and potent inhibitor of wild-type Axl. Compound (21) will serve as a useful compound for further in vivo studies.

Indoor mold levels and current asthma among school-aged children in Saskatchewan, Canada.

Current knowledge regarding the association between indoor mold exposures and asthma is still limited. The objective of this case-control study was to investigate the relationship between objectively measured indoor mold levels and current asthma among school-aged children. Parents completed a questionnaire survey of health history and home environmental conditions. Asthma cases had a history of doctor-diagnosed asthma or current wheeze without a cold in the past 12 months. Controls were age- and sex-matched to cases. Vacuumed dust samples were collected from the child's indoor play area and mattress. Samples were assessed for mold levels and quantified in colony-forming units (CFU). Sensitization to mold allergens was also determined by skin testing. Being a case was associated with family history of asthma, pet ownership, and mold allergy. Mold levels (CFU/m2 ) in the dust samples of children's mattress and play area floors were moderately correlated (r = 0.56; P < 0.05). High mold levels (≥30 000 CFU/m2 ) in dust samples from play [adjusted odds ratio (aOR) = 2.6; 95% CI: 1.03-6.43] and mattress (aOR) = 3.0; 95% CI: 1.11-8.00) areas were significantly associated with current asthma. In this study high levels of mold are a risk factor for asthma in children.

Environmental drivers of diurnal visits by transient predatory fishes to Caribbean patch reefs.

Video cameras recorded the diurnal visitation rates of transient (large home range) piscivorous fishes to coral patch reefs in The Bahamas and identified 11 species. Visits by bar jack Caranx ruber, mutton snapper Lutjanus analis, yellowtail snapper Ocyurus chrysurus, barracuda Sphyraena barracuda and cero Scomberomorus regalis were sufficiently frequent to correlate with a range of biophysical factors. Patch-reef visitation rates and fish abundances varied with distance from shore and all species except S. regalis were seen more frequently inshore. This pattern is likely to be caused by factors including close proximity to additional foraging areas in mangroves and on fore-reefs and higher abundances close to inshore nursery habitats. Visitation rates and abundances of C. ruber, L. analis, O. chrysurus and S. regalis also varied seasonally (spring v. winter), possibly as fishes responded to temperature changes or undertook spawning migrations. The abundance of each transient predator species on the patch reefs generally exhibited limited diurnal variability, but L. analis was seen more frequently towards dusk. This study demonstrates that the distribution of transient predators is correlated spatially and temporally with a range of factors, even within a single lagoon, and these drivers are species specific. Transient predators are considered an important source of mortality shaping reef-fish assemblages and their abundance, in combination with the biomass of resident predators, was negatively correlated with the density of prey fishes. Furthermore, transient predators are often targeted by fishers and understanding how they utilize seascapes is critical for protecting them within reserves.

Design, synthesis and optimization of novel Alk5 (activin-like kinase 5) inhibitors.

Using SBDD, a series of 4-amino-7-azaindoles were discovered as a novel class of Alk5 inhibitors that are potent in both Alk5 enzymatic and cellular assays. Subsequently a ring cyclization strategy was utilized to improve ADME properties leading to the discovery of a series of 1H-imidazo[4,5-c]pyridin-2(3H)-one drug like Alk5 inhibitors.

Discovery of potent, reversible MetAP2 inhibitors via fragment based drug discovery and structure based drug design-Part 2.

Methionine aminopeptidase-2 (MetAP2) is an enzyme that cleaves an N-terminal methionine residue from a number of newly synthesized proteins. This step is required before they will fold or function correctly. Pre-clinical and clinical studies with a MetAP2 inhibitor suggest that they could be used as a novel treatment for obesity. Herein we describe the discovery of a series of pyrazolo[4,3-b]indoles as reversible MetAP2 inhibitors. A fragment-based drug discovery (FBDD) approach was used, beginning with the screening of fragment libraries to generate hits with high ligand-efficiency (LE). An indazole core was selected for further elaboration, guided by structural information. SAR from the indazole series led to the design of a pyrazolo[4,3-b]indole core and accelerated knowledge-based fragment growth resulted in potent and efficient MetAP2 inhibitors, which have shown robust and sustainable body weight loss in DIO mice when dosed orally.