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 impact of coronavirus disease 2019 pandemic on dental school assessments - Current status and future perspectives.

PURPOSE: To evaluate course directors' feedback on the assessment methods used during the coronavirus disease 2019 (COVID-19) pandemic and identify effective approaches for future assessments in dental education. METHODS: Course directors at the US dental schools were surveyed for changes in assessments implemented during the early stages of the pandemic (March-July 2020) using the Qualtrics platform. The survey questions addressed assessment methods utilized in didactic, preclinical, and clinical arenas pre-COVID-19 (before March 2020) and during the early phase of the pandemic (between March and July 2020) and identified any sustained changes in assessments post-COVID-19. Of the 295 responses for the type of courses directed, 48%, 22%, and 30% responses were for didactic, pre-clinical, and clinical assessments, respectively. Chi-square tests and 95% confidence intervals were used to assess quantitative differences. RESULTS: Computer-based un-proctored and remote- proctored assessments increased whereas paper-based in-person proctored assessments decreased during an early pandemic. For pre-clinical and clinical courses, objective-structured clinical exams and case-based assessments increased whereas, for didactic courses, the number of presentations, short-answer, and multiple-choice questions-based assessments increased. Specimen-based assessments and patient-based encounters decreased significantly in didactic and clinical courses, respectively. Manikin-based exams increased in clinical but not in pre-clinical courses. Survey respondents disagreed that alternative assessments helped students learn better, resulted in better course evaluations, or were an equivalent replacement for pre-COVID-19 assessments. Interestingly, 49% of respondents indicated a likelihood of continuing alternative assessments whereas 36% were unlikely and 15% were neutral. CONCLUSIONS: A combination of effective pre-pandemic and innovative alternative assessments developed during the pandemic may be the new normal in the dental education curriculum.

Evaluating dental students' perspectives on the concurrent teaching of didactic and case-based courses.

PURPOSE: To evaluate student perception of integrating biomedical and clinical sciences based on survey of dental students on the concurrent teaching of a didactic systems-based course and a case-based course. METHODS: First-year to fourth-year students (DS1-DS4) students were surveyed for their experiences in concurrent teaching. Student response rate for the survey was 55% (229/420). Pearson's Chi-squared tests and Kruskal-Wallis rank sum tests were used to assess statistical significance (p < 0.05). RESULTS: Of the students surveyed, 83% strongly agreed or agreed that concurrent teaching of the didactic and case-based courses helped them better understand the biomedical science background and the clinical ramifications (p < 0.001). On average, 75% percent strongly agreed or agreed that concurrent teaching kept them engaged, motivated, think critically, apply the course content and prepare for clinical practice (p < 0.001). Of the students surveyed, 69% support expanding concurrent teaching to all four years (p < 0.001). Mean responses from DS1 and DS4 students differed for questions relating to understanding of biomedical sciences, critical thinking and application to clinic (p < 0.01). Qualitative data showed that students enjoyed the reinforcement of concepts and application to clinical scenarios. CONCLUSIONS: Concurrent teaching of didactic and case-based learning courses, thus showing clinical relevance of biomedical sciences in the first year of dental curriculum, is perceived by students as an effective method of educating dental students. Such integrative learning process with horizontal and vertical integration and concurrent curriculum is even more relevant with the implementation of the integrated national board dental examination.

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.

Scaffold repurposing of fendiline: Identification of potent KRAS plasma membrane localization inhibitors.

KRAS plays an essential role in regulating cell proliferation, differentiation, migration and survival. Mutated KRAS is a major driver of malignant transformation in multiple human cancers. We showed previously that fendiline (6) is an effective inhibitor of KRAS plasma membrane (PM) localization and function. In this study, we designed, synthesized and evaluated a series of new fendiline analogs to optimize its drug properties. Systemic structure-activity relationship studies by scaffold repurposing led to the discovery of several more active KRAS PM localization inhibitors such as compounds 12f (NY0244), 12h (NY0331) and 22 (NY0335) which exhibit nanomolar potencies. These compounds inhibited oncogenic KRAS-driven cancer cell proliferation at single-digit micromolar concentrations in vitro. In vivo studies in a xenograft model of pancreatic cancer revealed that 12h and 22 suppressed oncogenic KRAS-expressing MiaPaCa-2 tumor growth at a low dose range of 1-5 mg/kg with no vasodilatory effects, indicating their potential as chemical probes and anticancer therapeutics.

Identification of EGFR and RAS Inhibitors using Caenorhabditis elegans.

The changes in the plasma membrane localization of the epidermal growth factor receptor (EGFR) and its downstream effector RAS have been implicated in several diseases including cancer. The free-living nematode C. elegans possesses an evolutionary and functionally conserved EGFR-RAS-ERK MAP signal cascade which is central for the development of the vulva. Gain of function mutations in RAS homolog LET-60 and EGFR homolog LET-23 induce the generation of visible nonfunctional ectopic pseudovulva along the ventral body wall of these worms. Previously, the multivulval (Muv) phenotype in these worms has been shown to be inhibited by small chemical molecules. Here we describe a protocol for using the worm in a liquid-based assay to identify inhibitors that abolish the activities of EGFR and RAS proteins. Using this assay, we show R-fendiline, an indirect inhibitor of K-RAS, suppresses the Muv phenotype expressed in the let-60(n1046) and let-23(sa62) mutant worms. The assay is simple, inexpensive, is not time consuming to setup, and can be used as an initial platform for the discovery of anticancer therapeutics.

Methods and timing of curricular integration in U.S. dental education in preparation for the Integrated National Board Dental Examination.

PURPOSE: To characterize methods, timing, faculty training, and barriers pertaining to curricular integration in preparation for the Integrated National Board Dental Examination (INBDE) at U.S. dental schools METHODS: Academic deans at 67 U.S. dental schools were emailed an online survey consisting of questions on the time frame and methods of curricular integration, faculty training, and barriers to curricular integration RESULTS: Approximately 86% of schools have implemented (∼39%) or will implement (∼61%) changes in curriculum in preparation for the INBDE. Curricular integration was completed in 3-4 years in most schools, with those schools with larger class sizes taking longer. While 56% of respondents reported avoiding a complete curricular overhaul, 79% reported course sequencing changes, adding, subtracting, or combining courses, and making some changes in existing courses. Curriculum committees had the most input in curricular changes, while individual instructors had the least influence. Sixty seven percent of respondents reported faculty being trained to teach in an integrated curriculum. Problem/case-based learning and clinical case presentations are being used more for incorporation of biomedical sciences in clinical curriculum; use of clinical examples was the most used mechanism to integrate clinical information in biomedical sciences curriculum. Eighty two percent of respondents indicated that lack of faculty time to prepare courses as the primary barrier for implementing an integrated curriculum. CONCLUSIONS: Our results show that most U.S. dental schools are implementing integrated teaching models to prepare their students for the INBDE. However, lack of faculty time and training need to be addressed for more successful curricular integration.

Integration of Basic and Clinical Sciences: Student Perceptions.

The integrated curriculum is becoming a popular concept among dental schools. The purpose of this study was to query dental students at the University of Texas Health Science Center at Houston - School of Dentistry (UTSD) to elucidate their level of interest in the integrated curriculum, perception of how much integration is currently occurring, and identify challenges to integration. To address this question, dental students at UTSD were invited to participate in a survey. Participants reported their perspectives on integration of sciences. All survey participants agreed that it is beneficial to integrate clinical and basic sciences and that basic science educators were incorporating clinical relevance in their regular teaching. The third and fourth year classes, classes that had been exposed to general as well as all specialty dentistry clinics, agreed that basic sciences are being incorporated into most clinical teaching. Top two barriers to integration identified by the students were lack of crossover knowledge of faculty, and insufficient time to explore connections between basic sciences and clinical sciences because of the volume of information that needs to be covered. In conclusion, student perception at UTSD is that overall basic and clinical sciences are being integrated throughout the curriculum.

Assessment of Critical Thinking in a First-Year Dental Curriculum.

We studied how students in a Doctor of Dental Surgery (DDS) program perceived the importance of critical thinking and the extent to which critical thinking was perceived to be included in each of 25 courses of the first-year curriculum at The University of Texas School of Dentistry at Houston (UTSD). Sixty-nine of the 102 second-year students who were invited participated in an online survey. The survey had three parts, with all statements of each part evaluated on a five-point Likert scale ranging from strongly agree to strongly disagree. The first two parts assessed the importance of critical thinking in dental education and the criteria by which critical thinking in didactic curriculum can be measured. In the third part of the survey, students evaluated how well each course of the first-year curriculum achieved critical thinking. More than 90% of the respondents strongly agreed/agreed that critical thinking is essential to making clinical decisions. Students strongly agreed/agreed that 19 of 25 of the courses incorporated critical thinking. However, when students were asked to rank the top five of the 25 courses, only two courses (Human Biology, Neuroscience) emerged above all others in their weighted ranks, with another seven courses standing out, leaving 16 courses with low weighted rankings for their inclusion of critical thinking. In summary, students agreed on the importance of critical thinking in dental education, and on the criteria by which the incorporation of critical thinking should be measured in didactic and pre-clinical courses.

Targeting plasma membrane phosphatidylserine content to inhibit oncogenic KRAS function.

The small GTPase KRAS, which is frequently mutated in human cancers, must be localized to the plasma membrane (PM) for biological activity. We recently showed that the KRAS C-terminal membrane anchor exhibits exquisite lipid-binding specificity for select species of phosphatidylserine (PtdSer). We, therefore, investigated whether reducing PM PtdSer content is sufficient to abrogate KRAS oncogenesis. Oxysterol-related binding proteins ORP5 and ORP8 exchange PtdSer synthesized in the ER for phosphatidyl-4-phosphate synthesized in the PM. We show that depletion of ORP5 or ORP8 reduced PM PtdSer levels, resulting in extensive mislocalization of KRAS from the PM. Concordantly, ORP5 or ORP8 depletion significantly reduced proliferation and anchorage-independent growth of multiple KRAS-dependent cancer cell lines, and attenuated KRAS signaling in vivo. Similarly, functionally inhibiting ORP5 and ORP8 by inhibiting PI4KIIIα-mediated synthesis of phosphatidyl-4-phosphate at the PM selectively inhibited the growth of KRAS-dependent cancer cell lines over normal cells. Inhibiting KRAS function through regulating PM lipid PtdSer content may represent a viable strategy for KRAS-driven cancers.

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.

HRAS-driven cancer cells are vulnerable to TRPML1 inhibition.

By serving as intermediaries between cellular metabolism and the bioenergetic demands of proliferation, endolysosomes allow cancer cells to thrive under normally detrimental conditions. Here, we show that an endolysosomal TRP channel, TRPML1, is necessary for the proliferation of cancer cells that bear activating mutations in HRAS Expression of MCOLN1, which encodes TRPML1, is significantly elevated in HRAS-positive tumors and inversely correlated with patient prognosis. Concordantly, MCOLN1 knockdown or TRPML1 inhibition selectively reduces the proliferation of cancer cells that express oncogenic, but not wild-type, HRAS Mechanistically, TRPML1 maintains oncogenic HRAS in signaling-competent nanoclusters at the plasma membrane by mediating cholesterol de-esterification and transport. TRPML1 inhibition disrupts the distribution and levels of cholesterol and thereby attenuates HRAS nanoclustering and plasma membrane abundance, ERK phosphorylation, and cell proliferation. These findings reveal a selective vulnerability of HRAS-driven cancers to TRPML1 inhibition, which may be leveraged as an actionable therapeutic strategy.

Integration of Basic and Clinical Sciences: Faculty Perspectives at a U.S. Dental School.

Although dental education has traditionally been organized into basic sciences education (first and second years) and clinical education (third and fourth years), there has been growing interest in ways to better integrate the two to more effectively educate students and prepare them for practice. Since 2012, The University of Texas School of Dentistry at Houston (UTSD) has made it a priority to improve integration of basic and clinical sciences, with a focus to this point on integrating the basic sciences. The aim of this study was to determine the perspectives of basic and clinical science faculty members regarding basic and clinical sciences integration and the degree of integration currently occurring. In October 2016, all 227 faculty members (15 basic scientists and 212 clinicians) were invited to participate in an online survey. Of the 212 clinicians, 84 completed the clinician educator survey (response rate 40%). All 15 basic scientists completed the basic science educator survey (response rate 100%). The majority of basic and clinical respondents affirmed the value of integration (93.3%, 97.6%, respectively) and reported regular integration in their teaching (80%, 86.9%). There were no significant differences between basic scientists and clinicians on perceived importance (p=0.457) and comfort with integration (p=0.240), but the basic scientists were more likely to integrate (p=0.039) and collaborate (p=0.021) than the clinicians. There were no significant differences between generalist and specialist clinicians on importance (p=0.474) and degree (p=0.972) of integration in teaching and intent to collaborate (p=0.864), but the specialists reported feeling more comfortable presenting basic science information (p=0.033). Protected faculty time for collaborative efforts and a repository of integrated basic science and clinical examples for use in teaching and faculty development were recommended to improve integration. Although questions might be raised about the respondents' definition of "integration," this study provides a baseline assessment of perceptions at a dental school that is placing a priority on integration.

Sphingomyelin Metabolism Is a Regulator of K-Ras Function.

K-Ras must localize to the plasma membrane (PM) for biological activity. We show here that multiple acid sphingomyelinase (ASM) inhibitors, including tricyclic antidepressants, mislocalized phosphatidylserine (PtdSer) and K-RasG12V from the PM, resulting in abrogation of K-RasG12V signaling and potent, selective growth inhibition of mutant K-Ras-transformed cancer cells. Concordantly, in nude mice, the ASM inhibitor fendiline decreased the rate of growth of oncogenic K-Ras-expressing MiaPaCa-2 tumors but had no effect on the growth of the wild-type K-Ras-expressing BxPC-3 tumors. ASM inhibitors also inhibited activated LET-60 (a K-Ras ortholog) signaling in Caenorhabditis elegans, as evidenced by suppression of the induced multivulva phenotype. Using RNA interference against C. elegans genes encoding other enzymes in the sphingomyelin (SM) biosynthetic pathway, we identified 14 enzymes whose knockdown strongly or moderately suppressed the LET-60 multivulva phenotype. In mammalian cells, pharmacological agents that target these enzymes all depleted PtdSer from the PM and caused K-RasG12V mislocalization. These effects correlated with changes in SM levels or subcellular distribution. Selected compounds, including sphingosine kinase inhibitors, potently inhibited the proliferation of oncogenic K-Ras-expressing pancreatic cancer cells. In conclusion, these results show that normal SM metabolism is critical for K-Ras function, which may present therapeutic options for the treatment of K-Ras-driven cancers.

AMPK and Endothelial Nitric Oxide Synthase Signaling Regulates K-Ras Plasma Membrane Interactions via Cyclic GMP-Dependent Protein Kinase 2.

K-Ras must localize to the plasma membrane and be arrayed in nanoclusters for biological activity. We show here that K-Ras is a substrate for cyclic GMP-dependent protein kinases (PKGs). In intact cells, activated PKG2 selectively colocalizes with K-Ras on the plasma membrane and phosphorylates K-Ras at Ser181 in the C-terminal polybasic domain. K-Ras phosphorylation by PKG2 is triggered by activation of AMP-activated protein kinase (AMPK) and requires endothelial nitric oxide synthase and soluble guanylyl cyclase. Phosphorylated K-Ras reorganizes into distinct nanoclusters that retune the signal output. Phosphorylation acutely enhances K-Ras plasma membrane affinity, but phosphorylated K-Ras is progressively lost from the plasma membrane via endocytic recycling. Concordantly, chronic pharmacological activation of AMPK → PKG2 signaling with mitochondrial inhibitors, nitric oxide, or sildenafil inhibits proliferation of K-Ras-positive non-small cell lung cancer cells. The study shows that K-Ras is a target of a metabolic stress-signaling pathway that can be leveraged to inhibit oncogenic K-Ras function.

Inhibition of Acid Sphingomyelinase Depletes Cellular Phosphatidylserine and Mislocalizes K-Ras from the Plasma Membrane.

K-Ras must localize to the plasma membrane for biological activity; thus, preventing plasma membrane interaction blocks K-Ras signal output. Here we show that inhibition of acid sphingomyelinase (ASM) mislocalizes both the K-Ras isoforms K-Ras4A and K-Ras4B from the plasma membrane to the endomembrane and inhibits their nanoclustering. We found that fendiline, a potent ASM inhibitor, reduces the phosphatidylserine (PtdSer) and cholesterol content of the inner plasma membrane. These lipid changes are causative because supplementation of fendiline-treated cells with exogenous PtdSer rapidly restores K-Ras4A and K-Ras4B plasma membrane binding, nanoclustering, and signal output. Conversely, supplementation with exogenous cholesterol restores K-Ras4A but not K-Ras4B nanoclustering. These experiments reveal different operational pools of PtdSer on the plasma membrane. Inhibition of ASM elevates cellular sphingomyelin and reduces cellular ceramide levels. Concordantly, delivery of recombinant ASM or exogenous ceramide to fendiline-treated cells rapidly relocalizes K-Ras4B and PtdSer to the plasma membrane. K-Ras4B mislocalization is also recapitulated in ASM-deficient Neimann-Pick type A and B fibroblasts. This study identifies sphingomyelin metabolism as an indirect regulator of K-Ras4A and K-Ras4B signaling through the control of PtdSer plasma membrane content. It also demonstrates the critical and selective importance of PtdSer to K-Ras4A and K-Ras4B plasma membrane binding and nanoscale spatial organization.

SIGNAL TRANSDUCTION. Membrane potential modulates plasma membrane phospholipid dynamics and K-Ras signaling.

Plasma membrane depolarization can trigger cell proliferation, but how membrane potential influences mitogenic signaling is uncertain. Here, we show that plasma membrane depolarization induces nanoscale reorganization of phosphatidylserine and phosphatidylinositol 4,5-bisphosphate but not other anionic phospholipids. K-Ras, which is targeted to the plasma membrane by electrostatic interactions with phosphatidylserine, in turn undergoes enhanced nanoclustering. Depolarization-induced changes in phosphatidylserine and K-Ras plasma membrane organization occur in fibroblasts, excitable neuroblastoma cells, and Drosophila neurons in vivo and robustly amplify K-Ras-dependent mitogen-activated protein kinase (MAPK) signaling. Conversely, plasma membrane repolarization disrupts K-Ras nanoclustering and inhibits MAPK signaling. By responding to voltage-induced changes in phosphatidylserine spatiotemporal dynamics, K-Ras nanoclusters set up the plasma membrane as a biological field-effect transistor, allowing membrane potential to control the gain in mitogenic signaling circuits.

Another surprise from Metformin: novel mechanism of action via K-Ras influences endometrial cancer response to therapy.

Metformin is an oral biguanide commonly used for the treatment of type II diabetes and has recently been demonstrated to possess antiproliferative properties that can be exploited for the prevention and treatment of a variety of cancers. The mechanisms underlying this effect have not been fully elucidated. Using both in vitro and in vivo models, we examined the effects of metformin on endometrial tumors with defined aberrations in the PI3K/PTEN/mTOR and MAPK signaling pathways to understand metformin mechanism of action and identify clinically useful predictors of response to this agent. In vitro assays of proliferation, cytotoxicity, and apoptosis were used to quantify the effects of metformin on endometrial cancer cell lines with mutations in the PI3K/PTEN/mTOR and MAPK signaling pathways. The in vivo effects of oral metformin on tumor progression were further examined using xenograft mouse models of endometrial cancer. K-Ras localization was analyzed by confocal microscopy using GFP-labeled oncogenic K-Ras and by immunoblot following subcellular fractionation. Metformin inhibited cell proliferation, induced apoptosis, and decreased tumor growth in preclinical endometrial cancer models, with the greatest response observed in cells harboring activating mutations in K-Ras. Furthermore, metformin displaces constitutively active K-Ras from the cell membrane, causing uncoupling of the MAPK signaling pathway. These studies provide a rationale for clinical trials using metformin in combination with PI3K-targeted agents for tumors harboring activating K-Ras mutations, and reveal a novel mechanism of action for metformin.