Development of a high-throughput TR-FRET screening assay for a fast-cycling KRAS mutant.

Mutations in the small GTPase protein KRAS are one of the leading drivers of cancers including lung, pancreatic, and colorectal, as well as a group of developmental disorders termed "Rasopathies". Recent breakthroughs in the development of mutant-specific KRAS inhibitors include the FDA approved drug Lumakras (Sotorasib, AMG510) for KRAS G12C-mutated non-small cell lung cancer (NSCLC), and MRTX1133, a promising clinical candidate for the treatment of KRAS G12D-mutated cancers. However, there are currently no FDA approved inhibitors that target KRAS mutations occurring at non-codon 12 positions. Herein, we focused on the KRAS mutant A146T, found in colorectal cancers, that exhibits a "fast-cycling" nucleotide mechanism as a driver for oncogenic activation. We developed a novel high throughput time-resolved fluorescence resonance energy transfer (TR-FRET) assay that leverages the reduced nucleotide affinity of KRAS A146T. As designed, the assay is capable of detecting small molecules that act to allosterically modulate GDP affinity or directly compete with the bound nucleotide. A pilot screen was completed to demonstrate robust statistics and reproducibility followed by a primary screen using a diversity library totaling over 83,000 compounds. Compounds yielding >50% inhibition of TR-FRET signal were selected as hits for testing in dose-response format. The most promising hit, UNC10104889, was further investigated through a structure activity relationship (SAR)-by-catalog approach in an attempt to improve potency and circumvent solubility liabilities. Overall, we present the TR-FRET platform as a robust assay to screen fast-cycling KRAS mutants enabling future discovery efforts for novel chemical probes and drug candidates.

Chronic neuroinflammation impairs waste clearance in the rat brain.

Background: Previous reports have established an association between impaired clearance of macromolecular waste from the brain parenchyma and a variety of brain insults for which chronic neuroinflammation is a common pathological feature. Here we investigate whether chronic neuroinflammation is sufficient to impair macromolecular waste clearance from the rat brain. Methods: Using a rodent model of chronic neuroinflammation induced by a single high-dose injection of lipopolysaccharide, the clearance kinetics of two fluorophore-conjugated dextran tracers were assayed at 8-weeks post-induction. The expression and distribution of amyloid ß and aquaporin-4 proteins within selected brain regions were assayed at 36-weeks post-induction, following open-field, novel object recognition, and contextual fear conditioning assays. Results: Chronic neuroinflammation significantly impaired the clearance kinetics of both dextran tracers and resulted in significantly elevated levels of amyloid ß within the hippocampus. Aquaporin-4 density on astrocytic endfeet processes was also reduced within multiple brain regions. These pathologies were associated with significantly enhanced contextual fear memory. Conclusion: Our results suggest that chronic neuroinflammation is sufficient to compromise the clearance of macromolecular waste from the brain parenchyma and may be the root cause of impaired waste clearance associated with a variety of brain pathologies.

Cognitive dual-task alters Local Dynamic Stability of lower extremity during common movements.

A dual-task paradigm is most commonly used in the field of biomechanics to understand the effect of multi-tasking or cognitive load on motor performance. The Local Dynamic Stability (LDS) is most commonly used to quantify motor performance, but there are still several unknown effects of this metric with varied task conditions and cognitive demands. Therefore, this study used motion capture to collect biomechanical data from 28 healthy collegiate participants during a walk and jog task both with and without a semantic fluency task to investigate the effects of task speed, limb dominance, and semantic fluency on LDS. This study showed that the change in ankle plantarflexion LDS during a jog was dependent on self-selected speed. Participants with slower jogging speeds increased ankle plantarflexion LDS during a dual-task, while individuals with faster jogging speeds decreased ankle plantarflexion LDS during a dual-task. This study also found that subjects compensate during gait by both increasing and decreasing LDS in different degrees of freedom of the lower extremity. This study did not find evidence of limb dominance effecting the change in LDS during a dual-task while walking or jogging. These findings reveal where healthy adults compensate for simple movement patterns while multitasking. Future work should further explore the role and relationship between trunk movement and lower extremity compensation, and could help give further context to how the LDS can be interpreted by researchers and clinicians alike.

Local dynamic stability of the lower-limb as a means of post-hoc injury classification.

Since most sporting injuries occur at the lower extremity (50% to 66%) and many of those injuries occur at the knee (30% to 45%), it is important to have robust metrics to measure risk of knee injury. Dynamic measures of knee stability are not commonly used in existing metrics but could provide important context to knee health and improve injury screening effectiveness. This study used the Local Dynamic Stability (LDS) of knee kinematics during a repetitive vertical jump to perform a post-hoc previous injury classification of participants. This study analyzed the kinematics from twenty-seven female collegiate division 1 (D1) soccer, D1 basketball, and club soccer athletes from Auburn University (height = 171 ± 8.9cm, weight = 66.3 ± 8.6kg, age = 19.8 ± 1.9yr), with 7 subjects having sustained previous knee injury requiring surgery and 20 subjects with no history of injury. This study showed that LDS correctly identified 84% of previously injured and uninjured subjects using a multivariate logistic regression during a fatigue jump task. Findings showed no statistical difference in kinematic position at maximum knee flexion during all jumps between previously injured and uninjured subjects. Additionally, kinematic positioning at maximum knee flexion was not indicative of LDS values, which would indicate that future studies should look specifically at LDS with respect to injury prevention as it cannot be effectively inferred from kinematics. These points suggest that the LDS preserves information about subtle changes in movement patterns that traditional screening methods do not, and this information could allow for more effective injury screening tests in the future.

Dietary fibers reduce obesity-related disorders: mechanisms of action.

PURPOSE OF REVIEW: Dietary fibers decrease risk of cardiovascular disease and obesity, but the most important mechanisms for fiber's protective properties are debated. The purpose of the review is to summarize the recent human studies that examine mechanisms how dietary fiber decreases risk of obesity-related disorders. RECENT FINDINGS: Dietary fiber has effects throughout the digestive tract that decrease risk of obesity-related diseases. Soluble, viscous fibers slow absorption of and decrease serum cholesterol. Intake of dietary fiber enhances satiety and reduces food intake at future meals. The importance of gut fermentation and changes in the gut microbiota and metabolites are linked to decrease risk for obesity-related disorders. Dietary fibers alter the gut microbiota and produce metabolites such as short-chain fatty acids that may explain fiber's role in obesity prevention and treatment. Dietary fiber encompasses many plant compounds, so conclusions that dietary fiber reduces or treats obesity-related disorders must be considered by the fiber was fed in the study. SUMMARY: Dietary fiber prevents and treats obesity-related disorders. Mechanisms for this protection include decreased absorption of macronutrients and enhanced satiety. Changes in the gut microbiota and short-chain fatty acids are emerging mechanisms to explain why high fiber diets protect against obesity and have a role in obesity treatment.

Discovery and Characterization of Peptide Inhibitors for Calcium and Integrin Binding Protein 1.

Calcium and integrin binding protein 1 (CIB1) is an EF-hand-containing, small intracellular protein that has recently been implicated in cancer cell survival and proliferation. In particular, CIB1 depletion significantly impairs tumor growth in triple-negative breast cancer (TNBC). Thus, CIB1 is a potentially attractive target for cancer chemotherapy that has yet to be validated by a chemical probe. To produce a probe molecule to the CIB1 helix 10 (H10) pocket and demonstrate that it is a viable target for molecular intervention, we employed random peptide phage display to screen and select CIB1-binding peptides. The top peptide sequence selected, UNC10245092, was produced synthetically, and binding to CIB1 was confirmed by isothermal titration calorimetry (ITC) and a time-resolved fluorescence resonance energy transfer (TR-FRET) assay. Both assays showed that the peptide bound to CIB1 with low nanomolar affinity. CIB1 was cocrystallized with UNC10245092, and the 2.1 Å resolution structure revealed that the peptide binds as an α-helix in the H10 pocket, displacing the CIB1 C-terminal H10 helix and causing conformational changes in H7 and H8. UNC10245092 was further derivatized with a C-terminal Tat-derived cell penetrating peptide (CPP) to demonstrate its effects on TNBC cells in culture, which are consistent with results of CIB1 depletion. These studies provide a first-in-class chemical tool for CIB1 inhibition in cell culture and validate the CIB1 H10 pocket for future probe and drug discovery efforts.

Application of a MYC degradation screen identifies sensitivity to CDK9 inhibitors in KRAS-mutant pancreatic cancer.

Stabilization of the MYC oncoprotein by KRAS signaling critically promotes the growth of pancreatic ductal adenocarcinoma (PDAC). Thus, understanding how MYC protein stability is regulated may lead to effective therapies. Here, we used a previously developed, flow cytometry-based assay that screened a library of >800 protein kinase inhibitors and identified compounds that promoted either the stability or degradation of MYC in a KRAS-mutant PDAC cell line. We validated compounds that stabilized or destabilized MYC and then focused on one compound, UNC10112785, that induced the substantial loss of MYC protein in both two-dimensional (2D) and 3D cell cultures. We determined that this compound is a potent CDK9 inhibitor with a previously uncharacterized scaffold, caused MYC loss through both transcriptional and posttranslational mechanisms, and suppresses PDAC anchorage-dependent and anchorage-independent growth. We discovered that CDK9 enhanced MYC protein stability through a previously unknown, KRAS-independent mechanism involving direct phosphorylation of MYC at Ser62 Our study thus not only identifies a potential therapeutic target for patients with KRAS-mutant PDAC but also presents the application of a screening strategy that can be more broadly adapted to identify regulators of protein stability.

An Osteoconductive Antibiotic Bone Eluting Putty with a Custom Polymer Matrix.

With the rising tide of antibiotic resistant bacteria, extending the longevity of the current antibiotic arsenal is becoming a necessity. Developing local, controlled release antibiotic strategies, particularly for difficult to penetrate tissues such as bone, may prove to be a better alternative. Previous efforts to develop an osteoconductive local antibiotic release device for bone were created as solid molded composites; however, intimate contact with host bone was found to be critical to support host bone regrowth; thus, an osteocondconductive antibiotic releasing bone void filling putty was developed. Furthermore, a controlled releasing polymer matrix was refined using pendant-functionalized diols to provide tailorable pharmacokinetics. In vitro pharmacokinetic and bioactivity profiles were compared for a putty formulation with an analogous composition as its molded counterpart as well as four new pendant-functionalized polymers. A best-fit analysis of polymer composition in either small cylindrical disks or larger spheres revealed that the new pendant-functionalized polymers appear to release vancomycin via both diffusion and erosion regardless of the geometry of the putty. In silico simulations, a valuable technique for diffusion mediated controlled release models, will be used to confirm and optimize this property.

The effect of measurement of the contralateral hip if the spine is not included in the bone mineral density analysis.

The aim of this study was to determine if measurement of the contralateral femora has an effect on osteoporosis diagnosis and treatment classification if the spine is not included in the bone mineral density (BMD) scan. The method used was the T-score discordance from the dual femur BMD scans of 537 women (mean age: 61.2 yr; standard deviation: 10.5; age range: 32-90 yr) who were evaluated to determine if inclusion of the contralateral hip in the BMD study made a difference in clinical diagnosis and treatment classification when the spine was not included in the BMD scan. Clinical diagnosis and treatment classification was based on the lowest T-score at each hip of three femur sites: the neck, the trochanter, and the total femur. The results of the diagnosis classification (i.e., normal, osteopenia, and osteoporosis) differed in the right versus the left femora in 28% of subjects at one or more sites, and in 14%, 15%, and 10% of subjects at the neck, trochanter, and total femur, respectively. Diagnosis discordance increased in subjects who were aged 65 yr and older. Treatment classification (T>or=-1.5; T<-1.5; T<-2.0) differed in the right versus the left femora in 33% of subjects at one or more sites, and in 18%, 14%, and 12% of subjects at the neck, trochanter, and total femur, respectively. Treatment discordance increased in subjects age 65 yr and older. Using the lowest T-score for clinical diagnosis classification, when the contralateral hip was considered, a clinical difference in diagnosis from normal-->osteopenia occurred in 3.9% of subjects, and from osteopenia-->osteoporosis in 1.3% of subjects. A clinical difference in treatment category from T>or=-1.5-->T<-1.5 occurred in 2.7% of subjects, and from T>or=-2-->T<-2 in 2.7% of subjects. In conclusion, inclusion of the bilateral hip in the BMD study made a clinical difference in diagnosis classification in 5.2% of subjects and in treatment classification in 5.4% of subjects. T-score differences between the contralateral hips increased with age. In the subgroup of subjects age 65 yr and older, a clinical difference in classification to a more severe diagnosis or treatment category occurred in 5.35% and 7.25% of subjects, respectively.