Targeting Ras-, Rho-, and Rab-family GTPases via a conserved cryptic pocket.

The family of Ras-like GTPases consists of over 150 different members, regulated by an even larger number of guanine exchange factors (GEFs) and GTPase-activating proteins (GAPs) that comprise cellular switch networks that govern cell motility, growth, polarity, protein trafficking, and gene expression. Efforts to develop selective small molecule probes and drugs for these proteins have been hampered by the high affinity of guanosine triphosphate (GTP) and lack of allosteric regulatory sites. This paradigm was recently challenged by the discovery of a cryptic allosteric pocket in the switch II region of K-Ras. Here, we ask whether similar pockets are present in GTPases beyond K-Ras. We systematically surveyed members of the Ras, Rho, and Rab family of GTPases and found that many GTPases exhibit targetable switch II pockets. Notable differences in the composition and conservation of key residues offer potential for the development of optimized inhibitors for many members of this previously undruggable family.

RASopathy mutations provide functional insight into the BRAF cysteine-rich domain and reveal the importance of autoinhibition in BRAF regulation.

BRAF is frequently mutated in human cancer and the RASopathy syndromes, with RASopathy mutations often observed in the cysteine-rich domain (CRD). Although the CRD participates in phosphatidylserine (PS) binding, the RAS-RAF interaction, and RAF autoinhibition, the impact of these activities on RAF function in normal and disease states is not well characterized. Here, we analyze a panel of CRD mutations and show that they increase BRAF activity by relieving autoinhibition and/or enhancing PS binding, with relief of autoinhibition being the major factor determining mutation severity. Further, we show that CRD-mediated autoinhibition prevents the constitutive plasma membrane localization of BRAF that causes increased RAS-dependent and RAS-independent function. Comparison of the BRAF- and CRAF-CRDs also indicates that the BRAF-CRD is a stronger mediator of autoinhibition and PS binding, and given the increased catalytic activity of BRAF, our studies reveal a more critical role for CRD-mediated autoinhibition in BRAF regulation.

Revealing the mechanism of action of a first-in-class covalent inhibitor of KRASG12C (ON) and other functional properties of oncogenic KRAS by 31P NMR.

Individual oncogenic KRAS mutants confer distinct differences in biochemical properties and signaling for reasons that are not well understood. KRAS activity is closely coupled to protein dynamics and is regulated through two interconverting conformations: state 1 (inactive, effector binding deficient) and state 2 (active, effector binding enabled). Here, we use 31P NMR to delineate the differences in state 1 and state 2 populations present in WT and common KRAS oncogenic mutants (G12C, G12D, G12V, G13D, and Q61L) bound to its natural substrate GTP or a commonly used nonhydrolyzable analog GppNHp (guanosine-5'-[(ß,γ)-imido] triphosphate). Our results show that GppNHp-bound proteins exhibit significant state 1 population, whereas GTP-bound KRAS is primarily (90% or more) in state 2 conformation. This observation suggests that the predominance of state 1 shown here and in other studies is related to GppNHp and is most likely nonexistent in cells. We characterize the impact of this differential conformational equilibrium of oncogenic KRAS on RAF1 kinase effector RAS-binding domain and intrinsic hydrolysis. Through a KRAS G12C drug discovery, we have identified a novel small-molecule inhibitor, BBO-8956, which is effective against both GDP- and GTP-bound KRAS G12C. We show that binding of this inhibitor significantly perturbs state 1-state 2 equilibrium and induces an inactive state 1 conformation in GTP-bound KRAS G12C. In the presence of BBO-8956, RAF1-RAS-binding domain is unable to induce a signaling competent state 2 conformation within the ternary complex, demonstrating the mechanism of action for this novel and active-conformation inhibitor.

Machine learning-driven multiscale modeling reveals lipid-dependent dynamics of RAS signaling proteins.

RAS is a signaling protein associated with the cell membrane that is mutated in up to 30% of human cancers. RAS signaling has been proposed to be regulated by dynamic heterogeneity of the cell membrane. Investigating such a mechanism requires near-atomistic detail at macroscopic temporal and spatial scales, which is not possible with conventional computational or experimental techniques. We demonstrate here a multiscale simulation infrastructure that uses machine learning to create a scale-bridging ensemble of over 100,000 simulations of active wild-type KRAS on a complex, asymmetric membrane. Initialized and validated with experimental data (including a new structure of active wild-type KRAS), these simulations represent a substantial advance in the ability to characterize RAS-membrane biology. We report distinctive patterns of local lipid composition that correlate with interfacially promiscuous RAS multimerization. These lipid fingerprints are coupled to RAS dynamics, predicted to influence effector binding, and therefore may be a mechanism for regulating cell signaling cascades.

A Top-Down Proteomic Assay to Evaluate KRAS4B-Compound Engagement.

Development of new targeted inhibitors for oncogenic KRAS mutants may benefit from insight into how a given mutation influences the accessibility of protein residues and how compounds interact with mutant or wild-type KRAS proteins. Targeted proteomic analysis, a key validation step in the KRAS inhibitor development process, typically involves both intact mass- and peptide-based methods to confirm compound localization or quantify binding. However, these methods may not always provide a clear picture of the compound binding affinity for KRAS, how specific the compound is to the target KRAS residue, and how experimental conditions may impact these factors. To address this, we have developed a novel top-down proteomic assay to evaluate in vitro KRAS4B-compound engagement while assessing relative quantitation in parallel. We present two applications to demonstrate the capabilities of our assay: maleimide-biotin labeling of a KRAS4BG12D cysteine mutant panel and treatment of three KRAS4B proteins (WT, G12C, and G13C) with small molecule compounds. Our results show the time- or concentration-dependence of KRAS4B-compound engagement in context of the intact protein molecule while directly mapping the compound binding site.

Inhibition of Ras/Raf/MEK/ERK Pathway Signaling by a Stress-Induced Phospho-Regulatory Circuit.

Ras pathway signaling plays a critical role in cell growth control and is often upregulated in human cancer. The Raf kinases selectively interact with GTP-bound Ras and are important effectors of Ras signaling, functioning as the initiating kinases in the ERK cascade. Here, we identify a route for the phospho-inhibition of Ras/Raf/MEK/ERK pathway signaling that is mediated by the stress-activated JNK cascade. We find that key Ras pathway components, the RasGEF Sos1 and the Rafs, are phosphorylated on multiple S/TP sites in response to JNK activation and that the hyperphosphorylation of these sites renders the Rafs and Sos1 unresponsive to upstream signals. This phospho-regulatory circuit is engaged by cancer therapeutics, such as rigosertib and paclitaxel/Taxol, that activate JNK through mitotic and oxidative stress as well as by physiological regulators of the JNK cascade and may function as a signaling checkpoint to suppress the Ras pathway during conditions of cellular stress.

A Monte Carlo-Based Iterative Extended Kalman Filter for Bearings-Only Tracking of Sea Targets.

In this paper, a Monte Carlo (MC)-based extended Kalman filter is proposed for a two-dimensional bearings-only tracking problem (BOT). This problem addresses the processing of noise-corrupted bearing measurements from a sea acoustic source and estimates state vectors including position and velocity. Due to the nonlinearity and complex observability properties in the BOT problem, a wide area of research has been focused on improving its state estimation accuracy. The objective of this research is to present an accurate approach to estimate the relative position and velocity of the source with respect to the maneuvering observer. This approach is implemented using the iterated extended Kalman filter (IEKF) in an MC-based iterative structure (MC-IEKF). Re-linearizing dynamic and measurement equations using the IEKF along with the MC campaign applied to the initial conditions result in significantly improved accuracy in the estimation process. Furthermore, an observability analysis is conducted to show the effectiveness of the designed maneuver of the observer. A comparison with the widely used UKF algorithm is carried out to demonstrate the performance of the proposed method.

Object Detection and Tracking with YOLO and the Sliding Innovation Filter.

Object detection and tracking are pivotal tasks in machine learning, particularly within the domain of computer vision technologies. Despite significant advancements in object detection frameworks, challenges persist in real-world tracking scenarios, including object interactions, occlusions, and background interference. Many algorithms have been proposed to carry out such tasks; however, most struggle to perform well in the face of disturbances and uncertain environments. This research proposes a novel approach by integrating the You Only Look Once (YOLO) architecture for object detection with a robust filter for target tracking, addressing issues of disturbances and uncertainties. The YOLO architecture, known for its real-time object detection capabilities, is employed for initial object detection and centroid location. In combination with the detection framework, the sliding innovation filter, a novel robust filter, is implemented and postulated to improve tracking reliability in the face of disturbances. Specifically, the sliding innovation filter is implemented to enhance tracking performance by estimating the optimal centroid location in each frame and updating the object's trajectory. Target tracking traditionally relies on estimation theory techniques like the Kalman filter, and the sliding innovation filter is introduced as a robust alternative particularly suitable for scenarios where a priori information about system dynamics and noise is limited. Experimental simulations in a surveillance scenario demonstrate that the sliding innovation filter-based tracking approach outperforms existing Kalman-based methods, especially in the presence of disturbances. In all, this research contributes a practical and effective approach to object detection and tracking, addressing challenges in real-world, dynamic environments. The comparative analysis with traditional filters provides practical insights, laying the groundwork for future work aimed at advancing multi-object detection and tracking capabilities in diverse applications.

Revealing KRas4b topology on the membrane surface.

KRas4b is a membrane-bound regulatory protein belonging to the family of small GTPases that function as a molecular switch, facilitating signal transduction from activated membrane receptors to intracellular pathways controlling cell growth and proliferation. Oncogenic mutations locking KRas4b in the active GTP state are responsible for nearly 85% of all Ras-driven cancers. Understanding the membrane-bound state of KRas4b is crucial for designing new therapeutic approaches targeting oncogenic KRas-driven signaling pathways. Extensive research demonstrates the significant involvement of the membrane bilayer in Ras-effector interactions, with anionic lipids playing a critical role in determining protein conformations The preferred topology of KRas4b for interacting with signaling partners has been a long-time question. Computational studies suggest a membrane-proximal conformation, while other biophysical methods like neutron reflectivity propose a membrane-distal conformation. To address these gaps, we employed FRET measurements to investigate the conformation of KRas4b. Using fully post-translationally modified KRas4b, we designed a Nanodisc based FRET assay to study KRas4b-membrane interactions. We suggest an extended conformation of KRas4b relative to the membrane surface. Measurement of FRET donor - acceptor distances reveal that a negatively charged membrane surface weakly favors closer association with the membrane surface. Our findings provide insights into the role of anionic lipids in determining the dynamic conformations of KRas4b and shed light on the predominant conformation of its topology on lipid headgroups.

Uncovering a membrane-distal conformation of KRAS available to recruit RAF to the plasma membrane.

The small GTPase KRAS is localized at the plasma membrane where it functions as a molecular switch, coupling extracellular growth factor stimulation to intracellular signaling networks. In this process, KRAS recruits effectors, such as RAF kinase, to the plasma membrane where they are activated by a series of complex molecular steps. Defining the membrane-bound state of KRAS is fundamental to understanding the activation of RAF kinase and in evaluating novel therapeutic opportunities for the inhibition of oncogenic KRAS-mediated signaling. We combined multiple biophysical measurements and computational methodologies to generate a consensus model for authentically processed, membrane-anchored KRAS. In contrast to the two membrane-proximal conformations previously reported, we identify a third significantly populated state using a combination of neutron reflectivity, fast photochemical oxidation of proteins (FPOP), and NMR. In this highly populated state, which we refer to as "membrane-distal" and estimate to comprise ∼90% of the ensemble, the G-domain does not directly contact the membrane but is tethered via its C-terminal hypervariable region and carboxymethylated farnesyl moiety, as shown by FPOP. Subsequent interaction of the RAF1 RAS binding domain with KRAS does not significantly change G-domain configurations on the membrane but affects their relative populations. Overall, our results are consistent with a directional fly-casting mechanism for KRAS, in which the membrane-distal state of the G-domain can effectively recruit RAF kinase from the cytoplasm for activation at the membrane.

Interacting Multiple Model Estimators for Fault Detection in a Magnetorheological Damper.

This paper proposes a novel estimator for the purpose of fault detection and diagnosis. The interacting multiple model (IMM) strategy is effective for estimating the behaviour of systems with multiple operating modes. Each mode corresponds to a distinct mathematical model and is subject to a filtering process. This paper applies various model-based filters in combination with the IMM strategy. One such estimator employs the recently introduced extended sliding innovation filter (ESIF) known as the IMM-ESIF. The ESIF is an extension of the sliding innovation filter for nonlinear systems based on the sliding mode concept. In the presence of modeling uncertainties, the ESIF has been proven to be more robust compared to methods such as the extended Kalman filter (EKF). The novel IMM-ESIF strategy is also compared with the IMM strategy, which incorporates the unscented Kalman filter (UKF), referred to herein as IMM-UKF. While EKF uses Taylor series approximation to linearize the system model, the UKF uses sigma point to calculate the system's mean and covariance. The methods were applied to an experimental magnetorheological (MR) damper setup, which was designed for testing control and estimation theory. Magnetorheological dampers exhibit a diverse array of applications in the automotive and aerospace sectors, with particular relevance to attenuating vibrations through adaptive suspension systems. Applied to a magnetorheological (MR) damper with distinct operating modes determined by the damper's current, the results showcase the effectiveness of IMM-ESIF. In mixed operational conditions, IMM-ESIF demonstrates a notable 80% to 90% reduction in estimation error compared to its counterparts. Furthermore, it exhibits a 4% to 5% enhancement in correctly classifying operational modes, establishing IMM-ESIF as a promising and efficient alternative for adaptive estimation in electromechanical systems. The improved accuracy in estimating the system's behaviour, even amidst uncertainties and mixed operational scenarios, signifies the potential of IMM-ESIF to significantly enhance the overall robustness and efficiency of estimations.

From tablet to table: How augmented reality influences food desirability.

Augmented reality (AR) technology has generated enormous industry investment and buzz, with the food and beverage sector quickly embracing this technology in an effort to enhance the customer experience. However, academic research has only just begun to empirically explore how and why this technology might influence consumer judgements and behaviors in such contexts. Across two field studies involving consequential behavior and two controlled laboratory studies, we find that AR's unique ability to visually superimpose objects onto a real-time environment increases consumers' ability to mentally simulate consuming a pictured food, which in turn increases their desire and purchase likelihood of the food item. Further, we find the increased mental simulation produced by AR is itself preceded and driven by an increased sense of personal relevance of the food items. Supplementary Information: The online version contains supplementary material available at 10.1007/s11747-022-00919-x.

Exploring CRD mobility during RAS/RAF engagement at the membrane.

During the activation of mitogen-activated protein kinase (MAPK) signaling, the RAS-binding domain (RBD) and cysteine-rich domain (CRD) of RAF bind to active RAS at the plasma membrane. The orientation of RAS at the membrane may be critical for formation of the RAS-RBDCRD complex and subsequent signaling. To explore how RAS membrane orientation relates to the protein dynamics within the RAS-RBDCRD complex, we perform multiscale coarse-grained and all-atom molecular dynamics (MD) simulations of KRAS4b bound to the RBD and CRD domains of RAF-1, both in solution and anchored to a model plasma membrane. Solution MD simulations describe dynamic KRAS4b-CRD conformations, suggesting that the CRD has sufficient flexibility in this environment to substantially change its binding interface with KRAS4b. In contrast, when the ternary complex is anchored to the membrane, the mobility of the CRD relative to KRAS4b is restricted, resulting in fewer distinct KRAS4b-CRD conformations. These simulations implicate membrane orientations of the ternary complex that are consistent with NMR measurements. While a crystal structure-like conformation is observed in both solution and membrane simulations, a particular intermolecular rearrangement of the ternary complex is observed only when it is anchored to the membrane. This configuration emerges when the CRD hydrophobic loops are inserted into the membrane and helices α3-5 of KRAS4b are solvent exposed. This membrane-specific configuration is stabilized by KRAS4b-CRD contacts that are not observed in the crystal structure. These results suggest modulatory interplay between the CRD and plasma membrane that correlate with RAS/RAF complex structure and dynamics, and potentially influence subsequent steps in the activation of MAPK signaling.

Tolypocladamides A-G: Cytotoxic Peptaibols from Tolypocladium inflatum.

Seven new peptaibols named tolypocladamides A-G have been isolated from an extract of the fungus Tolypocladium inflatum, which inhibits the interaction between Raf and oncogenic Ras in a cell-based high-throughput screening assay. Each peptaibol contains 11 amino acid residues, an octanoyl or decanoyl fatty acid chain at the N-terminus, and a leucinol moiety at the C-terminus. The peptaibol sequences were elucidated on the basis of 2D NMR and mass spectral fragmentation analyses. Amino acid configurations were determined by advanced Marfey's analyses. Tolypocladamides A-G caused significant inhibition of Ras/Raf interactions with IC50 values ranging from 0.5 to 5.0 µM in a nanobioluminescence resonance energy transfer (NanoBRET) assay; however, no interactions were observed in a surface plasmon resonance assay for binding of the compounds to wild type or G12D mutant Ras constructs or to the Ras binding domain of Raf. NCI 60 cell line testing was also conducted, and little panel selectivity was observed.

Outcomes of PEG placement by acute care surgeons compared to those placed by gastroenterology.

BACKGROUND: Percutaneous endoscopic gastrostomy (PEG) tubes are placed by gastroenterologists (GI) and surgeons throughout the country. At Rhode Island Hospital, before July of 2017, all PEGs were placed by GI. In July of 2017, in response to a growing need for PEGs, acute care surgeons (ACS) also began performing PEGs at the bedside in ICUs. The purpose of this study was to review and compare outcomes of PEG tubes placed by ACS and GI. METHODS: Retrospective chart review of patients who received a PEG placed by ACS or GI at the bedside in any ICU from December 2016 to September 2019. Charts were reviewed for the following outcomes: Success rates of placing PEG, duration of procedure, major complications, and death. Secondary outcomes included discharge disposition, and rates of comfort measures only after PEG. RESULTS: In 2017, 75% of PEGs were placed by GI and 25% surgery. In 2018, 47% were placed by GI and 53% by surgery. In 2019, 33% were placed by GI and 67% by surgery. There was no significant difference in success rates between surgery (146/156 93.6%) and GI (173/185 93.5%) (p 0.97). On average, GI performed the procedure faster than surgery [Median 10 (7-16) min vs 16 (13-21) mins, respectively, p < 0.001]. There were no significant differences between groups in any of the PEG outcomes or complications investigated. CONCLUSION: Bedside PEG tube placement appears to be a safe procedure in the ICU population. GI and Surgery had nearly identical success rates in placing PEGs. GI performed the procedure faster than surgery. There were no significant differences in the reviewed patient outcomes or complications between PEGs placed by ACS or GI. Of note, when a complication occurred, ACS PEG patients typically were managed in the OR while GI tended to re-PEG patients highlighting a potential difference in management that should be further investigated.

Selinexor is a novel inhibitor of DNA damage response in Merkel cell carcinoma.

Merkel cell carcinoma (MCC) is a highly lethal cutaneous carcinoma, which in ~80% of cases in the USA is aetiologically linked to Merkel cell polyomavirus (MCPyV). Immune checkpoint inhibitors (ICIs) can successfully treat ~50% of patients with metastatic MCC, but some MCCs are refractory to ICIs, possibly due to altered DNA damage response (DDR). Selinexor, an anticancer therapy that is currently approved in combination with chemotherapy for multiple myeloma, downregulates the small T and large T tumour antigens in MCC through selective inhibition of nuclear exportin 1 (XPO1). We examined the effect of varying doses of selinexor on DDR protein expression in MCPyV-positive and MCPyV-negative MCC cells. Selinexor was found to inhibit DDR protein expression in both MCPyV-positive and MCPyV-negative cells. Addition of selinexor alone or combined with ICI may be a promising treatment for MCC, but further in vivo research and clinical trials are required to validate these findings.

Second harmonic generation detection of Ras conformational changes and discovery of a small molecule binder.

Second harmonic generation (SHG) is an emergent biophysical method that sensitively measures real-time conformational change of biomolecules in the presence of biological ligands and small molecules. This study describes the successful implementation of SHG as a primary screening platform to identify fragment ligands to oncogenic Kirsten rat sarcoma (KRas). KRas is the most frequently mutated driver of pancreatic, colon, and lung cancers; however, there are few well-characterized small molecule ligands due to a lack of deep binding pockets. Using SHG, we identified a fragment binder to KRasG12D and used 1H 15N transverse relaxation optimized spectroscopy (TROSY) heteronuclear single-quantum coherence (HSQC) NMR to characterize its binding site as a pocket adjacent to the switch 2 region. The unique sensitivity of SHG furthered our study by revealing distinct conformations induced by our hit fragment compared with 4,6-dichloro-2-methyl-3-aminoethyl-indole (DCAI), a Ras ligand previously described to bind the same pocket. This study highlights SHG as a high-throughput screening platform that reveals structural insights in addition to ligand binding.

Formulation of the Alpha Sliding Innovation Filter: A Robust Linear Estimation Strategy.

In this paper, a new filter referred to as the alpha sliding innovation filter (ASIF) is presented. The sliding innovation filter (SIF) is a newly developed estimation strategy that uses innovation or measurement error as a switching hyperplane. It is a sub-optimal filter that provides a robust and stable estimate. In this paper, the SIF is reformulated by including a forgetting factor, which significantly improves estimation performance. The proposed ASIF is applied to several systems including a first-order thermometer, a second-order spring-mass-damper, and a third-order electrohydrostatic actuator (EHA) that was built for experimentation. The proposed ASIF provides an improvement in estimation accuracy while maintaining robustness to modeling uncertainties and disturbances.

How consumer digital signals are reshaping the customer journey.

Marketers are adopting increasingly sophisticated ways to engage with customers throughout their journeys. We extend prior perspectives on the customer journey by introducing the role of digital signals that consumers emit throughout their activities. We argue that the ability to detect and act on consumer digital signals is a source of competitive advantage for firms. Technology enables firms to collect, interpret, and act on these signals to better manage the customer journey. While some consumers' desire for privacy can restrict the opportunities technology provides marketers, other consumers' desire for personalization can encourage the use of technology to inform marketing efforts. We posit that this difference in consumers' willingness to emit observable signals may hinge on the strength of their relationship with the firm. We next discuss factors that may shift consumer preferences and consequently affect the technology-enabled opportunities available to firms. We conclude with a research agenda that focuses on consumers, firms, and regulators.

Membrane-bound KRAS approximates an entropic ensemble of configurations.

KRAS4B is a membrane-anchored signaling protein and a primary target in cancer research. Predictions from molecular dynamics simulations that have previously shaped our mechanistic understanding of KRAS signaling disagree with recent experimental results from neutron reflectometry, NMR, and thermodynamic binding studies. To gain insight into these discrepancies, we compare this body of biophysical data to back-calculated experimental results from a series of molecular simulations that implement different subsets of molecular interactions. Our results show that KRAS4B approximates an entropic ensemble of configurations at model membranes containing 30% phosphatidylserine lipids, which is not significantly shaped by interactions between the globular G-domain of KRAS4B and the lipid membrane. These findings revise our understanding of KRAS signaling and promote a model in which the protein samples the accessible conformational space in a near-uniform manner while being available to bind to effector proteins.