Regulation of GTPase function by autophosphorylation.

A unifying feature of the RAS superfamily is a conserved GTPase cycle by which these proteins transition between active and inactive states. We demonstrate that autophosphorylation of some GTPases is an intrinsic regulatory mechanism that reduces nucleotide hydrolysis and enhances nucleotide exchange, altering the on/off switch that forms the basis for their signaling functions. Using X-ray crystallography, nuclear magnetic resonance spectroscopy, binding assays, and molecular dynamics on autophosphorylated mutants of H-RAS and K-RAS, we show that phosphoryl transfer from GTP requires dynamic movement of the switch II region and that autophosphorylation promotes nucleotide exchange by opening the active site and extracting the stabilizing Mg2+. Finally, we demonstrate that autophosphorylated K-RAS exhibits altered effector interactions, including a reduced affinity for RAF proteins in mammalian cells. Thus, autophosphorylation leads to altered active site dynamics and effector interaction properties, creating a pool of GTPases that are functionally distinct from their non-phosphorylated counterparts.

Signalling change: signal transduction through the decades.

The past few years have marked significant anniversaries in signal transduction, including the identification of classic growth factors and morphogens, the notion of protein modification through phosphorylation and the characterization of protein interaction domains. Here, six researchers reflect on the context in which these discoveries were made, and how our concept of cell signalling has evolved during the past three decades.

Early life conditions reduce similarity between reproductive partners in HPA axis response to stress.

Social environments modulate endocrine function, yet it is unclear whether individuals can become like their social partners in how they physiologically respond to stressors. This social transmission of hypothalamic-pituitary-adrenal (HPA) axis reactivity could have long-term consequences for health and lifespan of individuals if their social partners react to stressors with an exaggerated HPA axis response. We tested whether glucocorticoid levels in response to stress of breeding partners changes after breeding depending on whether partners had similar or dissimilar postnatal conditions. We manipulated postnatal conditions by mimicking early life stress in zebra finch chicks (Taeniopygia guttata) via postnatal corticosterone exposure. When they reached adulthood, we created breeding pairs where the female and male had experienced either the same or different early life hormonal treatment (corticosterone or control). Before and after breeding, we obtained blood samples within 3 min and after 10 min or 30 min of restraint stress (baseline, cort10, cort30). We found that corticosterone levels of individuals in response to restraint were affected by their own and their partner's early life conditions, but did not change after breeding. However, across all pairs, partners became more similar in cort30 levels after breeding, although differences between partners in cort10 remained greater in pairs with a corticosterone-treated female. Thus, we show that HPA axis response to stressors in adulthood can be modulated by reproductive partners and that similarity between partners is reduced when females are postnatally exposed to elevated glucocorticoids.

Efficacy, Feasibility, and Safety of the X-Tack Endoscopic HeliX Tacking System: A Multicenter Experience.

BACKGROUND AND AIMS: The application of endoscopic suturing has revolutionized defect closures. Conventional over-the-scope suturing necessitates removal of the scope, placement of the device, and reinsertion. A single channel, single sequence, through-the-scope suturing device has been developed to improve this process. This study aims to describe the efficacy, feasibility, and safety of a through-the-scope suturing device for gastrointestinal defect closure. METHODS: This was a retrospective multicenter study involving 9 centers of consecutive adult patients who underwent suturing using the X-Tack Endoscopic HeliX Tacking System (Apollo Endosurgery). The primary outcomes were technical success and long-term clinical success. Secondary outcomes included adverse events, recurrence, and reintervention rates. RESULTS: In all, 56 patients (mean age 53.8, 33 women) were included. Suturing indications included fistula repair (n=22), leak repair (n=7), polypectomy defect closure (n=12), peroral endoscopic myotomy (POEM) site closure (n=7), perforation repair (n=6), and ulcers (n=2). Patients were followed at a mean duration of 74 days. Overall technical and long-term clinical success rates were 92.9% and 75%, respectively. Both technical and clinical success rates were 100% for polypectomies, POEM-site closures, and ulcers. Success rates were lower for the repair of fistulas (95.5% technical, 54.5% clinical), leaks (57.1%, 28.6%), and perforations (100%, 66.7%). No immediate adverse events were noted. CONCLUSION: This novel, through-the-scope endoscopic suturing system, is a safe and feasible method to repair defects that are ≤3 cm. The efficacy of this device may be better suited for superficial defects as opposed to full-thickness defects. Larger defects will need more sutures and probably a double closure technique to provide a reinforcement layer.

Biochemical and biophysical characterization of the RAS family small GTPase protein DiRAS3.

DiRAS3, also called ARHI, is a RAS (sub)family small GTPase protein that shares 50-60% sequence identity with H-, K-, and N-RAS, with substitutions in key conserved G-box motifs and a unique 34 amino acid extension at its N-terminus. Unlike the RAS proto-oncogenes, DiRAS3 exhibits tumor suppressor properties. DiRAS3 function has been studied through genetics and cell biology, but there has been a lack of understanding of the biochemical and biophysical properties of the protein, likely due to its instability and poor solubility. To overcome this solubility issue, we engineered a DiRAS3 variant (C75S/C80S), which significantly improved soluble protein expression in E. coli. Recombinant DiRAS3 was purified by Ni-NTA and size exclusion chromatography (SEC). Concentration dependence of the SEC chromatogram indicated that DiRAS3 exists in monomer-dimer equilibrium. We then produced truncations of the N-terminal (ΔN) and both (ΔNC) extensions to the GTPase domain. Unlike full-length DiRAS3, the SEC profiles showed that ΔNC is monomeric while ΔN was monomeric with aggregation, suggesting that the N and/or C-terminal tail(s) contribute to dimerization and aggregation. The 1H-15N HSQC NMR spectrum of ΔNC construct displayed well-dispersed peaks similar to spectra of other GTPase domains, which enabled us to demonstrate that DiRAS3 has a GTPase domain that can bind GDP and GTP. Taken together, we conclude that, despite the substitutions in the G-box motifs, DiRAS3 can switch between nucleotide-bound states and that the N- and C-terminal extensions interact transiently with the GTPase domain in intra- and inter-molecular fashions, mediating weak multimerization of this unique small GTPase.

Rac activation and inactivation control plasticity of tumor cell movement.

Tumor cells exhibit two different modes of individual cell movement. Mesenchymal-type movement is characterized by an elongated cellular morphology and requires extracellular proteolysis. In amoeboid movement, cells have a rounded morphology, are less dependent on proteases, and require high Rho-kinase signaling to drive elevated levels of actomyosin contractility. These two modes of cell movement are interconvertible. We show that mesenchymal-type movement in melanoma cells is driven by activation of the GTPase Rac through a complex containing NEDD9, a recently identified melanoma metastasis gene, and DOCK3, a Rac guanine nucleotide exchange factor. Rac signals through WAVE2 to direct mesenchymal movement and suppress amoeboid movement through decreasing actomyosin contractility. Conversely, in amoeboid movement, Rho-kinase signaling activates a Rac GAP, ARHGAP22, that suppresses mesenchymal movement by inactivating Rac. We demonstrate tight interplay between Rho and Rac in determining different modes of tumor cell movement, revealing how tumor cells switch between different modes of movement.

Synthesis of Pt3Zn1 and Pt1Zn1 intermetallic nanocatalysts for dehydrogenation of ethane.

Pt3Zn1 and Pt1Zn1 intermetallic nanoparticles supported on SiO2 were synthesized by combining atomic layer deposition (ALD) of ZnO, incipient wetness impregnation (IWI) of Pt, and appropriate hydrogen reduction. The formation of Pt1Zn1 and Pt3Zn1 intermetallic nanoparticles was observed by both X-ray diffraction (XRD) and synchrotron X-ray absorption spectroscopy (XAS). STEM images showed that the 2-3 nm Pt-based intermetallic nanoparticles were uniformly dispersed on a SiO2 support. The relationships between Pt-Zn intermetallic phases and synthesis conditions were established. In situ XAS measurements at Pt L3 and Zn K edges during hydrogen reduction provided a detailed image of surface species evolution. Owing to a combined electronic and geometric effect, Pt1Zn1 exhibited much higher reactivity and stability than Pt3Zn1 and Pt in both the direct dehydrogenation and oxidative dehydrogenation of ethane to ethylene reactions.

Multivalent assembly of KRAS with the RAS-binding and cysteine-rich domains of CRAF on the membrane.

Membrane anchoring of farnesylated KRAS is critical for activation of RAF kinases, yet our understanding of how these proteins interact on the membrane is limited to isolated domains. The RAS-binding domain (RBD) and cysteine-rich domain (CRD) of RAF engage KRAS and the plasma membrane, unleashing the kinase domain from autoinhibition. Due to experimental challenges, structural insight into this tripartite KRAS:RBD-CRD:membrane complex has relied on molecular dynamics simulations. Here, we report NMR studies of the KRAS:CRAF RBD-CRD complex. We found that the nucleotide-dependent KRAS-RBD interaction results in transient electrostatic interactions between KRAS and CRD, and we mapped the membrane interfaces of the CRD, RBD-CRD, and the KRAS:RBD-CRD complex. RBD-CRD exhibits dynamic interactions with the membrane through the canonical CRD lipid-binding site (CRD ß7-8), as well as an alternative interface comprising ß6 and the C terminus of CRD and ß2 of RBD. Upon complex formation with KRAS, two distinct states were observed by NMR: State A was stabilized by membrane association of CRD ß7-8 and KRAS α4-α5 while state B involved the C terminus of CRD, ß3-5 of RBD, and part of KRAS α5. Notably, α4-α5, which has been proposed to mediate KRAS dimerization, is accessible only in state B. A cancer-associated mutation on the state B membrane interface of CRAF RBD (E125K) stabilized state B and enhanced kinase activity and cellular MAPK signaling. These studies revealed a dynamic picture of the assembly of the KRAS-CRAF complex via multivalent and dynamic interactions between KRAS, CRAF RBD-CRD, and the membrane.

The Self-Association of the KRAS4b Protein is Altered by Lipid-Bilayer Composition and Electrostatics.

KRAS is a peripheral membrane protein that regulates multiple signaling pathways, and is mutated in ≈30 % of cancers. Transient self-association of KRAS is essential for activation of the downstream effector RAF and oncogenicity. The presence of anionic phosphatidylserine (PS) lipids in the membrane was shown to promote KRAS self-assembly, however, the structural mechanisms remain elusive. Here, we employed nanodisc bilayers of defined lipid compositions, and probed the impact of PS concentration on KRAS self-association. Paramagnetic NMR experiments demonstrated the existence of two transient dimer conformations involving alternate electrostatic contacts between R135 and either D153 or E168 on the "α4/5-α4/5" interface, and revealed that lipid composition and salt modulate their dynamic equilibrium. These dimer interfaces were validated by charge-reversal mutants. This plasticity demonstrates how the dynamic KRAS dimerization interface responds to the environment, and likely extends to the assembly of other signaling complexes on the membrane.

Neural programming of seasonal physiology in birds and mammals: A modular perspective.

Most animals in the temperate zone exhibit robust seasonal rhythms in neuroendocrine, physiological and behavioral processes. The integration of predictive and supplementary environmental cues (e.g., nutrients) involves a series of discrete, and interconnected brain regions that span hypothalamic, thalamic, mesencephalic, and limbic regions. Species-specific adaptive changes in these neuroendocrine structures and cellular plasticity have likely evolved to support seasonal life-history transitions. Despite significant advances in our understanding of ecological responses to predictive and supplementary environmental cues, there remains a paucity of literature on how these diverse cues impact the underlying neural and cellular substrates. To date, most scientific approach has focused on neuroendocrine responses to annual changes in daylength, referred to as photoperiod, due to the robust physiological changes to light manipulations in laboratory settings. In this review, we highlight the relatively few animal models that have been effectively used to investigate how predictive day lengths, and supplementary cues are integrated across hypothalamic nuclei, and discuss key findings of how seasonal rhythms in physiology are governed by adaptive neuroendocrine changes. We discuss how specific brain regions integrate environmental cues to form a complex multiunit or 'modular' system that has evolved to optimize the timing of seasonal physiology. Overall, the review aims to highlight the existence of a modular network of neural regions that independently contribute to timing seasonal physiology. This paper proposes that a multi-modular neuroendocrine system has evolved in which independent neural 'units' operate to support species-specific seasonal rhythms.

Initial multicenter experience using a novel endoscopic tack and suture system for challenging GI defect closure and stent fixation (with video).

BACKGROUND AND AIMS: Closure of endoscopic resection defects can be achieved with through-the-scope clips, over-the-scope clips, or endoscopic suturing. However, these devices are often limited by their inability to close large, irregular, and difficult-to-reach defects. Thus, we aimed to assess the feasibility and safety of a novel through-the-scope, suture-based closure system developed to overcome these limitations. METHODS: This was a retrospective multicenter study involving 8 centers in the United States. Primary outcomes were feasibility and safety of early use of the device. Secondary outcomes were assessment of need for additional closure devices, prolonged procedure time, and technical feasibility of performing the procedure with an alternative device(s). RESULTS: Ninety-three patients (48.4% women) with mean age 63.6 ± 13.1 years were included. Technical success was achieved in 83 patients (89.2%), and supplemental closure was required in 24.7% of patients (n = 23) with a mean defect size of 41.6 ± 19.4 mm. Closure with an alternative device was determined to be impossible in 24.7% of patients because of location, size, or shape of the defect. The use of the tack and suture device prolonged the procedure in 8.6% of cases but was considered acceptable. Adverse events occurred in 2 patients (2.2%) over a duration of follow-up of 34 days (interquartile range, 13-93.5) and were mild and moderate in severity. No serious adverse events or procedure-related deaths occurred. CONCLUSIONS: The novel endoscopic through-the-scope tack and suture system is safe, efficient, and permits closure of large and irregularly shaped defects that were not possible with established devices.

Puncture performance tests reveal distinct feeding modes in pinniped teeth.

Marine mammals have undergone a dramatic series of morphological transformations throughout their evolutionary history that facilitated their ecological transition to life in the water. Pinnipeds are a diverse clade of marine mammals that evolved from terrestrial carnivorans in the Oligocene (∼27 million years ago). However, pinnipeds have secondarily lost the dental innovations emblematic of mammalian and carnivoran feeding, such as a talonid basin or shearing carnassials. Modern pinnipeds do not masticate their prey, but can reduce prey size through chopping behavior. Typically, small prey are swallowed whole. Nevertheless, pinnipeds display a wide breadth of morphology of the post-canine teeth. We investigated the relationship between dental morphology and pinniped feeding by measuring the puncture performance of the cheek-teeth of seven extant pinniped genera. Puncture performance was measured as the maximum force and the maximum energy required to puncture a standardized prey item (Loligo sp.). We report significant differences in the puncture performance values across the seven genera, and identify three distinct categories based on cheek-teeth morphology and puncture performance: effective, ineffective and moderate puncturers. In addition, we measured the overall complexity of the tooth row using two different metrics, orientation patch count rotated (OPCR) and relief index (RFI). Neither metric of complexity predicted puncture performance. Finally, we discuss these results in the broader context of known pinniped feeding strategies and lay the groundwork for subsequent efforts to explore the ecological variation of specific dental morphologies.

Dethiosulfovibrio faecalis sp. nov., a novel proteolytic, non-sulphur-reducing bacterium isolated from a marine aquaculture solid waste bioreactor.

A new Dethiosulfovibrio strain, designated F2BT, was isolated from an anaerobic digester for treating solid waste from a marine recirculating aquaculture system. The motile, Gram-negative, non-spore-forming curved rods were 2-7 µm long and 1 µm in diameter. Growth occurred at temperatures ranging from 20 to 40 °C with a maximum rate of growth at 30 °C. The pH range for growth was pH 6.0-8.0, with a maximum rate of growth at pH 7.5. This isolate was halotolerant growing in NaCl concentrations ranging from 0 to 1.6 M with a maximum rate of growth at 0.4 M. Similarly to the five described Dethiosulfovibrio species, this obligate anaerobe isolate was fermentative, capable of utilizing peptides, amino acids and some organic acids for growth, but unlike described strains in the genus did not reduce thiosulphate or elemental sulphur to hydrogen sulphide during fermentation of organic substrates. The G+C content of 55 mol% is similar to the described Dethiosulfovibrio species. The average nucleotide identity analysis between whole genome sequences showed less than 93.15% sequence similarity between strain F2BT and the five other described Dethiosulfovibrio species. Differences in the physiological and phylogenetic characteristics between the new strain and other Dethiosulfovibrio specied indicate that F2BT represents a novel species of this genus and the epithet Dethiosulfovibrio faecalis sp. nov. is proposed. The type strain is F2BT (=DSM 112078T=KCTC25378T).

Nanoluciferase complementation-based bioreporter reveals the importance of N-linked glycosylation of SARS-CoV-2 S for viral entry.

The ongoing COVID-19 pandemic has highlighted the immediate need for the development of antiviral therapeutics targeting different stages of the SARS-CoV-2 life cycle. We developed a bioluminescence-based bioreporter to interrogate the interaction between the SARS-CoV-2 viral spike (S) protein and its host entry receptor, angiotensin-converting enzyme 2 (ACE2). The bioreporter assay is based on a nanoluciferase complementation reporter, composed of two subunits, large BiT and small BiT, fused to the S receptor-binding domain (RBD) of the SARS-CoV-2 S protein and ACE2 ectodomain, respectively. Using this bioreporter, we uncovered critical host and viral determinants of the interaction, including a role for glycosylation of asparagine residues within the RBD in mediating successful viral entry. We also demonstrate the importance of N-linked glycosylation to the RBD's antigenicity and immunogenicity. Our study demonstrates the versatility of our bioreporter in mapping key residues mediating viral entry as well as screening inhibitors of the ACE2-RBD interaction. Our findings point toward targeting RBD glycosylation for therapeutic and vaccine strategies against SARS-CoV-2.

Microbiome for the Electrosynthesis of Chemicals from Carbon Dioxide.

The price for renewable electricity is rapidly decreasing, and the availability of such energy is expected to increase in the coming years. This is a welcomed outcome considering that mitigation of climate disruption due to the use of fossil carbon is reaching a critical stage. However, the economy will remain dependent on carbon-based chemicals and the problem of electricity storage persists. Therefore, the development of electrosynthetic processes that convert electricity and CO2 into chemicals and energy dense fuels, perhaps even food, would be desirable. Electrochemistry has been applied to the manufacture of many valuable products and at a large industrial scale, but it is difficult to produce multicarbon chemicals from CO2 by chemistry alone. Being that the biological world possesses expertise at the construction of C-C bonds, it is being examined in conjunction with electrochemistry to discover new ways of synthesizing chemicals from electricity and CO2. One approach is microbial electrosynthesis. This Account describes the development of a microbial electrosynthesis system by the authors. A biocathode consisting of a carbon-based electrode and a microbial community produced short chain fatty acids, primarily acetate. The device works by electrolysis of water, but microbes facilitate electron transfer from the cathode while reducing CO2 by the Wood-Ljungdahl pathway possessed by an Acetobacterium sp. While this acetogenic microorganism dominates the microbiome growing on the cathode surface, 13 total species of microbes overall were ecologically selected on the cathode and genomes for each have been assembled. The combined species may contribute to the stability of the microbiome, a common feature of naturally selected microbial communities. The microbial electrosynthesis system was demonstrated to operate continuously at a cathode for more than 2 years and could also be used with intermittent power, thus demonstrating the stability of the microbiome living at the cathode. In addition to the description of reactor design and startup procedures, the possible mechanisms of electron transfer are described in this Account. While mysteries remain to be solved, much evidence indicates that the microbiome may facilitate electron transfer by supplying catalyst(s) external to the bacterial cells and onto the cathode surface. This may be in the form of a hydrogen-producing catalyst that enhances hydrogen generation by an inert carbon-based electrode. Through the enrichment of the electrosynthetic microbiome along with several modifications in reactor design and operation, the productivity and efficiency were improved. In addition to the intrinsic value of the current products, coupling the process with a secondary stage might be used to produce more valuable products from the acetic acid stream such as lipids, biocrude oil, or higher value food supplements. Alternatively, additional work on the mechanism of electron transfer, reactor design/operation, and modification of the microbes through synthetic biology, particularly to enhance carbon efficiency into higher value chemicals, are the needed next steps to advance microbial electrosynthesis so that it may be used to transform renewable electrons and CO2 directly into products and help solve the problem of climate disruption.

Catalytic Applications of Vanadium: A Mechanistic Perspective.

The chemistry of vanadium has seen remarkable activity in the past 50 years. In the present review, reactions catalyzed by homogeneous and supported vanadium complexes from 2008 to 2018 are summarized and discussed. Particular attention is given to mechanistic and kinetics studies of vanadium-catalyzed reactions including oxidations of alkanes, alkenes, arenes, alcohols, aldehydes, ketones, and sulfur species, as well as oxidative C-C and C-O bond cleavage, carbon-carbon bond formation, deoxydehydration, haloperoxidase, cyanation, hydrogenation, dehydrogenation, ring-opening metathesis polymerization, and oxo/imido heterometathesis. Additionally, insights into heterogeneous vanadium catalysis are provided when parallels can be drawn from the homogeneous literature.

Survey of the perceived treatment gap in status epilepticus care across Sub-Saharan countries from the perspective of healthcare providers.

BACKGROUND: Status epilepticus (SE) is a serious condition disproportionately affecting Sub-Saharan African (SSA) countries. Little is known about healthcare provider experiences. This study investigated the healthcare provider perspective of SE care. METHODS: A pilot questionnaire was developed for healthcare professionals in SSA countries. It was distributed online at a conference concerning epilepsy care and local coordinators distributed the questionnaire in their networks. It was available online between 16th Jan and 1st Feb 2021. The unvalidated questionnaire questioned practitioner demographics, experience, confidence in SE care, common etiologies encountered, anticipated prognosis in their setting, available treatments, and barriers to care. We assessed practitioner perceptions not their knowledge base around SE care. Thematic analysis was used for open-ended questions. RESULTS: Fifty nine responses were received from 11 countries. Respondents (44% nurses, 46% doctors) reported poor level of adequate SE training (mean self-reported confidence in training 2.9/10 (0/10 very inadequate and 10/10 very adequate training). Delays in arriving at hospital were common with 15 (32%) taking three or more hours and 28 (62%) proposing transport issues and distance were the main reasons for delay. Urban location was significantly associated with clinician confidence. Less than 20% used prehospital benzodiazepine treatment. 46 (78%) stated benzodiazepines were first-line hospital drug management, and 52 (88%) indicated alternative second-line hospital treatments were available. CONCLUSION: A substantial perceived treatment gap in the management of SE in SSA is identified by staff working in SSA. Key issues are around staff training, patient education, medication access, and compliance.

A comparative analysis: international variation in PET-CT service provision in oncology-an International Cancer Benchmarking Partnership study.

OBJECTIVE: To explore differences in position emission tomography-computed tomography (PET-CT) service provision internationally to further understand the impact variation may have upon cancer services. To identify areas of further exploration for researchers and policymakers to optimize PET-CT services and improve the quality of cancer services. DESIGN: Comparative analysis using data based on pre-defined PET-CT service metrics from PET-CT stakeholders across seven countries. This was further informed via document analysis of clinical indication guidance and expert consensus through round-table discussions of relevant PET-CT stakeholders. Descriptive comparative analyses were produced on use, capacity and indication guidance for PET-CT services between jurisdictions. SETTING: PET-CT services across 21 jurisdictions in seven countries (Australia, Denmark, Canada, Ireland, New Zealand, Norway and the UK). PARTICIPANTS: None. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): None. RESULTS: PET-CT service provision has grown over the period 2006-2017, but scale of increase in capacity and demand is variable. Clinical indication guidance varied across countries, particularly for small-cell lung cancer staging and the specific acknowledgement of gastric cancer within oesophagogastric cancers. There is limited and inconsistent data capture, coding, accessibility and availability of PET-CT activity across countries studied. CONCLUSIONS: Variation in PET-CT scanner quantity, acquisition over time and guidance upon use exists internationally. There is a lack of routinely captured and accessible PET-CT data across the International Cancer Benchmarking Partnership countries due to inconsistent data definitions, data linkage issues, uncertain coverage of data and lack of specific coding. This is a barrier in improving the quality of PET-CT services globally. There needs to be greater, richer data capture of diagnostic and staging tools to facilitate learning of best practice and optimize cancer services.

NMR in integrated biophysical drug discovery for RAS: past, present, and future.

Mutations in RAS oncogenes occur in ~ 30% of human cancers, with KRAS being the most frequently altered isoform. RAS proteins comprise a conserved GTPase domain and a C-terminal lipid-modified tail that is unique to each isoform. The GTPase domain is a 'switch' that regulates multiple signaling cascades that drive cell growth and proliferation when activated by binding GTP, and the signal is terminated by GTP hydrolysis. Oncogenic RAS mutations disrupt the GTPase cycle, leading to accumulation of the activated GTP-bound state and promoting proliferation. RAS is a key target in oncology, however it lacks classic druggable pockets and has been extremely challenging to target. RAS signaling has thus been targeted indirectly, by harnessing key downstream effectors as well as upstream regulators, or disrupting the proper membrane localization required for signaling, by inhibiting either lipid modification or 'carrier' proteins. As a small (20 kDa) protein with multiple conformers in dynamic equilibrium, RAS is an excellent candidate for NMR-driven characterization and screening for direct inhibitors. Several molecules have been discovered that bind RAS and stabilize shallow pockets through conformational selection, and recent compounds have achieved substantial improvements in affinity. NMR-derived insight into targeting the RAS-membrane interface has revealed a new strategy to enhance the potency of small molecules, while another approach has been development of peptidyl inhibitors that bind through large interfaces rather than deep pockets. Remarkable progress has been made with mutation-specific covalent inhibitors that target the thiol of a G12C mutant, and these are now in clinical trials. Here we review the history of RAS inhibitor development and highlight the utility of NMR and integrated biophysical approaches in RAS drug discovery.

Activation of a Classic Hunger Circuit Slows Luteinizing Hormone Pulsatility.

INTRODUCTION: The central regulation of fertility is carefully coordinated with energy homeostasis, and infertility is frequently the outcome of energy imbalance. Neurons in the hypothalamus expressing neuropeptide Y and agouti-related peptide (NPY/AgRP neurons) are strongly implicated in linking metabolic cues with fertility regulation. OBJECTIVE: We aimed here to determine the impact of selectively activating NPY/AgRP neurons, critical regulators of metabolism, on the activity of luteinizing hormone (LH) pulse generation. METHODS: We employed a suite of in vivo optogenetic and chemogenetic approaches with serial measurements of LH to determine the impact of selectively activating NPY/AgRP neurons on dynamic LH secretion. In addition, electrophysiological studies in ex vivo brain slices were employed to ascertain the functional impact of activating NPY/AgRP neurons on gonadotropin-releasing hormone (GnRH) neurons. RESULTS: Selective activation of NPY/AgRP neurons significantly decreased post-castration LH secretion. This was observed in males and females, as well as in prenatally androgenized females that recapitulate the persistently elevated LH pulse frequency characteristic of polycystic ovary syndrome (PCOS). Reduced LH pulse frequency was also observed when optogenetic stimulation was restricted to NPY/AgRP fiber projections surrounding GnRH neuron cell bodies in the rostral preoptic area. However, electrophysiological studies in ex vivo brain slices indicated these effects were likely to be indirect. CONCLUSIONS: These data demonstrate the ability of NPY/AgRP neuronal signaling to modulate and, specifically, reduce GnRH/LH pulse generation. The findings suggest a mechanism by which increased activity of this hunger circuit, in response to negative energy balance, mediates impaired fertility in otherwise reproductively fit states, and highlight a potential mechanism to slow LH pulsatility in female infertility disorders, such as PCOS, that are associated with hyperactive LH secretion.