RAS oncogenes are the most commonly mutated oncogenes in human cancer, and RAS-mutant cancers represent a major burden of human disease. Though these oncogenes were discovered decades ago, recent years have seen major advances in understanding of their structure and function, including the therapeutic and prognostic significance of diverse isoforms. Targeting of these mutations has proven difficult, despite some successes with inhibition of RAS effector signalling. More recently, direct RAS inhibition has been achieved in a trial setting. While this has yet to be translated to everyday clinical practice, this development carries much promise. This review summarizes the diverse approaches that have been taken to RAS inhibition and then focuses on the most recent developments in direct inhibition of KRAS(G12C). The three RAS genes, Kirsten rat sarcoma viral oncogene homolog (K-RAS), neuroblastoma RAS viral (v-ras) oncogene homolog (N-RAS) and Harvey rat sarcoma viral oncogene homolog (H-RAS), are the most commonly mutated oncogenes in human cancer with approximately one-third of all cancers driven by these oncoproteins, including 40%-50% of colorectal cancer and over 90% of pancreatic cancers,4. The discovery of oncogenes and the elucidation of intracellular signalling pathways heralded an era of targeted therapy that has greatly improved the outlook for many cancers,6. New agents targeting receptor kinases and their downstream mediators demonstrated the ability to stabilise and shrink tumours, with side effects that were frequently milder than those associated with standard cytotoxic chemotherapy. Throughout this enormous paradigm shift in cancer therapeutics, however, RAS stood apart, dominant and seemingly undruggable. The development of direct RAS inhibitors proved very challenging. RAS has a high affinity towards GDP and GTP and a lack of deep hydrophobic pockets which would allow binding of small molecules. Subtle differences in structure and variable activation of RAS proteins added greatly to the complexity and attention largely focused on downstream inhibition of the transduced signalling pathways. In recent years, however, there have been some very promising developments in direct RAS targeting which would suggest that this has real potential as a therapeutic avenue. Here we aim to review current efforts at RAS inhibition in the context of both RAS family biology and the historical efforts which attempted, largely without success, to perturb its role as a major oncogenic driver.Since the discovery of RAS oncogenes as the transforming genes of oncogenic retroviruses. Three RAS genes, H-RAS, K-RAS and N-RAS, encode four protein isoforms: H-RAS, K-RAS4A, K-RAS4B and N-RAS. K-RAS4B is the predominant isoform and is referred to simply as K-RAS in this article. RAS proteins are small GTPases, of about 21kD molecular weight, and are monomeric proteins that have a central role in cell differentiation, adhesion, migration, proliferation and survival. RAS proteins convey signals from growth factors and extracellular components, and are upstream of signalling pathways including the ERK pathway and the PI3K/mTOR survival pathway. As illustrated in The RAS superfamily of genes has about 36 members, which encode for 39 proteins. The consequence of this aberrantly activated RAS is prolonged oncogenic signalling rather than short, controlled bursts of activation. There is subsequent activation of downstream signalling molecules such as PI3K, RAF and Rin1. By contrast, mutations in other RAS codons, or any nonsense mutations, are likely to inhibit rather than enhance the activity of RAS, and do not provide a survival advantage. As well as uncontrolled proliferation, the mutagenic RAS oncogene has been implicated in tumour immune resistance by causing intrinsic - as opposed to adaptive - upregulation of programmed death ligand 1.The normal function of RAS, as described above, can be deranged by mutations which unbalance this equilibrium. Single point missense mutations in codons G12 (most commonly), G13 or Q61 are responsible for converting proto-oncogenes to oncogenes. These mutations favour GTP binding and lead to constitutive activation of RAS, with reduction or loss of GTPase activity. These codons are implicated as their amino acid residues are found in the cavity where GTPase catalytic activity operates. For example, in pancreatic adenocarcinoma, where RAS mutations are virtually ever-present, precursor lesions - pancreatic intraepithelial neoplasia (PanIN) - contain RAS mutations which increase in frequency as they progress stepwise to malignancy. Though generally cooperative with other oncogenes during malignant transformation, these mutations are capable of neoplastic growth in the absence of further genetic abnormalities,14. K-RAS is the isoform most frequently mutated and in addition to being almost inevitably mutated in pancreatic cancers, K-RAS mutations are present in approximately half of colorectal cancers and a third of lung cancers. H-RAS mutations are found in salivary gland cancers , cervical cancers (9%) and urinary tract cancers (9%). N-RAS mutations are found in melanoma (17%), haematologic malignancies (10%) and thyroid cancers (7%). Not only are RAS mutations implicated in carcinogenesis, but also they are strongly associated with treatment resistance,17, and have been shown to be adverse prognostic markers in cancer. RAS-mutated colorectal cancer will not benefit from EGFR-directed treatment with cetuximab or panitumumab. Similarly, in lung cancer RAS status has been shown to be an independent predictor for EGFR tyrosine kinase inhibition.Approximately 25%-30% of cancers contain mutations in one of the RAS isoforms, and they are considered an early genetic event in tumour progression. In addition, given variation in the frequency of isoform mutations within different cancer types, and in the specific mutations involved, there may not be a single effective RAS inhibitor for all RAS-mutated cancers. Historical efforts to target RAS which have made it to the clinic could be broadly summarized as those focusing on RAS plasma membrane localisation and those attempting to indirectly block RAS by inhibiting the downstream effector signalling.Given the central role of RAS both in carcinogenesis and tumour progression, the RAS oncoprotein is an important therapeutic target. . Two farnesyltransferase inhibitors (FTIs), lonafarnib and tipifarnib, were investigated in Phase III trials either as monotherapy or in combination with chemotherapy in a number of different RAS-mutated tumours. Despite Phase I and II clinical trials showing some antitumour activity and low toxicity, no improvement in overall survival was reported in Phase III trials-23. One reason for lack of “pan-RAS” efficacy for the FTI class is that K-RAS and N-RAS membrane localisation can be achieved in the absence of farnesyltransferases, via geranylgeranyl transferases. Attempts were made to target these enzymes with geranylgeranyltransferase inhibitors, but these were ineffective and associated with toxicity. In contrast, H-RAS is not a substrate for geranylgeranyl transferase and therefore its membrane localization could be suppressed solely by FTIs. Tipifarnib has demonstrated preclinical activity against a wide panel of H-RAS-mutated head and neck squamous cell carcinoma xenograft models and is undergoing clinical development in advanced head and neck cancers harbouring activating H-RAS mutations (NCT02383927). These efforts highlight the differences between RAS isoforms and the need for a tailored, isoform-specific approach to clinical trial design.In order to carry out their role, RAS proteins must become membrane-bound. This involves a complex series of post-translational modifications. Three enzymatic steps are necessary for RAS to associate with membranes - (1) prenylation of the CAAX box by farnesyltransferase (FTase); (2) cleavage of the terminal AAX residues by RAS converting enzymes RCE1; and (3) methylation of the cysteine residues of the CAAX box by isoprenylcysteine carboxyl methyltransferase ICMT. Farnesylation, the addition of farnesyl groups to RAS, is a critical step in creating a hydrophobic domain in RAS that allows the protein to associate with the plasma membrane, and therefore to be biologically active. Farnesyltransferases (FTases) are the enzymes responsible for this step, and they were an early target in efforts to inhibit RAS function. A further target is isoprenylcysteine carboxyl methyltransferase (ICMT), an enzyme at the endoplasmic reticulum which increases RAS membrane affinity. It lacks homology with other protein methyltransferases, which adds to its specificity as a target. While agents that target ICMT have been isolated, and have demonstrated promising results in vitro against cancer cell lines, they have not been tested in the clinical setting. Palmitoylation, the modification by the fatty acid palmitate, is necessary for the membrane interactions of H-RAS and N-RAS. Depalmitoylation inhibitors and palmitoyl acyltransferases have been described as having activity against RAS, but uncertainty regarding their specificity and concern about off-target effects, have impeded their further clinical development. Many other proteins are modified by prenylation and farnesylation to ensure their correct subcellular localization, which makes off-target effects unavoidable even for highly specific inhibitors. Moreover, RAS can also signal from endomembranes  in addition to the plasma membrane,30. It is unclear how localization inhibitors affect RAS signalling from different subcellular compartments.Salirasib is an S-trans, trans-farnesylthiosalicylic acid and a novel oral RAS inhibitor which competes with farnesylated RAS for binding sites on membranes. A recent trial demonstrated encouraging activity in patients with advanced solid tumours including a subset with K-RAS mutations. These approaches have largely failed, perhaps mainly due to the inherent complexity and redundancy within these networks, but also due to a lack of specificity in the selection of inhibitors as well as isoform homogeneity within each subpopulation. In addition, the concept of linear pathways is misleading and has given way to that of signalling networks, whereby activated kinases interact via RAS (or other GTPases) with a large variety of signalling molecules resulting in highly interconnected networks.Given the historic difficulties in directly targeting RAS, many efforts focused instead on inhibiting the downstream signal transduction pathways, either at a single point or as a combined approach targeting different nodal points. RAS effector families are involved in cancer initiation and maintenance, and it was hoped that inhibition of downstream proteins within these pathways could be an effective means of countering RAS-mediated oncogenesisEGFR: There is substantial crosstalk between EGFR tyrosine kinase and RAS. With EGFR upstream of RAS, inactivation of these receptor tyrosine kinases can in theory reduce RAS activation, and this linear model explains the lack of clinical activity for EGFR inhibitors in colorectal cancer in the setting of K-RAS or N-RAS-mutant tumours,33. This has also been demonstrated experimentally, through activation of the RAS signalling pathway by introduction of an activated K-RAS allele, confirming that as mutated RAS is constitutively active, disruption of signalling from EGFR impairs the therapeutic effect of anti-EGFR monoclonal antibodies. Similarly in advanced non-small cell lung cancer (NSCLC), EGFR inhibition is insufficient to produce a response in the setting of RAS-mutant disease. It is possible, however, that the specific RAS mutation plays a significant role in determining whether upstream inhibition of EGFR may be effective. We have seen in K-RAS G12D mutation-specific advanced solid tumours that a response can be achieved with pan-ERBB/EGFR inhibitors Afatinib and neratinib,37. EGFR and pan-ERBB inhibitors have shown promise in preclinical studies in combination with both direct covalent RAS inhibitors and MEK inhibitors. This synergistic effect between EGFR inhibitors and covalent RAS inhibitors appears to be the result of EGFR inhibition leading to a reduced amount of GTP-bound RAS, therefore leaving RAS in the unbound GDP state open to targeting by direct inhibition.ERK pathway: Directly downstream of RAS signaling are the ERK and PI3K signaling pathways. Active GTP-bound RAS results in RAF dimerization and phosphorylation, RAF kinase activity and, ultimately, phosphorylation of its substrates MEK1 and MEK2. The terminal kinases of this pathway, ERK1 and ERK2, act as growth promoting transcription factors. The RAS-RAF-MEK-ERK cascade is targeted with RAF kinase inhibitors, ERK inhibitors or MEK inhibitors. Eleven RAF kinase inhibitors have reached clinical evaluation and four are approved for use by the US Food and Drug Administration (FDA). Vemurafenib and dabrafenib are two ATP-competitive RAF inhibitors that are approved for use in BRAF-mutant metastatic melanoma. Further clinical evidence in both lung cancer and colorectal cancer has shown benefit for these agents when BRAF is mutated,40. The current agents used in clinical practice, dabrafenib and vemurafenib, act on RAF monomers. However, in RAS-mutant cancer, RAF inhibition has been unsuccessful and the clinical experience has been very negative. The reason for this is that clinically used RAF inhibitors enhance RAF kinase homo-and heterodimerization, leading to the paradoxical activation of ERK signalling. Homo- and heterodimerization of the RAF kinases BRAF and CRAF significantly increases their catalytic activities. The binding of RAF molecules to active RAS drives RAF dimerization by inducing conformational changes, dephosphorylation of inhibitory residues, and brings RAF molecules into proximity of each other. Due to allosteric interactions between protomers in the RAF dimer, inhibitor binding to the first protomer in a dimer strongly decreases the affinity of the second protomer to the inhibitor. In this constellation the drug-bound RAF protomer allosterically activates the drug-free protomer causing paradoxical pathway activation and drug resistance. As oncogenic RAS proteins are effective drivers of RAF kinase dimerization, RAS mutations lead to intrinsic or acquired resistance to RAF inhibitors. Overcoming dimerization-induced resistance to RAF inhibitors could lead to effective anti-RAS therapy.. There are a number of newer agents that target RAF dimers rather than the monomer isoform all of which appear to demonstrate less paradoxical upregulation of the ERK pathway,46. Belvarafenib and LXH-254 are pan-RAF inhibitors, effective against the monomer and dimer isoform, which are under clinical evaluation both as monotherapy and in combinations for RAS-mutant advanced solid tumours.Another interesting drawback to targeting wildtype RAF in RAS-mutant disease is that inhibition of wildtype RAF can paradoxically upregulate the ERK pathway in the setting of RAS mutations leading to downstream phosphorylation of MEK and ERK. In RAS-mutant tumours, MEK inhibition as monotherapy has failed to demonstrate meaningful benefits largely due to the induction of feedback loops similar to those when RAF inhibitors are use in this setting. Trametinib, Cobimetinib and Binimetinib are allosteric, non-ATP competitive inhibitors of MEK1 and MEK2, and are used in combination with RAF inhibitors in the treatment of melanoma. A number of trials have failed to show clinical benefit for MEK inhibition in advanced KRAS-mutant pancreatic, colorectal and non-small cell lung cancer-51. One possible explanation for this is that inhibition of the ERK pathway induces autophagy, a process of cellular recycling that protects cells from the cytocidal effects of pathway inhibition. Pancreatic cancer cells in particular utilize autophagy for growth and as a means of resistance to ERK inhibition. Combining downstream MEK inhibition with hydroxychloroquine - an inhibitor of autophagy - displayed synergistic anti-proliferative effects against pancreatic ductal adenocarcinoma (PDA) cell lines and promoted striking regression of PDA xenografts from 2 patients with PDA which was superior to (standard of care) gemcitabine plus nab-paclitaxel chemotherapy. Highly encouraging clinical responses were also seen with this commercially available drug combination and clinical trials are ongoing ,54.ERK upregulation through MEK appears to be the predominant method of resistance to BRAF directed monotherapy and we have seen both in preclinical and clinical studies that concurrent inhibition of BRAF and MEK can decrease acquired resistance and delay progressionERK pathway: With ERK being the final kinase in the RAF-MEK-ERK pathway it would appear to be an attractive target to inhibit in RAS or RAF mutant tumours. Based on previous preclinical studies of the compound SCH-772984, a dual ERK1/2 inhibitor which demonstrated a reduction of phosphorylated ERK in a number of RAS-mutant cancer cell lines, an oral version MK-8353 was developed for clinical testing. In a Phase I study, however, with 26 patients with advanced K-RAS or N-RAS mutated tumours, no objective responses were seen. It is now being tested in combination with a MEK inhibitor (selumetinib) and the anti-PD-1 inhibitor pembrolizumab in patients with RAS-mutant cancers. In K-RAS-mutant tumour models, ERK inhibitors such as GDC-0944 have shown efficacy in combination with the MEK inhibitor cobimetinib. Phase I studies of the combination were stopped prematurely due to toxicity. The ERK inhibitor was studied as monotherapy in a Phase I trial and appeared tolerable. In this study, 14 patients had K-RAS-mutant advanced malignancies and of those 4 had stable disease and 10 had progression. ERK pathway suppression detected with NanoString gene expression was observed more commonly in those with BRAF-mutant tumours compared to those with KRAS-mutant tumours.. This in combination with nab-paclitaxel has been examined in a Phase 1 study in patients with advanced pancreas cancer and results are awaited (NCT02608229). Other studies are underway examining its role as monotherapy and in combination with other agents in patients with genetic alteration in the ERK pathway . LY-3214996 is a selective inhibitor of ERK1 and ERK2. Unfortunately, however, in a Phase I study of this agent, only one patient with advanced RAS-mutant cancer had stable disease and the remaining patients had progressive disease. KO-947, similarly a selective ERK1/2 inhibitor, demonstrated potent and sustained reduction in phosphorylated ERK in vitro in RAS-mutant cell lines. This is now in clinical studies for patients with advanced RAS- or RAF-mutant tumours (NCT03051035).Ulixertinib is another ATP competitive ERK1/2 inhibitor that has shown clinical efficacy in N-RAS-mutant melanomaPI3K pathway: The other major target of RAS effector signalling is the PI3K-AKT-mTOR pathway. PI3K is implicated in RAS-dependent cancer initiation and maintenance. While there are many inhibitors of this signalling pathway under investigation, they have demonstrated little activity as monotherapy in RAS-mutant cancers. KRAS and BRAF mutations are predictive of resistance to mTOR inhibition. Moreover, inhibition of mTOR may lead to the upregulation and activation of MEK-ERK pathways. Therefore, the dual inhibition of the PI3K and the ERK pathway would appear to be a reasonable therapeutic target. With this in mind combination strategies have been explored in RAS mutant disease using both mTOR inhibitors along with RAF/MEK inhibitors. Direct PI3K inhibitors have also been trialled with MEK inhibitors. However, in clinical trials, this combination was both toxic and only minimally efficacious.. Unfortunately no AKT inhibitors are approved for use in clinical practice. The evaluation of AKT inhibition with MEK inhibitors is under investigation, but similar challenging side effect profiles have been seen to that of combined mTOR and MEK inhibition.AKT, which plays a key role in the activation of mTOR following its interaction with one of the 3 main PI3Ks, is amplified in the setting of RAS-mutant cancers,69. Finally, there are selectivity and toxicity challenges. The “switch region”, which changes conformation upon GTP binding and recruits effector proteins, is highly conserved across other G-proteins in the body. Therefore, any agents which target this region in RAS, would be associated with risk of toxicity elsewhere.Attempts at direct RAS inhibition have been hampered by numerous challenges. Firstly, the activity of RAS is tightly governed by GEFs and GAPs, which control the high-affinity, picomolar interactions between GDP, GTP and RAS. Putative inhibitors struggle to overcome this affinity. Secondly, there are structural challenges. An effective inhibitor would typically require a deep hydrophobic pocket for binding. These pockets were long understood to be absent, although recent research demonstrates that they may arise dynamically as RAS goes through the GDP/GTP cycle and compounds that bind to RAS at the RAF binding site and inhibit RAS/RAF complex formation. However, none were sufficiently potent to be considered for further investigation. More recently, however, direct targeting of RAS has been achieved for the G12C K-RAS mutation. This has been realised through the development of a small molecule inhibitor that binds covalently to the cysteine residue that results from the specific G12C mutation and has been shown to inhibit oncogenic RAS,73. The inherently reactive nature of cysteine which is found at codon 12 of K-RAS(G12C) can be exploited for covalent small molecule inhibitors and the idea of targeting cysteine is one that is commonly exploited in drug discovery. Another important feature of targeting this cysteine is that wild-type K-RAS lacks the cysteine in the active site unlike the mutant K-RAS(G12C).A number of agents investigated were compounds that competed directly with GDP for the nucleotide binding site of RASet al. initially identified this novel allosteric binding pocket behind switch II referred to as the switch-II pocket, which led to the development of the first compounds to irreversibly target G12C. These compounds bind to K-RAS(G12C) in the GDP-bound inactive state, blocking SOS-catalysed nucleotide exchange and ultimately inhibiting K-RAS(G12C) association with RAF. This switch II pocket is present in the GDP-bound inactive form of K-RAS only. Therefore, targeting of G12C needs to occur in the GDP-bound state. K-RAS(G12C) in its steady state is in the active GTP-bound state, but the presence of a high level of GTPase activity leaves it open to covalent attack. The identification of this pocket led to a search for covalent inhibitors of K-RAS. The first was a molecule ARS-853, and its development led to the proof of concept that this specific isoform of K-RAS could be targeted using a covalent inhibitor.Ostrem . This particular G12C inhibitor has succeeded where others had not as its potency and selectivity was optimised through an interaction with a previously unexploited groove His95. Preclinical studies have demonstrated responses and regression of K-RAS mutant tumours treated with AMG510. The results of the Phase I study of sotorasib (AMG510) demonstrated promising anticancer activity in patients with advanced solid tumours harbouring K-RAS(G12C) mutations. 129 patients were treated on the dose escalation study, 59 with NSCLC, 42 with colorectal cancer and 28 with other solid tumours. Sotorasib appeared to be well tolerated with 11.6% of grade 3 or 4 toxicity. 32.2% of the NSCLC had an objective response with a total of 88.1% having a response or stable disease. The median progression free survival was 6.3 months. In the colorectal cohort 7.1% had a confirmed response with 73.8% having a response or stable disease with a median PFS of 4 months. This study represented the first clinical trial demonstrating objective response to direct KRAS inhibition. On December 8, 2020 the FDA granted Breakthrough Therapy designation for its investigational K-RAS(G12C) inhibitor, sotorasib, for the treatment of patients with locally advanced or metastatic NSCLC with K-RAS(G12C) mutations, as determined by an FDA-approved test, following at least one prior systemic therapy. Preclinical studies have also demonstrated that sotorasib was able to clear colon cancer from mice when given in combination with checkpoint inhibitors. There is a Phase 1/2 study under way of sotorasib in solid tumours which will include a combination arm of sotorasib with an anti(PD-1/L1) (NCT03600883).A number of advances in the area ultimately led to the development of AMG510 (sotorasib). A Phase 1/2 study of adagrasib monotherapy in patients with pretreated NSCLC demonstrated an overall response rate of 45% and a high disease control rate. This multiple-expansion-cohort trial is ongoing, investigating the use of adagrasib combined with pembrolizumab in NSCLC, afatinib in NSCLC or cetuximab in colorectal cancer. Research on this agent in nonclinical models has also demonstrated mechanisms of resistance, including KRAS nucleotide cycling and pathways that induce feedback reactivation or bypass KRAS dependence.Adagrasib (MRTX849) is an additional agent under investigation in this field. It is a potent, highly selective inhibitor of KRAS(G12C)K-RAS(G12C) mutations only account for a small proportion of KRAS mutations that are found in cancer and are primarily found in lung cancer. As these irreversible allosteric inhibitors block RAS signalling by exclusively binding to the cysteine residue that results from the specific mutation, this limits their application to the particular allele they target. To target KRAS(G12D) and KRAS(G12V) different approaches are needed as these mutants lack the cysteines needed in the active state.In vitro studies have shown that Compound 3144, a molecule that binds a conserved residue Asp38 in switch-I, can block RAS effector binding. This compound suppresses the growth of KRAS(G13D) tumours in vivo. A major concern, however, is that pan-inhibition of RAS potentially may lead to considerable toxicity as normal cellular function is reliant on RAS signalling in non-cancerous cells. Models by which the deletion of all three RAS isoforms is carried out are not compatible with life, and therefore a pan inhibition of RAS in humans is likely to result in significant off-target toxicity. Nevertheless, it has been suggested that if such a compound were optimised for greater potency and specificity, this would be a viable approach.Attempting to design and develop drugs specifically targeting each individual RAS mutation would be extremely challenging and time-consuming, so direct targeting of ligand binding sites conserved on all RAS proteins  has been thought to be one potential method of inhibiting RAS across all mutation and tumour types. . GTP binding and activation of RAS leads to conformational changes in it, allowing it to bind effectors in RAS-binding domains. Mutations in RAS that are relevant to cancer usually lead to RAS permanently in the GTP bound active state. Efforts have been made to block this nucleotide exchange in an attempt to stop RAS transitioning to the active GTP bound form.Nucleotide exchange inhibition: In order to cycle between active GTP-bound and inactive GDP-bound states, RAS possesses intrinsic guanine nucleotide exchange and GTP hydrolysis activities. This cycling and exchange are accelerated by GEF and by GAPs, both of which change the activation state of RAS through covalent modifications. Upon activation of GEFs, nucleotide binding is destabilized and GDP is released. As GTP is much more prevalent than GDP in the cell, this loss of GDP leads to a transient formation of RAS-GTP, the active state. Of these, SOS is the best known and studied of the RAS-specific GEFs and this has led to the development of a number of strategies and attempts to block it and ultimately inhibit RAS. SOS1 binds RAS at its catalytic binding site and thereby promotes exchange of GDP for GTP. RAS-GTP can also bind at an allosteric site on SOS1 to enhance GEF activity. Genetic inactivation of SOS1 has been shown to decrease the survival of RAS-mutant tumor cells, but not in RAS wild type cells that are not reliant on RAS signaling. Inhibition of SOS1 has been thought of as an attractive mechanism of RAS inhibition over direct RAS inhibitors as it does not appear to depend on targeting specific mutations.An alternative to blocking the direct nucleotide site is to inhibit the proteins that regulate the nucleotide exchange process. Normal RAS activation requires nucleotide exchange, processing, membrane localization and effector binding. Targeting any of these steps can be used to indirectly inhibit RAS. GEFs are responsible for releasing GDP from RAS allowing it to be replaced by GTP and ultimately leading to activation of RAS. In mammals, three families of RAS-specific GEFs exist: SOS, RASGRF1/Cdc25Mm and GRP/Cal-DAG-GEFin vitro and in vivo. This orally bioavailable agent appears to reduce RAS-GTP level and inhibits ERK pathway signaling, thereby limiting the growth of tumor cells driven by RAS. Whilst most RAS variants appear to show reduction in cell proliferation when exposed to BI-3406, certain variants appear less sensitive. Mutations in codon 61 due to the resultant molecular conformation appear to have low intrinsic GTPase activity and are subsequently less sensitive to SOS1 inhibition. Initial studies of SOS1 inhibitor BI-3406 suggest that it may benefit as many as 80%-90% of RAS-driven cancers.BI-3406 is a potent and selective SOS1:K-RAS interaction inhibitor that potently decreased the formation of GTP-bound RAS and reduced cell proliferation of RAS-driven cancers both SOS1 is phosphorylated by ERK, a kinase in the ERK pathway downstream of RAS, ultimately leading to the reduction of its GEF activity. It is thought that efforts to treat RAS-driven cancers with MEK inhibitors have failed in part as inhibition of MEK reduces the activity of ERK1/2, resulting in the release of a negative feedback loop, thus increasing the activity of SOS1-dependent formation of GTP-bound RAS. Combination therapy of a MEK inhibitor with BI-3406 blocks this negative feedback by reducing levels of phospho-MEK and phospho-ERK leading to sustained pathway inhibition and potentiating the benefit of SOS1 inhibition. A Phase I clinical trial of this combination to assess safety, tolerability and preliminary efficacy, as well as another SOS1 inhibitor, BI-1701963 in combination with Trametinib is ongoing (NCT040111458).. Mutations in PTPN11, which encodes SHP2, cause “rasopathies” and are found in about 50% of patients with Noonan syndrome. Although not fully defined, SHP2 appears to act as a scaffold protein, binding GRB2 and SOS1 in close proximity to RAS and ultimately thereby increasing RAS nucleotide exchange. Research has explored the role of SHP2 inhibitors in the treatment of various cancers. In the preclinical setting, the allosteric SHP2 inhibitor SHP099 has been shown to inhibit myeloid leukaemia cell lines, and to elicit a response in colorectal cancer cell lines. Of note, in the colorectal cancer cells, these responses seemed limited to cells that were sensitive to lapatinib, and therefore dependent on EGFR signalling. By contrast, RAS- or BRAF-mutant cells were generally resistant to SHP099.Although much less defined than SOS, another protein involved in the nucleotide exchange process is SHP2. SHP2 is a non-receptor protein tyrosine phosphatase that is required for the full activation of the ERK pathwayEffective and safe inhibition of RAS was considered a holy grail for cancer researchers decades ago, and it remains so today. While extraordinary advances have been made in our understanding of RAS and carcinogenesis - as well as methods of targeting its downstream effectors - the direct inhibition of RAS does not yet have a role in everyday practice. However, our deeper understanding of molecular pathogenesis is the foundation on which that future progress will be built, and effective treatments are now close.. Therefore, those designing clinical trials should carefully consider the need for isoform and mutation-specific approaches.The unmet need for proven therapies in this setting is clear, given the prevalence of RAS mutations in common cancers, many of which are highly fatal. Our recognition of the structure and clinical significance of diverse RAS isoforms represents a major step forward, of relevance to all future efforts at RAS targeting. Their importance is clear when considering candidate inhibitors of RAS plasma membrane localization, which are now reaching the clinic. Even individual isoforms, like K-RAS, have a range of possible mutations with different responses to targeted therapies.The development of inhibitors of RAS effector signalling, which are in widespread clinical practice, is an example of the concrete achievements of research in this area. This success has been tempered over time by failures, many of which can be attributed to misconceptions of RAS signalling pathways as unidirectional and linear. A more nuanced appreciation of these as delicately balanced signalling networks can help researchers refine their approach, and may warrant increased focus on drug combinations, as seen in trials involving inhibitors of MEK and autophagy. However, these studies also raise questions - why did response rates to Sotorasib vary between cancers despite identical mutations, and what provoked early progression in some patients after an initial response? It is suggested that tumour heterogeneity or driver mutations alternative to K-RAS(G12C) may be the key to understanding this and that, as with inhibition of RAS effector signalling, drug combinations may be the next step. In addition, optimizing monotherapy by developing inhibitors with higher affinity for inactive KRAS(G12C) or the use of pulsatile therapy has been suggested as approaches to maximise therapeutic effect.Possibly showing greater promise than any of these initiatives, however, is efforts at direct targeting of RAS. The past 12 months has seen breakthroughs in this field that have been sought for years, vindicating decades of basic scientific endeavour. Successes here belie outdated dogma describing RAS as “undruggable”, demonstrating that drug exposure associated with preclinical efficacy can be achieved safely in patientsIn summary, a more textured understanding of RAS pathogenesis emerging from decades of basic scientific research has led to a more refined approach to RAS inhibition, which is now beginning to bear fruit. The coming months and years will hopefully take these efforts “over the line” to the routine use of effective therapeutics in the clinic setting, but it will be a long time before we have fully tapped the potential of RAS inhibition.


Screw loosening is a widely reported issue after spinal screw fixation and triggers several complications after lumbar interbody fusion. Osteoporosis is an essential risk factor for screw loosening. Hounsfield units (HU) value is a credible indicator during bone mineral density (BMD) evaluation. As compared with the general evaluation of BMD, we hypothesized that specific measurements of HU at the precise location of the future screw insertion may be a better predictor of screw loosening.Clinical data of 56 patients treated by oblique lumbar interbody fusion (OLIF) of the L4-L5 segments with an anterior lateral single rod (ALSR) screw fixation were reviewed in this study. Vertebral bodies with ≥ 1 mm width radiolucent zones around the screw were defined as screw loosening. HU in the insertional screw positions, the central transverse plane, and the average values of three and four planes were measured. Regression analyses identified independent risk factors for screw loosening separately. The area under the receiver operating characteristic curve (AUC) was computed to evaluate predictive performance.The local HU values were significantly lower in the loosening group, regardless of the selected measuring methods. The AUC of screw loosening prediction was higher in the insertional screw positions’ HU than other frequently used methods.The HU value measured in the insertional screw position is a better predictor of ALSR screw loosening than other methods. The risk of screw loosening should be reduced by optimizing the trajectory of the screw based on the measurement of HU in preoperative CT.• Osteoporosis is an essential risk factor for screw loosening, and Hounsfield units (HU) are a credible predictor during bone mineral density (BMD) evaluation.• The HU value measured in the insertional screw position is a better predictor of screw loosening than other frequently used HU measurement methods.• The risk of screw loosening might potentially be reduced by optimizing the trajectory of the screw based on the measurement of HU in preoperative CT. Screw fixation is a standard surgical method in spinal surgery –3. With Measurements of vertebral Hounsfield unit (HU) value on preoperative computed tomography (CT) is a credible indicator to evaluate spinal BMD and diagnose osteoporosis , 8, 9. HHowever, HU measurement methods still have inherent drawbacks when evaluating the risk of screw loosening. The damage of local cancellous microstructures triggers the loss of screw-bone integration and resulting in a higher risk of screw loosening , 15. ConApproval for the current study protocol was obtained from the ethics committees of our hospital (2020-554). Informed consent was waived for this retrospective study. We retrospectively reviewed demographic and radiographic data of 56 patients who underwent OLIF with ALSR screw fixation and without posterior decompression in the L4-L5 motion segment from May 2017 to August 2019. The average follow-up period of these patients was 12.2 months (within 11 to 13.5 months). The OLIF operation in these patients was performed by a senior spine surgeon. Screw types and sizes were completely identical. All screws were placed in a single attempt, and all screws penetrated the contralateral cortex.The inclusion criteria were as follows: (1) Patients who underwent OLIF with ALSR screw fixation for lumbar degenerative diseases, including spinal stenosis and grade 1 and grade 2 degenerative spondylolisthesis; (2) Patients who underwent lumbar CT three times, including 1 week before, 1 week after, and approximately 1 year (within 11 to 13.5 months) after OLIF surgery. The exclusion criteria were as follows: (1) Patients with a history of lumbar surgery; (2) Patients with primary or metastatic spinal tumors, lumbar tuberculosis, rheumatic immune diseases, and secondary osteoporosis caused by medication or other metabolic diseases; (3) Patients with grade 3 and grade 4 degenerative spondylolisthesis or any grades of spondylolysis ; (4) Patients with additional posterior approach decompression; (5) Patients who underwent lumbar revision surgery within the clinical follow-up period of 12 months for complications other than screw loosening; (6) Patients who underwent intraoperative screw replacement.All patients underwent lumbar CT three times in the radiology center of our hospital, including 1 week before, 1 week after, and around 1 year (within 11 to 13.5 months) after OLIF surgery. Imaging data from different CT scans play different roles in this study. Specifically, all HU value measurement procedure was performed in the preoperative CT scan, screw trajectories and corresponding insertional screw position in the preoperative CT were judged according to the instant postoperative CT scan (1 week after OLIF operation), and finally, the screw loosening status was judged based on the 1 year’s postoperative CT scan. The tube voltage was set as 120 kV, and this parameter was identical to studies on the same topic , 25, 26.The measurement of these imaging data was independently performed by an experienced radiologist  for more than 10 years (He independently produced more than 2000 reports about MSK diseases)). Screw loosening was confirmed by computational tomography (CT) during the 1-year postoperative follow-up , 27, 28,To judge the interobserver and intraobserver reliability, 10 patients were randomly selected. One week after the measurement of these imaging data, imaging data of these selected patients were remeasured by the radiologist and a spine surgeon  during the standardized training). The intraclass correlation efficiency (ICC) was computed to identify the repeatability of measured continuous variables (ICC ≥ 0.8 represents excellent reliability) . The kapt-test was used for continuous variables, and the chi-square test was used for categorical variables. We performed binary logistic regression to identify independent risk factors for screw loosening. To investigate whether demographic data affect the risk of screw loosening, patients’ sex, BMI, and age were enrolled in regression analyses. In contrast, because excellent consistency between HU values measured by different methods existed (according to the computation of ICC), HU values were included in the regression analyses separately. Univariate analyses of each potential risk factor were performed, and the variables that achieved a significance level of p < 0.1 were entered into multivariate analyses [p < 0.05 were considered independent risk factors in the multivariate analyses. A p value less than 0.05 indicated a significant difference [p value less than 0.05 indicated a significant difference.Statistical analyses for cranial and caudal side screw loosening were performed separately. When comparing the difference between screw loosening status (nonloosening and loosening) in both cranial and caudal vertebral bodies. The independent samples Student’s analyses –35. Varifference –35. FinaA total of 56 patients  with an average age of 56.57 ± 11.96 years treated by L4-L5 segment OLIF with ALSR screw fixation were recorded. There were no significant differences in patients’ BMI and sex in the nonloosening and screw loosening groups. The overall incidence rate of screw loosening was 35.71% (40/112), the screw loosening rate of the vertebral body on the cranial side was 42.86% (24/56) and that of the caudal vertebral body was 28.57% (16/56). Patients in the cranial screw loosening group were older than those in the nonloosening group, but there was no significant difference in age between the caudal side’s nonloosening and screw loosening groups  of different HU measurement methods. The current results proved that HU values directly measured from the insertional screw position were a better predictor  than other methods during screw loosening prediction.The T score measured by DXA is a commonly used indicator of osteoporosis , 36. TheAdmittedly, the overall screw loosening rate was 35.71% 40/112), higher than that of other studies whose patients were fixed by bilateral pedicle screws (BPS) , 39, 40./112, higMeanwhile, studies have also repeatedly proven that the fixation stability of BPS is better than that of ALSR screw fixation –44. MoreScrew fixation is a feasible method to construct instant postoperative stability of the surgical segment in LIF patients, and the surgical segment's stability could provide an ideal biomechanical environment for osteogenesis during the LIF process. In other words, in patients with screw loosening, loss of bone-screw integration will lead to poor instant postoperative stability, resulting in cage migration, subsidence, and nonunion that will adversely affect patients’ prognosis and increase the rate of revision surgery , 46, espAlthough studies proved that the measurement of HU values in the pedicle region could provide a good predictive performance of pedicle screw loosening , 25, 47,Indeed, this study has several limitations. First, this is a retrospective study; the conclusion of this study should be re-estimated by prospective clinical follow-up. In addition, although most of the same studies have set the follow-up period at 1 year, a longer follow-up is still necessary to further validate our findings. Additionally, the indications of patients included in this study were limited to degenerative spinal stenosis and slight degenerative spondylolisthesis, and patients with posterior decompression were also excluded from this study. Finally, the confounding effect of metal artifacts cannot be eliminated in current patients. Therefore, the conclusions in this study should be reverified in future studies on patients with different diagnoses and different surgical procedures. In future studies, an artifact-removed CT scan should be performed to reverify the current conclusion.Compared with frequently used HU measurement methods, the insertional screw position’s HU value could better represent the BMD in the screw-bone integrated area. The elimination of regional BMD differences in the cancellous area by using this method could optimize the predictive performance of HU during the evaluation of screw loosening risk. In addition, preoperative optimization of the ALSR screw trajectory  should reduce the risk of screw loosening and related complications.


The primary Antarctic contribution to modern sea-level rise is glacial discharge from the Amundsen Sea sector of the West Antarctic Ice Sheet. The main processes responsible for ice mass loss include: (1) ocean-driven melting of ice shelves by upwelling of warm water onto the continental shelf; and (2) atmospheric-driven surface melting of glaciers along the Antarctic coast. Understanding the relative influence of these processes on glacial stability is imperative to predicting sea-level rise. Employing a beryllium isotope-based reconstruction of ice-shelf history, we demonstrate that glaciers flowing into the Amundsen Sea Embayment underwent melting and retreat between 9 and 6 thousand years ago. Despite warm ocean water influence, this melting event was mainly forced by atmospheric circulation changes over continental West Antarctica, linked via a Rossby wave train to tropical Pacific Ocean warming. This millennial-scale glacial history may be used to validate contemporary ice-sheet models and improve sea-level projections. Ice loss from the Amundsen Sea sector of West Antarctica is rapidly accelerating. Here, the authors reveal that this region also underwent thinning and retreat from 9 to 6 thousand years ago, due to atmospheric connections with a warming tropical Pacific. Recent WAIS mass loss has been focused within the Amundsen Sea Embayment (ASE), which may indicate the beginning of marine ice sheet instability3. Ice mass loss since the 1990s is attributed to the ocean-driven melting of ice shelves by the upwelling of relatively warm Circumpolar Deep Water (CDW) onto the West Antarctic continental shelf4. Precipitation and warming over West Antarctica was also more elevated in the 1990s than at any time over the last 200 years, linked to higher tropical Pacific Ocean temperatures6. Atmospheric rivers (narrow bands with enhanced water vapour flux) associated with this tropical-polar teleconnection represent 100% of summer surface melt in Marie Byrd Land7, presenting an additional contribution to global sea-level rise from the atmospheric-driven melting of glaciers along the West Antarctic coast. Significant uncertainty therefore remains for the permanence of increasing ice loss, projected contributions to sea level, and the dominant driving mechanisms behind mass balance change1. Integrated models predicting the timing and rate of future Antarctic ice mass loss rely on validation from long-term (102–103 yr) records of past ice-sheet change9, and therefore require glacial reconstructions since the Last Glacial Maximum (LGM)10.Increasing ice loss and sea-level contribution from Antarctica since the early twenty-first century is caused by rapid thinning, retreat, and acceleration of major outlet glaciers of the West Antarctic Ice Sheet (WAIS)11  isotopes, a proxy for glacial processes, to provide a 10.3 kyr history of the Cosgrove Ice Shelf (CIS) in the eastern ASE , 9Be abundance ([9Be]reactive) and 10Be/9Be ratios in marine sediments collected during the IB Oden Southern Ocean cruise (OSO-0910)16 (“Methods”). Kasten cores KC-15, KC-16 and KC-17 were collected just offshore the modern terminus of the CIS in Ferrero Bay  bet-17 Fig.  yet plat-17 Fig. ; (3) bet-15 Fig. ; (4) abo bet-17 F10Be]reactive and 10Be/9Be ratios in all three cores  transitioning from the Early Holocene to Mid-Holocene, 10Be/9Be increases between ~9.8 kyr BP and ~5.9 kyr BP from ~0.46 to ~2.33; (3) then through the Late Holocene, 10Be/9Be ratios are relatively constant at ~2.3. During the Late Holocene, 10Be, 9Be, TOC, and TN potentially display millennial-scale variability, increasing after ~1.4 kyr BP 20. Beryllium-9, on the other hand, is released during the chemical weathering of silicate minerals18 and is enriched in waters near high fluvial or aeolian inputs e.g., equatorial Atlantic surface waters in proximity to Saharan dust plumes20. Away from regions of high surface inputs, such as the South Pacific, Be follows a general profile of nutrient scavenging with a depletion in the surface mixed layer (~750 atoms/g) and an enrichment in deeper waters (~2000 atoms/g), reflecting the high scavenging potential of Be21. The 10Be/9Be of Pacific seawater has an average ratio of ~1 × 10−7 but ranges from ~0.4 × 10−7 to ~3.1 × 10−7 in equatorial surface waters of the Atlantic and Pacific due to additional inputs of 9Be and 10Be, respectively20. The disparate sources of 10Be and 9Be led to fluctuations in the 10Be/9Be of seawater, recorded in marine sediments and ferromanganese crusts, over Quaternary climate cycles, due to large regional variability in fluvial inputs between glacial and interglacial periods22.The cosmogenic isotope 10Be/9Bereactive due to the input of fluvial 9Be, and another sourced from entraining seawater, defined by high 10Be concentrations and 10Be/9Be ratios23. The high 10Be/9Be of seawater is augmented in polar regions by the input of 10Be from atmospheric sources24 and/or the melting of sea ice ([10Be] = ~0.25–0.65 ×104 atoms/g)25 and ice sheets ([10Be] = ~1–3 × 105 atoms/g)26, which act to accumulate meteoric 10Be overs years to millennia, leading to exceptionally high surface water ratios which have been observed in the Deep Canadian Basin (~1.4 × 10−7)25 and Drake Passage (~3.3 × 10−7)20, respectively. The export of meltwater signals further offshore may be enhanced by the calving of icebergs and their subsequent passage off the continental shelf27.The Be isotope composition of marine sediments on the continental shelf is controlled by the relative mixing of two reactive phases, one sourced from the coast, defined by low 10Be and 9Be in ice sheets has allowed Be in glaciomarine sediments to become a powerful proxy of glacial dynamics31. Open marine sediments close to the calving line of ice shelves are defined by higher Be concentrations and 10Be/9Be ratios relative to other glacial environments due to efficient scavenging of Be by diatoms from seawater defined by higher overall ratios as previously discussed28. Lower 10Be concentrations and 10Be/9Be ratios can be found in sub-ice shelf sediments, induced by lower productivity and limited advection of open marine material into the ice-shelf cavity28. Finally, sediments in proximity to the grounding line receive 9Be from the basal zone during glacial erosion leading to exceptionally low 10Be/9Be ratios31. Here, we relate Be isotope variation in Ferrero Bay to three main processes: (1) environmental setting; (2) depositional processes; and (3) meltwater contribution  indicate two phases of grounding line retreat within Ferrero Bay terminating by 124 (131) and 105 (99) cmbsf, corresponding to ~10.7 kyr BP and ~9.6 kyr BP, respectively17.Bathymetric surveys reveal that grounded ice occupied Ferrero Bay during the LGM as part of the larger ice stream in the Cosgrove-Abbot Trough, which reached the continental shelf edge north of the Abbot Ice Shelf33 Fig. . The Amu10Be/9Be indicate that the KC-15 and KC-17 core sites remained proximal to the grounding line. Subsequently, an increasing trend in [10Be]reactive and 10Be/9Be is observed between ~9.8 kyr BP (~111 cmbsf) and ~5.9 kyr BP (~71 cmbsf) suggesting a gradual retreat of the grounding line and the development of a more distal environmental setting , low Be concentrations and ng Figs. a and 4a..1 Figs. a and 4a.22, much like the percentage of TOC35. From the bottom of the cores to ~111 cmbsf in KC-15 and KC-17, TOC is likely diluted by the input of coarse-grained clastic material from the grounding line with a high sand content of 27–46% (ref. 17). From ~111 cmbsf, TOC is largely controlled by steadily increasing accumulation rates  of fine-grained (% sand ≦ 12.5%) sediment up-section, which allows for a more rapid passage of organic carbon through the near-surface zone, reducing its degradation35. Reactive 10Be, and to a lesser extent 9Be, displays a strong correlation to TOC  in the overlying water column or the advection of biogenic material into the ice-shelf cavity. However, the diatoms were not counted at the same sampling interval and other organic plankton such as dinoflagellates are not included in previous studies17, limiting a direct comparison between TOC, Be abundance, and productivity.The relative change in Be concentration across these various glacial environments is likely to be controlled by depositional processes such as dilution, scavenging efficiency, and/or extraction efficiencyTOC Fig. –S5, sugg10Be and relative 9Be is largely controlled by extraction efficiency, scavenging efficiency, and/or dilution, generally defined by a 1:1 relationship. Changes in the gradient of this relationship above or below one is driven by an additional input of 9Be or 10Be, respectively37. The relative 10Be vs. relative 9Be relationship of sediments from Baffin Bay and offshore Wilkes Land are defined by a gradient less than one  the calving of ice shelves and melting of icebergs, the meltwater of which is subsequently advected under the CIS Fig. , or (2) e33 Fig. . During nts Fig. .33  the grounding line was proximal to KC-15 prior to ~9.8 kyr BP during the Early Holocene, with a permanent ice-shelf extending beyond KC-17 into the ASE; (2) the grounding line retreated from the Early-to-Mid-Holocene between ~9.8 kyr BP and ~5.9 kyr BP, associated with melting of the CIS and its paleo-ice stream; (3) the grounding line and the CIS remained stable for the rest of the Mid-Holocene until ~2.3 kyr BP; (4) the CIS retreated past KC-15 to its modern position during the Late Holocene, establishing open marine conditions in Ferrero Bay. In the following section, we relate our findings to the wider context of the Amundsen Sea sector of the WAIS and the possible mechanisms driving West Antarctic climatic change during the Holocene.18 and Pine Island Glacier16 from 9 to 6 cal kyr BP and 8 to 6 cal kyr BP, indicating a widespread melting event within the Amundsen Sea sector of the WAIS . The driving mechanism of sea-level rise acceleration has been attributed to ocean-driven melting of ice shelves that buttress glacial flow12. However, atmospheric rivers associated with tropical-polar teleconnections present a mechanism for substantial contribution to seasonal surface melt over continental West Antarctica as well7. Greater atmospheric moisture content and poleward expansion of the Hadley cells under rising greenhouse gas emissions suggest atmospheric river climatology is already beginning to resemble that of the Mid-Holocene45, possibly linked to extensive melt events along the Ross and Amundsen Sea coasts since the 1990s44. Our study suggests that future warming and increasing precipitation brought by enhanced atmospheric river activity could contribute to further surface melting and significant glacial discharge into the ASE. This information is vital for validating numerical models, thereby improving future predictions of sea-level rise54 which is currently estimated to be as much as 1 m from Antarctica by the end of the twenty-first century9. Here, we demonstrate that Be isotopes can be successfully employed alongside other sediment and biological proxies to reconstruct past changes in depositional environment, ice shelf extent, and meltwater input—a parameter that is completely missing from most glacial records since the LGM27. However, uncertainty remains owing to the multiple sources and processes influencing the Be isotopes systematics of glacimarine sediments27. Future work will need to constrain the Be abundance in sea ice, ice sheets, and shelf waters, whilst also determining how Be is incorporated into sediments from the grounding line to the continental slope.Presently, glaciers flowing into the ASE are the main Antarctic contribution to global sea-level rise, averaging 0.23 ± 0.02 mm yr17. Three Kasten cores were collected from Ferrero Bay during the IB Oden OSO-0910 expedition from 2009 to 2010: KC-15 , a 1.3 m sediment core from the innermost fjord; KC-16 , a 0.4 m sediment core from a structural high; and, KC-17 , a 1.4 m core recovered from the outer bay  was separated from marine sediments using the technique previously presented29. Approximately 0.1 g of sediment was dried and crushed before being leached with 0.04 M hydroxylamine hydrochloride in 25% acetic acid for 7 h at 80 °C. An aliquot of the leached solution was measured for reactive 9Be using a Thermo® ELEMENT XR high resolution inductively coupled plasma mass spectrometer (HR-ICP-MS) at the Atmosphere and Ocean Research Institute (AORI), the University of Tokyo (UTokyo), after spiking with 5 µl of indium (1 µg/g) solution55. The remaining solution was spiked with 1 ml of a 0.097 mg/ml 9Be carrier before purification using two solvent extractions of acetylacetone in the presence of EDTA followed by precipitation of Be(OH)2 with NH4 (ref. 57). The resulting hydroxide was converted to BeO powder using a microwave ceramic crucible58 before being mixed with niobium, inserted into a copper cathode, and measured by a National Electrostatic Corporation accelerator mass spectrometer (AMS) at the UTokyo Micro Analysis Laboratory, Tandem Accelerator (MALT)59.Reactive Be . Carbon-14 ages were corrected for local effects61 and a marine reservoir effect (ΔR) of 900 ± 100 years (ref. 62) prior to calibration using the Marine13 curve63. Undatable was run for 106 iterations using a bootstrapping percentage of 20% and a Gaussian SAR uncertainty factor of 0.1 (ref. 60).Radiocarbon analysis of two marine carbonate samplesSupplementary Information


Background: Ice is used in healthcare facilities for medical purposes and consumption by the medical staff and the patients, but some studies have revealed significant microbial contamination of ice machines leading to nosocomial outbreaks or pseudooutbreaks and infections by opportunistic pathogens, including the fungi Candida, the bacteria Pseudomonas aeruginosa, and nontuberculous mycobacteria (NTM). Although ice machines are complex devices that are prone to contamination, very little is known about their potential as vectors of infections for populations at risk in hospitals. Only few studies document efficient maintenance regimes, specifically cleaning procedures and microbial indicators that would ensure their safe use. Method: In this prospective study, combined samples of water and ice, and drain biofilm samples were collected from 36 ice and cold-water distribution machines of a recently built hospital, for a total of 72 samples. Physicochemical parameters  were measured in water, and several opportunistic pathogens  and biological indicators , total and viable bacteria and enterococci) were monitored in water and ice and biofilm. Culture methods were used for HPCs, Candida spp, P. aeruginosa, and enterococci, and total and viable bacterial populations were estimated using flow cytometry. NTM were monitored by quantitative polymerase chain reaction (qPCR). Results: We observed clear differences between the machines in terms of biological contamination, with frequent detection of NTM and presumed Candida. Thus, NTM were detected in the 36 samples of ice and water with concentrations from 0.5 to 2×104 gene copies/mL. Among the several species of fungi detected in the ice machines, some were identified as C. parapsilosis and C. guilliermondii, which are organisms of concern in healthcare facilities. Factors affecting the level of contamination in ice machines include the location of the machines and water quality . Depending on the location in the building and the model of ice machine sampled, the biological indicators measurements indicated more or less significant contamination. No link was established between environmental strains recovered from the machines and clinical infections. Conclusions: Monitoring results showed that ice machines, while subject to few regulations and controls, can be reservoirs of unsuspected opportunistic pathogens that could lead to nosocomial infections of vulnerable patients. Cleaning procedures should be based on the disinfection of resistant opportunistic pathogens, such as Candida and NTM, and the use of general indicators, such as HPCs, should be questioned.Funding: NoneDisclosures: None


MYH6 variant in an Icelandic cohort, and two common deletions in the protocadherin α cluster (PCDHA delCNVs) are significantly associated with CoA and BAV. Here, analysis of a non-Icelandic white CHD cohort (n = 166) recovered rare MYH6 variants in 10.9% of CoA and 32.7% of BAV/CoA patients, yielding odds ratios of 18.6 (p = 2.5 × 10−7) and 20.5 (p = 7.4 × 10−5) for the respective association of MYH6 variants with CoA and BAV/CoA. In combination with the PCHDA delCNVs, they accounted for a third of CoA cases. Gene expression datasets for the human and mouse embryonic heart showed that both genes are predominantly expressed in the atria, not the ventricle. Moreover, cis-eQTLs analysis showed the PCHDA delCNV is associated with reduced atrial expression of PCHDA10, a gene in the delCNV interval. Together, these findings showed that PCDHA/MYH6 variants account for a substantial fraction of CoA cases. An atrial rather than ventricular hemodynamic model for CoA is indicated, consistent with the known early atrial functional dominance of the human embryonic heart.Coarctation of the aorta (CoA) and bicuspid aortic valve (BAV) often cooccur and are genetically linked congenital heart defects (CHD). While CoA is thought to have a hemodynamic origin from ventricular dysfunction, we provide evidence pointing to atrial hemodynamics based on investigating the genetic etiology of CoA. Previous studies have shown a rare  Development of the cardiovascular system is regulated by blood flow, with hemodynamic perturbation likely contributing to various congenital heart defects (CHD), such as coarctation of the aorta (CoA), bicuspid aortic valve (BAV), or other related CHD with left ventricular outflow tract obstructions (LVOTO) ,2,3,4. CMYH6 founder mutation (p.Arg721Trp) associated with CoA and also BAV, and was reported to account for 20% of all CoA cases in the Icelandic population [MYH6 variants to CoA in other populations has not been investigated, although rare pathogenic MYH6 variants have been reported in a family with HLHS [PCDHA delCNV) associated with the pathogenesis of LVOTO lesions [MYH6/PCDHA and their combined impact on CoA and BAV. We further examined the temporospatial profile of MYH6 and PCDHA gene expression in the human heart, with further validation using cis-eQTL analysis tracked by the PCHDA delCNV.A genetic etiology for CoA, BAV, and other LVOTO lesions are well described , and is pulation . Howeverith HLHS . As we r lesions , here weOne hundred sixty-six subjects with LVOTO were recruited with informed consent from Children’s Hospital of Pittsburgh (CHP) as previously described . PersonaMYH6 variants were recovered [Whole exome sequencing (WES) data from the CHP patients was processed as previously described and high-quality ecovered . Only prMYH6 variants in the case cohort: (1) variant coverage is at >10x in >90% of samples; (2) SNV or indel genotype quality score ≥ 20 or 60, respectively, and read depth ≥ 10x; (3) minor allele frequency (MAF) of variants < 2 × 10−4 across all samples in gnomAD exome v2.1.1 [PCDHA delCNVs were recovered from the WES data as previously described [For ensuring compatibility of the WES data with the GnomAD database, the following variant quality controls were included for recovery of rare pathogenic e v2.1.1 . This moe v2.1.1 ,16,17. Tescribed .MYH6 rare damaging variants in a case cohort with comparison to control cohort (gnomAD exome v2.1.1) using only high-confidence pathogenic variants as described above. For the control cohort (gnomAD exome v2.1.1), only MYH6 variants with high-quality calls  and with coverage at >10x in >90% of samples were retained for downstream analyses. The rare predicted pathogenic MYH6 variants were extracted as described above. The total number of alleles evaluated in MYH6 was taken as the median of the allele numbers recovered for all rare damaging MYH6 variants as previously described [p value and the odds ratio (OR) with 95% confidence intervals  [MYH6, which is not expected to be disease-related. This showed no significant increase in burden in cases vs. controls (p = 0.49).The Genome Aggregation Database (gnomAD) exome v2.1.1  or ExAC  databases integrating large-scale exome sequencing projects with variable ancestry backgrounds were used as controls for gene burden analysis, similar to other published studies ,17,18. Wescribed ,16. Fishreshold) ,17. SimiPCDHA delCNV (esv3606958 in 1KG) in a European-American population. Samples from a population with non-Finnish European or American ancestry  were extracted from the reference panel. A measure of LD (D′) and r2 was calculated using the Haploview 4.2 software (-maxdistance 500 -dprime -memory 5000) [r2 = 1) were recovered as tagSNPs. This yielded four tagSNPs  for the 16.8 kb PCDHA delCNV in the European-American population. TagSNPs for the 13.6kb delCNV were not found due to its rarity.The reference panel from the 1000 Genomes Project (1KG) phase 3 ,20 was ary 5000) . SNPs inn = 49) in the European-American subjects. The definition of eGenes in GTEx database refer to genes with at least one SNP in cis that is significantly associated with expression differences in that gene with a false discovery rate (FDR) of <0.05 [Data files were directly downloaded from the GTEx v8 portal ,23 contaPcdha cluster were combined to generate the expression of the entire Pcdha cluster. Downstream analyses were performed as described previously [Publicly available raw single cell RNA sequencing (scRNAseq) data of the early mouse embryonic hearts from E8.5 to E10.5  were doweviously .The flow diagram of bioinformatic analyses involved in this study was found in MYH6 rare damaging variants in LVOTO cohorts was statistically assessed using Fisher’s exact test, and odds ratio (OR) with 95% confidence interval (CI) was calculated. p values < 0.00714 (Bonferroni corrected) were considered statistically significant.Statistical analyses were performed using the R package. Gene-based burden analysis that test the excess of MYH6 with CoA and BAV in the Icelandic population [MYH6 variants in the whole exome sequencing data of a cohort of 166 white patients recruited in Pittsburgh with CoA, BAV, and other related LVOTO CHD. Eleven rare potentially pathogenic MYH6 variants were recovered in 10 LVOTO patients in the Pittsburgh LVOTO cohort , 20.47 for BAV/CoA (p = 7.43 × 10−5), and 8.20 for all LVOTO cases combined (p = 9.12 × 10−7)  was recovered in a patient with CoA and BAV (CoA/BAV)  . While cnchanged  Given thith HLHS . We noteCoA/BAV) . This pavariants A. OveralPCDHA delCNVs with LVOTO, including CoA and BAV [MYH6 Arg721Trp mutation was observed in 20% of CoA cases in the Icelandic cohort [MYH6 variants accounted for only 10.9% of CoA cases, while PCDHA delCNV was found in 20% of CoA cases, and together they accounted for over 30% of all CoA cases  linked to the delCNV in a wide range of human tissues, including the heart  [PCDHA delCNV [PCDHA delCNV spanning PCDHA8-10 (r2 = 1) in 1KG non-Finnish European-American population (n = 751), indicating their utility for genotyping subjects for the 16.8 kb PCDHA delCNV. Three of the tagSNPs, which are intron variants in PCDHA8-10, were among the top 5 cis-eQTLs found, while tagSNP rs115070123 was absent in GTEx. The 13.6 kb PCDHA delCNV was not examined as no tagSNPs were available for this delCNV, likely due to its much lower prevalence.To assess the impact of the tricles) . As GTExA delCNV . These fPCDHA expression, including atria tissue of the heart (PCDHA10 (slope = −0.593 and p = 3.8 × 10−8). This is observed for atrial tissue (PCHDA8-10, the three PCDHA genes in the 16.8 kb PCDHA delCNV interval, while genes flanking either side, PCHDA5-7 and 11–12, showed elevated expression, indicating possible compensatory increase in expression (Pcdha knockout mice that similarly showed increased expression of Pcdha genes flanking either side of deletions in the Pcdha gene cluster [PCDHA gene cluster in the human heart is atrial specific, with no expression detected in the ventricle.Examination of the GTEX database for the three tagSNPs showed they had a strong negative effect on he heart . Using tl tissue A,B, and l tissue A,B. Exampression C. These  cluster . OverallMYH6 variants with CoA and BAV previously reported in the Icelandic population, confirming the role of rare pathogenic MYH6 variants in malformation of the aorta and aortic valve. While we did not recover the Icelandic founder mutation p.Arg721Trp, another missense mutation was recovered in the same amino acid residue, supporting its functional importance. In contrast to 20% of CoA cases being explained by the MYH6 variant in the Icelandic population, only 10.9% of CoA cases in the Pittsburgh cohort were associated with rare MYH6 variants. However, rare MYH6 variants together with the common PCDHA delCNVs were associated with nearly a third of CoA cases in the Pittsburgh cohort.Our findings replicated the significant association of MYH6 and PCDHA are both expressed predominantly in the atria. This is supported by the eQTL analysis, which showed association of the 16.8 kb PCDHA delCNV with reduced expression of PCDHA10, gene in the deletion interval, in human atrial tissue. We note mice with mutation in Pcdha9, the mouse ortholog of human PCDHA10, can exhibit BAV [MYH6, a sarcomeric heavy chain myosin, or PCDHA mediating cell-cell adhesion, can cause CoA via expression in the atria. Thus, each gene is likely to have importance for force generation in the atria, rather than ventricle as suggested by the current ventricular hemodynamic model of CoA (Our in silico analysis of transcriptomic data from the human and mouse embryonic heart showed ibit BAV . Thus, ml of CoA A.It is likely that deficiency of either gene in the early embryonic heart may cause decreased atrial contractility with reduced blood flow that can lead to CoA B. This aMYH6 variants and common PCDHA delCNVs together accounting for nearly a third of CoA cases. Given atrial dominance of the human and mouse embryonic heart and with MYH6 and PCDHA both being predominantly atrial specific, this suggests the genetic etiology of CoA may be further elucidated by examining other genes regulating atrial contractility in the embryonic heart.In summary, we showed that common and rare variants both contribute to the genetic etiology of CoA, with rare MYH6 variants in CoA and other LVOTO CHD. This may explain the failure to detect significant association of MYH6 variants with HLHS. The small sample size also may account for the paucity of CoA cases with co-occurrence of rare MYH6 variants with the common PCDHA delCNVs.A major limitation in this study is the relatively small cohort size, which could have underestimated the contribution of rare


Interest in exploiting allosteric sites for the development of new therapeutics has grown considerably over the last two decades. The chief driving force behind the interest in allostery for drug discovery stems from the fact that in comparison to orthosteric sites, allosteric sites are less conserved across a protein family, thereby offering greater opportunity for selectivity and ultimately tolerability. While there is significant overlap between structure-based drug design for orthosteric and allosteric sites, allosteric sites offer additional challenges mostly involving the need to better understand protein flexibility and its relationship to protein function. Here we examine the extent to which structure-based drug design is impacting allosteric drug design by highlighting several targets across a variety of target classes. The majority of naturally occurring biological processes exploit a variety of types of allosteric regulation. This regulation originates in response to various stimuli such as post-translational modifications, point mutations, molecular interactions, and other environmental factors, which ultimately control the specific function of a protein . Knowledvia protein scaffolding evolved gradually. Also, a gradual increase in the rate of enzymatic function was observed along the evolution of the regulatory partner TPX2. This study also explained that the source of allosteric control is encoded within the kinase rather than in the activation partner with an example of equally observed activation of Aurora A by INCENP, the activation partner of a close homolog Aurora B. The authors ruled out the coevolution of Aurora A and TPX2 by observing an experimentally similar allosteric activation of Aurora A using a generic synthetic substrate and by the fact that none of the TPX2 forms allosterically regulated the precanonical ancestral Aurora A forms even at very high concentrations. Specifically, for proteins controlled by phosphorylation, In recent groundbreaking work, The traditional definition of allostery involves bidirectional communication from a functional site of a protein to a secondary site through conformational changes of the protein. More recently, it has come to also include sites involved in other protein regulation though not necessarily conformationally linked to the orthosteric site. Here we adopt the functional definition proposed by via allosteric mechanisms , MD simulation, normal mode analysis, elastic network, computational analysis, computational modeling, docking and .1 years . Of the Lastly, we quantified the interest in allosteric sites by protein family. To do this we filtered the PAS with standard keywords for several target families. The counts of abstracts from the PAS for the five most common target families in the PAS, kinases (keyword: “kinase”), GPCRs (keywords: “GPCR” and “G-protein coupled receptor”), proteases (keywords: “protease” and “peptidase”), phosphatases (keyword: “phosphatase”), and transcription factors (keyword: “transcription factor”), are shown in The growth in allostery in Pubmed described above is mirrored in a number of different areas relevant to drug discovery. In particular, the allosteric structural database  has captDue to the increase in interest discussed above and the challenging computational problems, allostery is a new frontier for the development of computational tools. Accordingly, there has been intense effort to develop computational tools to understand allostery . The purBy avoiding the structurally conserved ATP binding site, allosteric kinase inhibitors have the potential to be highly selective and therefore can be excellent candidates for kinase drug discovery. In large part, this explains why the kinase family appears most frequently in the context of the PAS and in allosteric structures in the PDB.). The type III allosteric pocket , binimetinib, selumetinib (IC50 = .008 µM), and cobimetinib (IC50 = .0009 µM) have been approved by the FDA (MEK1 is a member of the mitogen-activated protein kinase (MAPK) pathway involved in many key cellular processes including proliferation and differentiation, stress response, and cell death . MEK1 an the FDA  and more the FDA .It has been nearly 25 years since the first type III allosteric MEK inhibitor, PD098059, was identified . This coA comparison of the co-crystal structure of the ATP analog, AMP-PNP (adenylyl imidodiphosphate) and to that of type-III allosteric inhibitors such as the approved drug cobimetinib shows that MEK1 adopts an inactive/locked/unphosphorylated conformation in which its αC-helix is shifted outward from its actual position . This αCIn addition to MEK1, type III allosteric inhibitors have been identified for other well-characterized kinases including AKT, EGFR, WNK (With-No-Lysine) and TRK (Tropomyosin receptor kinase) . The repapo crystal structure (6cn9)   shows noe (6cn9) . The pube (6cn9) ) reveale50 = .042 µM, Unfortunately, the advancement of WNK463 as a potential therapeutic was withdrawn due to its poor safety profile . In gene50 values of 27 µM  and 7 (Kd = 65 µM), led to MEK1 cocrystal structures showing that these fragments bind to the same allosteric site as other type III allosteric inhibitors. Further substructure searches for close analogs of example 6 (Kd = 70 µM) led to example 8 (Kd = 28 µM). In addition, analogs of example 4 (Kd = 45 µM) yielded the most potent molecule example 9 (Kd = 0.03 µM), which also showed good bioavailability. High resolution co-crystal structures of both examples 8 and 9 revealed that the methoxy-phenyl group of example 9 and the iodo-phenyl group of example 8 occupy the same position as the 2,4-dihalogen group of cobimetinib  and absence (3zls) of the approved type-III allosteric inhibitor drug cobimetinib  Zhao et. They obMore recently, Fleischmann and coworkers developed a biosensor platform called KinCon to track different kinase conformational states in real time . They apProtein tyrosine phosphatases balance the role of tyrosine kinases by catalyzing the removal of the phosphate group from previously phosphorylated tyrosine residues. Because phosphorylation plays a significant role in virtually every intracellular pathway, they are an important drug target class for many indications . While sAllosteric inhibitors have been extensively pursued for two of the family members: SHP2 and PTP1B. Despite having structurally similar phosphatase domains, the modes of allosteric inhibition of these two targets are very different . The knovia molecular dynamics as early as 2006  (vice versa. They found that the S-loop (residues 198–209) transmits the signal to a3 and finally to a7. They subsequently found that the presence of a bound allosteric inhibitor decreased the flexibility of both the S-loop and the WPD loop.Allosteric PTP1B inhibitors were first reported in 2004 . The mec as 2006  and many as 2006 . One of  as 2006  (residue286–298)  to neigh286–298) . For exaWhile subsequent experimental work is generally consistent with this model, more details have been revealed in the last decade that warrant more detailed molecular dynamics studies. For example, in separate NMR studies It is worth noting that the only other tyrosine phosphatase with the equivalent of a7 is the T-cell protein-tyrosine phosphatase (TCPTP), which may explain the selectivity of this class of inhibitors across the phosphatase family. Recently, it has been shown that TCPTP also depends on the stable binding of a7 to achieve its maximal catalytic efficiency . A seconThe biggest challenge with optimizing current PTP1B allosteric inhibitors has been achieving the necessary potency. Indeed, despite extensive effort, breaking the 1 µM barrier with these compounds has been difficult. This raises an important question for PTP1B which is likely be common to many allosteric campaigns: which allosteric sites are amenable to drug-like potency along with the necessary allosteric effects on the orthosteric site? Thus, PTP1B provides a good challenge for druggability assessment at both the orthosteric and allosteric sites.Bakan and coworkers use PTP1B along with several other proteins to describe a simulation based approach to druggability assessment . BrieflyFor PTP1B, their calculations yielded a maximal affinity of .0003–.0009 μM at the orthosteric site. Because the catalytic site has evolved in large part to recognize a phosphate, the acetate probe is the dominant probe, as expected. In comparison, the calculated maximal affinity at the allosteric site is in the range of 9–18 µM. Because the allosteric site is largely hydrophobic, isopropanol was the dominant fragment. Further, the allosteric site of PTP1B had the lowest calculated maximal affinity of all the proteins/sites studied: for the other proteins considered, MDM2, LFA, EG5, and p38, the next weakest maximal affinity, .047 µM, is for a 3rd site on EG5. It is noteworthy that many of the sites in the other proteins used in this study were also allosteric. Thus, this approach predicts that gaining sufficient affinity at the PTP1B allosteric site is difficult with the caveat that the starting structure for the PTP1B simulation did not have a7 and leaves open the possibility of a compound cooperatively interacting with a7 rather than displacing it showing greater potency.Surprisingly, their approach highlighted two additional sites on PTP1B that are predicted to have greater maximal affinities than the known allosteric site: the IRK (insulin receptor kinase) interaction site  are a family of transcription factors consisting of 48 members. They are central players in many physiological processes and have been the targets for numerous drug discovery efforts and ultimately approved drugs . Most ofBecause NHRs directly interact with numerous partners that regulate their function, they offer the potential to find modulators outside their traditional orthosteric site . Indeed,Consistent with the fact that RORg shows considerable basal activity, an observation from the many co-crystal structures of RORg is that helix 12 is stabilized in the active conformation even without a bound agonist. Through an analysis of RORg crystal structures, Li and coworkers  identifiapo , agonispo (5vb3 ), inverspo (5vb3 ) and allpo (5vb3 ). Their apo simulation compared to only <.1% of the time for the agonist bound structure. This is consistent with the observation that RORg has basal activity but increased activity in the presence of agonist. They further elucidate the significance of the HYF triad by comparing simulations with examples 13 and 14 which are matched agonist/antagonist pairs  low pH-induced conformational changes in the propeptide partially exposes the catalytic site of a procathepsin B, which is a unimolecular event and ii) two procathepsin B molecules with partially exposed catalytic sites come in proximity and cleave the other propeptide.The human cathepsin family comprises 11 members of which the majority are endopeptidases : cathepsin B and X are also carboxypeptidases, H is also an aminopeptidase, and C is a dipeptidyl peptidase . All cat92. In fact, chondroitin sulfate (CS) and other GAGs allosterically bind cathepsin K with different affinities and strongly couple to its collagenase activity  have also been shown to promote the activation of cathepsins even at pH 6.5activity . GAG conactivity . In addiactivity .Cathepsin K is considered a promising target for the treatment of osteoporosis due to its key role in bone turnover and remodeling. Due to this function, pharmaceutical companies have worked on the development of cathepsin K inhibitors, but none of these inhibitors have been approved by the FDA to date . Among tvia allosteric sites by GAGs. Finally, their ectosteric sites specifically control its collagenase activity without affecting the catalytic site. Novinec and coworkers contributed significantly to the identification and understanding of allosteric sites in cathepsins employing both experimental and computational approaches. They discovered the first small molecule collagenase inhibitor, example 19  . They usDynaMAD algorithm combining mini-fingerprint searching and compound mapping methods to identify two selective inhibitors for cathepsin K.All other virtual screening efforts have focused on catalytic site binders including covalent binders for cathepsin K. Wang et al. and Ravikumar et al. have performed a virtual screen combining a ligand-based pharmacophore model and molecular docking for the catalytic site . Schrödei values of .098 µM and 84 μM, respectively (50 = .8 µM) with no cytotoxicity  are a family of enzymes that transfer the methyl group from S-adenosylmethionine (SAM) to one or two of the guanidine nitrogens of an arginine residue of a substrate protein. PRMTs participate in several cellular processes, including phase separation, DNA damage repair, transcriptional regulation, and RNA metabolism. Hence, the PRMTs have a profound effect on human diseases such as cancer and cardiovascular diseases . In part50 = 2.5 µM) binds at the dimer interface site which is distinct from both the SAM and substrate peptide binding pockets . These compounds proved to be selective against 31 other methytransferases and ∼250 non-epigenetic targets. They successfully solved the co-crystal structure of example 23 with PRMT3 (4RYL)   which sh3 (4RYL) . It occu3 (4RYL) .Collective studies have suggested that the conserved α-helix across the class I PRMTs is critical for catalytic activity. The authors claimed that compound binding at the interface pocket induces a conformational stabilization at the N-terminus of the conserved α-helix by flipping the sidechain of R396 from its interaction with E422 out of the pocket, thereby preventing proper positioning of the substrate in a catalytically competent conformation . Intrigu50 of .016 µM in a biochemical methylation assay with a very slow on-rate of ka∼1,000 M−1 s−1 in binding kinetics  of PRMT6 by screening a diverse library of 5,000 compounds (50 > 100 μM). The cocrystal structures of PRMT6 with example 26 (6W6D) and example 25 (5WCF) revealed that the allosteric inhibitors are bound to a similar site as the allosteric pocket of PRMT5, described above, positioned inside the β-barrel domain and flexible loop  form those that lead to drug-like affinity. This challenge is exemplified with the effort on the PTP1b allosteric site near α7. Despite significant effort, no inhibitor at this site has broken the 1 µM barrier. In cases such as this, identifying when and where deeper sub-pockets can be induced will be critical. A related challenge is to identify cryptic pockets that are not at all apparent without a bound ligand.A third important challenge unique to allosteric sites is identifying those sites that are functionally relevant. Several methods, such as molecular dynamics, normal mode analysis and elastic network models, have been developed to understand the conformational coupling between two sites. The PTP1B and RORg examples demonstrate that some progress has been made in this area. As more examples emerge, new tools are developed and large independent assessments are made of available methods, they may become more commonly used prospectively.via conformational coupling with the orthosteric site. For example, allosteric sites can be functionally relevant because they block a necessary scaffolding interaction as discussed in cathepsin K or preventing a post-translational modification. Thus, a fourth challenge is to identify these types of sites. Identifying these sites will necessarily involve a larger view of the protein in the context of its physiological environment.Not all allosteric sites, however, work Addressing these problems computationally will be challenging. To fully do so will require a view beyond the 3D structure of an isolated target of interest. Fortunately, there is a vast amount of publicly available information to address these problems. This data includes sequence data from hundreds of genomes, disease relevant mutational data derived by comparing diseased to normal tissues, data on cell wide post translational modifications under a variety of stimulations, hundreds of thousands of crystal structures of proteins, and large easily accessible datasets of small molecule modulators for many protein families. Only by integrating all this data can we hope to develop the holistic view of a protein and its family to select the best strategy to target a protein allosterically, the best molecules to interrogate the allosteric site and the best assays to identify the right modulators.


Habitability of exoplanet’s deepest oceans could be limited by the presence of high-pressure ices at their base. New work demonstrates that efficient chemical transport within deep planetary ice mantles is possible through significant salt incorporation within the high-pressure ice. The largest amount of liquid water in our universe by volume might be found in the gigantic oceans of water-rich exoplanets and icy moons. These vast ocean worlds might host some of the most promising environments for extra-terrestrial life to emerge and prosper. Nonetheless, the incredible pressures found within the water-rich layers will freeze the oceans from the bottom into dense high-pressure (HP) ices, separating the liquid layer from the nutrient-rich rocky interior  when compared to these worlds that may contain up to several 10 s wt% of H2O. This would result in up to thousands of kilometers thick water-rich envelopes, called “hydrospheres”, covering a rocky-metal core  and mini-Neptunes (1.8–4·REarth radius). Mini-Neptune’s water-rich fluid envelopes are expected to be at several thousands of Kelvin, making them less relevant for astrobiology. Ocean exoplanets, such as the Trappist 1 e, f, g and h planets9, are prime targets for habitability within their temperate oceans, possibly in equilibrium with their atmosphere, allowing for potential characterization and biosignature detection by upcoming ground and space based observatories10. The icy worlds of the outer solar systems , scaled-down versions of ocean exoplanets, also host large liquid water oceans below their frozen surfaces. Interest in the geophysical and astrobiological attributes of these icy moons has motivated the most ambitious planetary exploration missions of the upcoming decade with NASA’s Europa Clipper11 and Dragonfly12, and ESA’s JUICE13. Furthermore, the search for biosignatures and habitable environments on exoplanets is one of the four main science goals of the newly launched James Webb Space Telescope6.The growing catalog of exoplanets has revealed the widespread existence of a type of planet unknown in our solar system: ocean worlds. These planets, first suggested by ref. y dry <0. wt% H2O 6 , the pressure within the hydrosphere of such planets can form dense HP water ices , stable below the liquid ocean6 Fig. . The pre16. This remains to be studied for thicker exoplanet hydrospheres, but efficient liquid formation at the base of the HP mantle seems to be the rule rather than the exception17. Secondly, chemical physics advocates for efficient chemical transport through HP ices mantle.This assumption is actively challenged by experimental and theoretical studies of icy mantle dynamics and HP ices chemical physics. Both suggest efficient transport of solutes from the rocky core to the upper ocean may be possible. First, dynamically, it has been demonstrated that efficient convection can happen within HP ice mantles in large icy moons like Titan and Ganymede, involving transport of liquid pockets from the hydrosphere-rock boundary to the ocean20. These new type of crystalline solids in salt-water systems are also called “solid solutions” as the amount of salt is not stoichiometrically fixed, like with common types of salt hydrates such as hydrohalite (NaCl•2H2O).To understand the importance of this new study by Hernandez et al., one needs to re-examine a pillar of undergraduate physical chemistry teachings: “ice grows chemically pure from a saline solution by expelling all the salt ions into the liquid”. This is generally true for low pressure common hexagonal ice (ice Ih). However, one remarkable discovery of ice physics of the last decade is that HP water ice with cubic crystalline structure above 20,000 atmospheres (phase VII and X) can incorporate significant amounts of simple ionic salts  up to several weight %, while maintaining the same a crystalline structure21. Furthermore the presence of ions in the lattice extend to higher pressure the stability of ice VII (molecular ice with H2O molecule linked by H-bonding) at the expense of ice X (higher-pressure ionic ice with hydrogens in an symmetric position between oxygens)23. Controversy remained regarding the maximum amount of salt soluble24, as well as the mechanism of salt ions inclusion being either interstitial (between H2O molecules) or substitutional (replacing H2O molecules). Furthermore, there is still uncertainty about solute incorporation effects on the lattice volume and density of salty ice. Experimental work by ref. 18 reported a reduction of the lattice volume, however, other recent experimental and theoretical work reported an increase22. How much salt can be incorporated and how it could either reduce or increase the volume of HP ice (and affect its density), has crucial implications for our ability to describe the interior structure and dynamics of ocean exoplanets. Too high a density increase due to high salt solubility, or possible immiscibility within the HP mantle would inhibit convection and solute vertical transport20. Finally, limited data exists above 300 K on incorporation into HP ices, conditions found in exoplanets. To determine if solute transport is possible through planetary HP ice mantle, one needs to know, over the relevant range of pressure and temperatures, the solute solubility and its effect on density. Until now, we were lacking such a unified physical model and thermodynamic representation.Several groups have reported intriguing properties of these salt-bearing ices. Salt incorporation distorts the HP ice crystalline lattice by breaking some of the hydrogen bound network8 provide a comprehensive study of NaCl incorporation in HP ice using state-of-the-art molecular dynamics simulations (first principle DFT-MD) over a wide range of conditions. By simulating crystallization of salty solutions, this extensive study binds together within a self-consistent physical model, several observations from previous studies more limited in scope and at lower temperatures. They report up to 2.5 wt% NaCl incorporation on substitutional sites that results in an increased lattice volume of salty HP ice. The study also provides a thermodynamic representation (equation of state) for salty HP ices that allows the authors to show that convective chemical transport through the dense salty HP ices is possible. Hernandez et al.8 demonstrate that the density increase due to NaCl incorporation does not hinder vertical transport through solid-state convection. Along with previous studies on convection of HP ice and melt vertical transport in mantles in smaller icy moons16, this substantiates the notion that HP ice mantles do not act as chemical barriers between the rocky core and the liquid water ocean. The authors also suggest solute recycling mechanism with diffuse return flow of salty ice across the icy mantle-ocean boundary due to crystallization of salty ice , or phosphates (PO43−). Secondly, it has been shown that salt solubility can decrease by an order of magnitude in lower HP ice, like ice VI20 expected in thick hydrospheres6, or at high temperatures24. Due to these transitions, solute-rich ice could release its solutes within the HP ice mantle, forming a deep reservoir  will directly test some of these conclusions by looking at the geophysical and surface signatures of the ocean composition and estimate the permeability of HP ice mantles (Fig. The study by Hernandez et al.les Fig. . These m