De novo GTP synthesis is a metabolic vulnerability for the interception of brain metastases.

Patients with brain metastases (BM) face a 90% mortality rate within one year of diagnosis and the current standard of care is palliative. Targeting BM-initiating cells (BMICs) is a feasible strategy to treat BM, but druggable targets are limited. Here, we apply Connectivity Map analysis to lung-, breast-, and melanoma-pre-metastatic BMIC gene expression signatures and identify inosine monophosphate dehydrogenase (IMPDH), the rate-limiting enzyme in the de novo GTP synthesis pathway, as a target for BM. We show that pharmacological and genetic perturbation of IMPDH attenuates BMIC proliferation in vitro and the formation of BM in vivo. Metabolomic analyses and CRISPR knockout studies confirm that de novo GTP synthesis is a potent metabolic vulnerability in BM. Overall, our work employs a phenotype-guided therapeutic strategy to uncover IMPDH as a relevant target for attenuating BM outgrowth, which may provide an alternative treatment strategy for patients who are otherwise limited to palliation.

A molten globule ensemble primes Arf1-GDP for the nucleotide switch.

The adenosine di-phosphate (ADP) ribosylation factor (Arf) small guanosine tri-phosphate (GTP)ases function as molecular switches to activate signaling cascades that control membrane organization in eukaryotic cells. In Arf1, the GDP/GTP switch does not occur spontaneously but requires guanine nucleotide exchange factors (GEFs) and membranes. Exchange involves massive conformational changes, including disruption of the core ß-sheet. The mechanisms by which this energetically costly switch occurs remain to be elucidated. To probe the switch mechanism, we coupled pressure perturbation with nuclear magnetic resonance (NMR), Fourier Transform infra-red spectroscopy (FTIR), small-angle X-ray scattering (SAXS), fluorescence, and computation. Pressure induced the formation of a classical molten globule (MG) ensemble. Pressure also favored the GDP to GTP transition, providing strong support for the notion that the MG ensemble plays a functional role in the nucleotide switch. We propose that the MG ensemble allows for switching without the requirement for complete unfolding and may be recognized by GEFs. An MG-based switching mechanism could constitute a pervasive feature in Arfs and Arf-like GTPases, and more generally, the evolutionarily related (Ras-like small GTPases) Rags and Gα GTPases.

Capturing RAS oligomerization on a membrane.

RAS GTPases associate with the biological membrane where they function as molecular switches to regulate cell growth. Recent studies indicate that RAS proteins oligomerize on membranes, and disrupting these assemblies represents an alternative therapeutic strategy. However, conflicting reports on RAS assemblies, ranging in size from dimers to nanoclusters, have brought to the fore key questions regarding the stoichiometry and parameters that influence oligomerization. Here, we probe three isoforms of RAS [Kirsten Rat Sarcoma viral oncogene (KRAS), Harvey Rat Sarcoma viral oncogene (HRAS), and Neuroblastoma oncogene (NRAS)] directly from membranes using mass spectrometry. We show that KRAS on membranes in the inactive state (GDP-bound) is monomeric but forms dimers in the active state (GTP-bound). We demonstrate that the small molecule BI2852 can induce dimerization of KRAS, whereas the binding of effector proteins disrupts dimerization. We also show that RAS dimerization is dependent on lipid composition and reveal that oligomerization of NRAS is regulated by palmitoylation. By monitoring the intrinsic GTPase activity of RAS, we capture the emergence of a dimer containing either mixed nucleotides or GDP on membranes. We find that the interaction of RAS with the catalytic domain of Son of Sevenless (SOScat) is influenced by membrane composition. We also capture the activation and monomer to dimer conversion of KRAS by SOScat. These results not only reveal the stoichiometry of RAS assemblies on membranes but also uncover the impact of critical factors on oligomerization, encompassing regulation by nucleotides, lipids, and palmitoylation.

Insight of the molecular mechanism of inhibitors located at different allosteric sites regulating the activity of wild type and mutant KRAS (G12).

As the important hub of many cellular signaling networks, KRAS (Kirsten rat sarcoma viral oncogene homologue) has been identified as a tumor biomarker. It is the frequently mutated oncogene in human cancers, and KRAS protein activation caused by mutations, such as G12D, has been found in many human tumors tissues. Although, there are two specific allosteric sites (AS1 and AS2) on the KRAS protein that can be used as the targets for inhibitor development, the difference of regulatory mechanisms between two individual allosteric sites still not be reported. Here, using molecular dynamics simulations combined with molecular mechanics generalized born surface area (MM/GBSA) analysis, we found that both of the inhibitors, located at AS1 and AS2, were able to reduce the binding free energy between wild type, mutant KRAS (G12/D/V/S/C) and GTP remarkably, however the effect of inhibitors on the binding free energy between wild type, mutant KRAS and GDP was limited. In addition, the degree of decrease of binding free energy between KRAS and GTP caused by inhibitors at AS2 was significantly greater than that caused by inhibitors at AS1. Further analysis revealed that both inhibitors at AS1 and AS2 were able to regulate the fluctuation of Switch Ⅰ and Switch Ⅱ to expand the pocket of the orthosteric site (GTP binding site), thereby reducing the binding of KRAS to GTP. Noteworthy there was significant differences in the regulatory preferences on Switch Ⅰ and Switch Ⅱ between two type inhibitor. The inhibitor at AS2 mainly regulated Switch Ⅱ to affect the pocket of the orthosteric site, while the inhibitor at AS1 mainly expand the pocket of the orthosteric site by regulating the fluctuation of Switch Ⅰ. Our study compared the differences between two type inhibitors in regulating the KRAS protein activity and revealed the advantages of the AS2 as the small molecule drug target, aiming to provide theoretical guidance for the research of novel KRAS protein inhibitors.

Deciphering the allosteric regulation of mycobacterial inosine-5'-monophosphate dehydrogenase.

Allosteric regulation of inosine 5'-monophosphate dehydrogenase (IMPDH), an essential enzyme of purine metabolism, contributes to the homeostasis of adenine and guanine nucleotides. However, the precise molecular mechanism of IMPDH regulation in bacteria remains unclear. Using biochemical and cryo-EM approaches, we reveal the intricate molecular mechanism of the IMPDH allosteric regulation in mycobacteria. The enzyme is inhibited by both GTP and (p)ppGpp, which bind to the regulatory CBS domains and, via interactions with basic residues in hinge regions, lock the catalytic core domains in a compressed conformation. This results in occlusion of inosine monophosphate (IMP) substrate binding to the active site and, ultimately, inhibition of the enzyme. The GTP and (p)ppGpp allosteric effectors bind to their dedicated sites but stabilize the compressed octamer by a common mechanism. Inhibition is relieved by the competitive displacement of GTP or (p)ppGpp by ATP allowing IMP-induced enzyme expansion. The structural knowledge and mechanistic understanding presented here open up new possibilities for the development of allosteric inhibitors with antibacterial potential.

RAS-ON inhibition overcomes clinical resistance to KRAS G12C-OFF covalent blockade.

Selective KRASG12C inhibitors have been developed to covalently lock the oncogene in the inactive GDP-bound state. Two of these molecules, sotorasib and adagrasib, are approved for the treatment of adult patients with KRASG12C-mutated previously treated advanced non-small cell lung cancer. Drug treatment imposes selective pressures leading to the outgrowth of drug-resistant variants. Mass sequencing from patients' biopsies identified a number of acquired KRAS mutations -both in cis and in trans- in resistant tumors. We demonstrate here that disease progression in vivo can also occur due to adaptive mechanisms and increased KRAS-GTP loading. Using the preclinical tool tri-complex KRASG12C-selective covalent inhibitor, RMC-4998 (also known as RM-029), that targets the active GTP-bound (ON) state of the oncogene, we provide a proof-of-concept that the clinical stage KRASG12C(ON) inhibitor RMC-6291 alone or in combination with KRASG12C(OFF) drugs can be an alternative potential therapeutic strategy to circumvent resistance due to increased KRAS-GTP loading.

Tumor-derived RHOA mutants interact with effectors in the GDP-bound state.

RHOA mutations are found at diverse residues in various cancer types, implying mutation- and cell-specific mechanisms of tumorigenesis. Here, we focus on the underlying mechanisms of two gain-of-function RHOA mutations, A161P and A161V, identified in adult T-cell leukemia/lymphoma. We find that RHOAA161P and RHOAA161V are both fast-cycling mutants with increased guanine nucleotide dissociation/association rates compared with RHOAWT and show reduced GTP-hydrolysis activity. Crystal structures reveal an altered nucleotide association in RHOAA161P and an open nucleotide pocket in RHOAA161V. Both mutations perturb the dynamic properties of RHOA switch regions and shift the conformational landscape important for RHOA activity, as shown by 31P NMR and molecular dynamics simulations. Interestingly, RHOAA161P and RHOAA161V can interact with effectors in the GDP-bound state. 1H-15N HSQC NMR spectra support the existence of an active population in RHOAA161V-GDP. The distinct interaction mechanisms resulting from the mutations likely favor an RHOAWT-like "ON" conformation, endowing GDP-bound state effector binding activity.

Probing the Electrostatic Effects of H-Ras Tyrosine 32 Mutations on Intrinsic GTP Hydrolysis Using Vibrational Stark Effect Spectroscopy of a Thiocyanate Probe.

The wildtype H-Ras protein functions as a molecular switch in a variety of cell signaling pathways, and mutations to key residues result in a constitutively active oncoprotein. However, there is some debate regarding the mechanism of the intrinsic GTPase activity of H-Ras. It has been hypothesized that ordered water molecules are coordinated at the active site by Q61, a highly transforming amino acid site, and Y32, a position that has not previously been investigated. Here, we examine the electrostatic contribution of the Y32 position to GTP hydrolysis by comparing the rate of GTP hydrolysis of Y32X mutants to the vibrational energy shift of each mutation measured by a nearby thiocyanate vibrational probe to estimate changes in the electrostatic environment caused by changes at the Y32 position. We further compared vibrational energy shifts for each mutation to the hydration potential of the respective side chain and demonstrated that Y32 is less critical for recruiting water molecules into the active site to promote hydrolysis than Q61. Our results show a clear interplay between a steric contribution from Y32 and an electrostatic contribution from Q61 that are both critical for intrinsic GTP hydrolysis.

Endopeptidase activities of Clostridium botulinum toxins in the development of this bacterium.

By-products like CO2 and organic acids, produced during Clostridium botulinum growth, appear to inhibit its development and reduce ATP production. A decrease in ATP production creates an imbalance in the ATP/GTP ratio. GTP activates CodY, which regulates BoNT expression. This toxin is released into the extracellular medium. Its light chains act as a specific endopeptidase, targeting SNARE proteins. The specific amino acids released enter the cells and are metabolized by the Stickland reaction, resulting in the synthesis of ATP. This ATP might then be used by histidine kinases to activate Spo0A, the main regulator initiating sporulation, through phosphorylation.

Lower ratio of IMPDH1 to IMPDH2 sensitizes gliomas to chemotherapy.

Gliomas are the most common primary tumors of the central nervous system, with approximately half of patients presenting with the most aggressive form of glioblastoma. Although several molecular markers for glioma have been identified, they are not sufficient to predict the prognosis due to the extensive genetic heterogeneity within glioma. Our study reveals that the ratio of IMPDH1 to IMPDH2 expression levels serves as a molecular indicator for glioma treatment prognosis. Patients with a higher IMPDH1/IMPDH2 ratio exhibit a worse prognosis, while those with a lower ratio display a more favorable prognosis. We further demonstrate that IMPDH1 plays a crucial role in maintaining cellular GTP/GDP levels following DNA damage compared to IMPDH2. In the absence of IMPDH1, cells experience an imbalance in the GTP/GDP ratio, impairing DNA damage repair capabilities and rendering them more sensitive to TMZ. This study not only introduces a novel prognostic indicator for glioma clinical diagnosis but also offers innovative insights for precise and stratified glioma treatment.

D3S-001, a KRAS G12C Inhibitor with Rapid Target Engagement Kinetics, Overcomes Nucleotide Cycling, and Demonstrates Robust Preclinical and Clinical Activities.

First-generation KRAS G12C inhibitors, such as sotorasib and adagrasib, are limited by the depth and duration of clinical responses. One potential explanation for their modest clinical activity is the dynamic "cycling" of KRAS between its guanosine diphosphate (GDP)- and guanosine triphosphate (GTP)-bound states, raising controversy about whether targeting the GDP-bound form can fully block this oncogenic driver. We herein report that D3S-001, a next-generation GDP-bound G12C inhibitor with faster target engagement (TE) kinetics, depletes cellular active KRAS G12C at nanomolar concentrations. In the presence of growth factors, such as epithelial growth factor and hepatocyte growth factor, the ability of sotorasib and adagrasib to inhibit KRAS was compromised whereas the TE kinetics of D3S-001 was nearly unaffected, a unique feature differentiating D3S-001 from other GDP-bound G12C inhibitors. Furthermore, the high covalent potency and cellular TE efficiency of D3S-001 contributed to robust antitumor activity preclinically and translated into promising clinical efficacy in an ongoing phase 1 trial (NCT05410145). Significance: The kinetic study presented in this work unveils, for the first time, that a GDP-bound conformation-selective KRAS G12C inhibitor can potentially deplete cellular active KRAS in the presence of growth factors and offers new insights into the critical features that drive preclinical and clinical efficacy for this class of drugs.

Serine 39 in the GTP-binding domain of Drp1 is involved in shaping mitochondrial morphology.

Continuous fusion and fission are critical for mitochondrial health. In this study, we further characterize the role played by dynamin-related protein 1 (Drp1) in mitochondrial fission. We show that a single amino acid change in Drp1 at position 39 from serine to alanine (S39A) within the GTP-binding (GTPase) domain results in a fused mitochondrial network in human SH-SY5Y neuroblastoma cells. Interestingly, the phosphorylation of Ser-616 and Ser-637 of Drp1 remains unaffected by the S39A mutation, and mitochondrial bioenergetic profile and cell viability in the S39A mutant were comparable to those observed in the control. This leads us to propose that the serine 39 residue of Drp1 plays a crucial role in mitochondrial distribution through its involvement in the GTPase activity. Furthermore, this amino acid mutation leads to structural anomalies in the mitochondrial network. Taken together, our results contribute to a better understanding of the function of the Drp1 protein.

The IMPDH cytoophidium couples metabolism and fetal development in mice.

The cytoophidium is an evolutionarily conserved subcellular structure formed by filamentous polymers of metabolic enzymes. In vertebrates, inosine monophosphate dehydrogenase (IMPDH), which catalyses the rate-limiting step in guanosine triphosphate (GTP) biosynthesis, is one of the best-known cytoophidium-forming enzymes. Formation of the cytoophidium has been proposed to alleviate the inhibition of IMPDH, thereby facilitating GTP production to support the rapid proliferation of certain cell types such as lymphocytes, cancer cells and pluripotent stem cells (PSCs). However, past studies lacked appropriate models to elucidate the significance of IMPDH cytoophidium under normal physiological conditions. In this study, we demonstrate that the presence of IMPDH cytoophidium in mouse PSCs correlates with their metabolic status rather than pluripotency. By introducing IMPDH2 Y12C point mutation through genome editing, we established mouse embryonic stem cell (ESC) lines incapable of forming IMPDH polymers and the cytoophidium. Our data indicate an important role of IMPDH cytoophidium in sustaining a positive feedback loop that couples nucleotide biosynthesis with upstream metabolic pathways. Additionally, we find that IMPDH2 Y12C mutation leads to decreased cell proliferation and increased DNA damage in teratomas, as well as impaired embryo development following blastocoel injection. Further analysis shows that IMPDH cytoophidium assembly in mouse embryonic development begins after implantation and gradually increases throughout fetal development. These findings provide insights into the regulation of IMPDH polymerisation in embryogenesis and its significance in coordinating cell metabolism and development.

Molecular Mechanism of Phosphorylation-Mediated Impacts on the Conformation Dynamics of GTP-Bound KRAS Probed by GaMD Trajectory-Based Deep Learning.

The phosphorylation of different sites produces a significant effect on the conformational dynamics of KRAS. Gaussian accelerated molecular dynamics (GaMD) simulations were combined with deep learning (DL) to explore the molecular mechanism of the phosphorylation-mediated effect on conformational dynamics of the GTP-bound KRAS. The DL finds that the switch domains are involved in obvious differences in conformation contacts and suggests that the switch domains play a key role in the function of KRAS. The analyses of free energy landscapes (FELs) reveal that the phosphorylation of pY32, pY64, and pY137 leads to more disordered states of the switch domains than the wild-type (WT) KRAS and induces conformational transformations between the closed and open states. The results from principal component analysis (PCA) indicate that principal motions PC1 and PC2 are responsible for the closed and open states of the phosphorylated KRAS. Interaction networks were analyzed and the results verify that the phosphorylation alters interactions of GTP and magnesium ion Mg2+ with the switch domains. It is concluded that the phosphorylation pY32, pY64, and pY137 tune the activity of KRAS through changing conformational dynamics and interactions of the switch domains. We anticipated that this work could provide theoretical aids for deeply understanding the function of KRAS.

Molecular mechanism for recognition of the cargo adapter Rab6GTP by the dynein adapter BicD2.

Rab6 is a key modulator of protein secretion. The dynein adapter Bicaudal D2 (BicD2) recruits the motors cytoplasmic dynein and kinesin-1 to Rab6GTP-positive vesicles for transport; however, it is unknown how BicD2 recognizes Rab6. Here, we establish a structural model for recognition of Rab6GTP by BicD2, using structure prediction and mutagenesis. The binding site of BicD2 spans two regions of Rab6 that undergo structural changes upon the transition from the GDP- to GTP-bound state, and several hydrophobic interface residues are rearranged, explaining the increased affinity of the active GTP-bound state. Mutations of Rab6GTP that abolish binding to BicD2 also result in reduced co-migration of Rab6GTP/BicD2 in cells, validating our model. These mutations also severely diminished the motility of Rab6-positive vesicles in cells, highlighting the importance of the Rab6GTP/BicD2 interaction for overall motility of the multi-motor complex that contains both kinesin-1 and dynein. Our results provide insights into trafficking of secretory and Golgi-derived vesicles and will help devise therapies for diseases caused by BicD2 mutations, which selectively affect the affinity to Rab6 and other cargoes.

Insights into the ATP / GTP selectivity of a GTPase, adenylosuccinate synthetase from Leishmania donovani.

Many GTPases have been shown to utilize ATP too as the phosphoryl donor. Both GTP and ATP are important molecules in the cellular environments and play multiple and discrete functional role within the cells. In our present study, we showed that one of the purine metabolic enzymes Adenylosuccinate synthetase from Leishmania donovani (LdAdSS) which belongs to the BioD-superfamily of GTPases can also carry out the catalysis by hydrolysing ATP instead of its cognate substrate GTP albeit with less efficiency. Biochemical and biophysical studies indicated its ability to bind to ATP too but at a higher concentration of ATP compared to that of GTP. Sequence analysis and molecular dynamic simulations suggested that residues of the switch loop and the G4-G5 (593SAXD596) connected motif of LdAdSS plays a role in determining the nucleotide specificity. Though the crucial interaction between Asp596 and the nucleotide is broken when ATP is bound, interactions between the Ala594 and the adenine ring of ATP could still hold ATP in the GTP binding site. The results of the present study suggested that though LdAdSS is GTP specific, it still shows ATP hydrolysing activity.

Tumour-selective activity of RAS-GTP inhibition in pancreatic cancer.

Broad-spectrum RAS inhibition has the potential to benefit roughly a quarter of human patients with cancer whose tumours are driven by RAS mutations1,2. RMC-7977 is a highly selective inhibitor of the active GTP-bound forms of KRAS, HRAS and NRAS, with affinity for both mutant and wild-type variants3. More than 90% of cases of human pancreatic ductal adenocarcinoma (PDAC) are driven by activating mutations in KRAS4. Here we assessed the therapeutic potential of RMC-7977 in a comprehensive range of PDAC models. We observed broad and pronounced anti-tumour activity across models following direct RAS inhibition at exposures that were well-tolerated in vivo. Pharmacological analyses revealed divergent responses to RMC-7977 in tumour versus normal tissues. Treated tumours exhibited waves of apoptosis along with sustained proliferative arrest, whereas normal tissues underwent only transient decreases in proliferation, with no evidence of apoptosis. In the autochthonous KPC mouse model, RMC-7977 treatment resulted in a profound extension of survival followed by on-treatment relapse. Analysis of relapsed tumours identified Myc copy number gain as a prevalent candidate resistance mechanism, which could be overcome by combinatorial TEAD inhibition in vitro. Together, these data establish a strong preclinical rationale for the use of broad-spectrum RAS-GTP inhibition in the setting of PDAC and identify a promising candidate combination therapeutic regimen to overcome monotherapy resistance.

Crystal structure of NRAS Q61K with a ligand-induced pocket near switch II.

The RAS isoforms (KRAS, HRAS and NRAS) have distinct cancer type-specific profiles. NRAS mutations are the second most prevalent RAS mutations in skin and hematological malignancies. Although RAS proteins were considered undruggable for decades, isoform and mutation-specific investigations have produced successful RAS inhibitors that are either specific to certain mutants, isoforms (pan-KRAS) or target all RAS proteins (pan-RAS). While extensive structural and biochemical investigations have focused mainly on K- and H-RAS mutations, NRAS mutations have received less attention, and the most prevalent NRAS mutations in human cancers, Q61K and Q61R, are rare in K- and H-RAS. This manuscript presents a crystal structure of the NRAS Q61K mutant in the GTP-bound form. Our structure reveals a previously unseen pocket near switch II induced by the binding of a ligand to the active form of the protein. This observation reveals a binding site that can potentially be exploited for development of inhibitors against mutant NRAS. Furthermore, the well-resolved catalytic site of this GTPase bound to native GTP provides insight into the stalled GTP hydrolysis observed for NRAS-Q61K.