2'-Deoxy Guanosine Nucleotides Alter the Biochemical Properties of Ras.

Ras proteins in the mitogen-activated protein kinase (MAPK) signaling pathway represent one of the most frequently mutated oncogenes in cancer. Ras binds guanosine nucleotides and cycles between active (GTP) and inactive (GDP) conformations to regulate the MAPK signaling pathway. Guanosine and other nucleotides exist in cells as either 2'-hydroxy or 2'-deoxy forms, and imbalances in the deoxyribonucleotide triphosphate pool have been associated with different diseases, such as diabetes, obesity, and cancer. However, the biochemical properties of Ras bound to dGNP are not well understood. Herein, we use native mass spectrometry to monitor the intrinsic GTPase activity of H-Ras and N-Ras oncogenic mutants, revealing that the rate of 2'-deoxy guanosine triphosphate (dGTP) hydrolysis differs compared to the hydroxylated form, in some cases by seven-fold. Moreover, K-Ras expressed from HEK293 cells exhibited a higher than anticipated abundance of dGNP, despite the low abundance of dGNP in cells. Additionally, the GTPase and dGTPase activity of K-RasG12C was found to be accelerated by 10.2- and 3.8-fold in the presence of small molecule covalent inhibitors, which may open opportunities for the development of Pan-Ras inhibitors. The molecular assemblies formed between H-Ras and N-Ras, including mutant forms, with the catalytic domain of SOS (SOScat) were also investigated. The results show that the different mutants of H-Ras and N-Ras not only engage SOScat differently, but these assemblies are also dependent on the form of guanosine triphosphate bound to Ras. These findings bring to the forefront a new perspective on the nucleotide-dependent biochemical properties of Ras that may have implications for the activation of the MAPK signaling pathway and Ras-driven cancers.

Temperature Regulates Stability, Ligand Binding (Mg2+ and ATP), and Stoichiometry of GroEL-GroES Complexes.

Chaperonins are nanomachines that harness ATP hydrolysis to power and catalyze protein folding, a chemical action that is directly linked to the maintenance of cell function through protein folding/refolding and assembly. GroEL and the GroEL-GroES complex are archetypal examples of such protein folding machines. Here, variable-temperature electrospray ionization (vT-ESI) native mass spectrometry is used to delineate the effects of solution temperature and ATP concentrations on the stabilities of GroEL and GroEL-GroES complexes. The results show clear evidence for destabilization of both GroEL14 and GroES7 at temperatures of 50 and 45 °C, respectively, substantially below the previously reported melting temperature (Tm ∼ 70 °C). This destabilization is accompanied by temperature-dependent reaction products that have previously unreported stoichiometries, viz. GroEL14-GroESy-ATPn, where y = 1, 2, 8 and n = 0, 1, 2, 8, that are also dependent on Mg2+ and ATP concentrations. Variable-temperature native mass spectrometry reveals new insights about the stability of GroEL in response to temperature effects: (i) temperature-dependent ATP binding to GroEL; (ii) effects of temperature as well as Mg2+ and ATP concentrations on the stoichiometry of the GroEL-GroES complex, with Mg2+ showing greater effects compared to ATP; and (iii) a change in the temperature-dependent stoichiometries of the GroEL-GroES complex (GroEL14-GroES7 vs GroEL14-GroES8) between 24 and 40 °C. The similarities between results obtained by using native MS and cryo-EM [Clare et al. An expanded protein folding cage in the GroEL-gp31 complex. J. Mol. Biol. 2006, 358, 905-911; Ranson et al. Allosteric signaling of ATP hydrolysis in GroEL-GroES complexes.Nat. Struct. Mol. Biol. 2006, 13, 147-152] underscore the utility of native MS for investigations of molecular machines as well as identification of key intermediates involved in the chaperonin-assisted protein folding cycle.

Mechanism-Based Inactivation of Mycobacterium tuberculosis Isocitrate Lyase 1 by (2R,3S)-2-Hydroxy-3-(nitromethyl)succinic acid.

The isocitrate lyase paralogs of Mycobacterium tuberculosis (ICL1 and 2) are essential for mycobacterial persistence and constitute targets for the development of antituberculosis agents. We report that (2R,3S)-2-hydroxy-3-(nitromethyl)succinic acid (5-NIC) undergoes apparent retro-aldol cleavage as catalyzed by ICL1 to produce glyoxylate and 3-nitropropionic acid (3-NP), the latter of which is a covalent-inactivating agent of ICL1. Kinetic analysis of this reaction identified that 5-NIC serves as a robust and efficient mechanism-based inactivator of ICL1 (kinact/KI = (1.3 ± 0.1) × 103 M-1 s-1) with a partition ratio <1. Using enzyme kinetics, mass spectrometry, and X-ray crystallography, we identified that the reaction of the 5-NIC-derived 3-NP with the Cys191 thiolate of ICL1 results in formation of an ICL1-thiohydroxamate adduct as predicted. One aspect of the design of 5-NIC was to lower its overall charge compared to isocitrate to assist with cell permeability. Accordingly, the absence of the third carboxylate group will simplify the synthesis of pro-drug forms of 5-NIC for characterization in cell-infection models of M. tuberculosis.

Self-Masked Aldehyde Inhibitors: A Novel Strategy for Inhibiting Cysteine Proteases.

Cysteine proteases comprise an important class of drug targets, especially for infectious diseases such as Chagas disease (cruzain) and COVID-19 (3CL protease, cathepsin L). Peptide aldehydes have proven to be potent inhibitors for all of these proteases. However, the intrinsic, high electrophilicity of the aldehyde group is associated with safety concerns and metabolic instability, limiting the use of aldehyde inhibitors as drugs. We have developed a novel class of self-masked aldehyde inhibitors (SMAIs) for cruzain, the major cysteine protease of the causative agent of Chagas disease-Trypanosoma cruzi. These SMAIs exerted potent, reversible inhibition of cruzain (Ki* = 18-350 nM) while apparently protecting the free aldehyde in cell-based assays. We synthesized prodrugs of the SMAIs that could potentially improve their pharmacokinetic properties. We also elucidated the kinetic and chemical mechanism of SMAIs and applied this strategy to the design of anti-SARS-CoV-2 inhibitors.

Covalent Inactivation of Mycobacterium tuberculosis Isocitrate Lyase by cis-2,3-Epoxy-Succinic Acid.

The isocitrate lyases (ICL1/2) are essential enzymes of Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis. At present, no ICL1/2 inhibitors have progressed to clinical evaluation, despite extensive drug discovery efforts. Herein, we surveyed succinate analogs against ICL1 and found that dicarboxylic acids constrained in their synperiplanar conformations, such as maleic acid, comprise uncompetitive inhibitors of ICL1 and inhibit more potently than their trans-isomers. From this, we identified cis-2,3 epoxysuccinic acid (cis-EpS) as a selective, irreversible covalent inactivator of Mtb ICL1 (kinact/Kinact= (5.0 ± 1.4) × 104 M-1 s-1; Kinact = 200 ± 50 nM), the most potent inactivator of ICL1 yet characterized. Crystallographic and mass spectrometric analysis demonstrated that Cys191 of ICL1 was S-malylated by cis-EpS, and a crystallographic "snapshot" of inactivation lent insight into the chemical mechanism of this inactivation. Proteomic analysis of E. coli lysates showed that cis-EpS selectively labeled plasmid-expressed Mtb ICL1. Consistently, cis-EpS, but not its trans-isomer, inhibited the growth of Mtb under conditions in which ICL function is essential. These findings encourage the development of analogs of cis-2,3-epoxysuccinate as antituberculosis agents.

Molecular assemblies of the catalytic domain of SOS with KRas and oncogenic mutants.

Ras is regulated by a specific guanine nucleotide exchange factor Son of Sevenless (SOS), which facilitates the exchange of inactive, GDP-bound Ras with GTP. The catalytic activity of SOS is also allosterically modulated by an active Ras (Ras-GTP). However, it remains poorly understood how oncogenic Ras mutants interact with SOS and modulate its activity. Here, native ion mobility-mass spectrometry is employed to monitor the assembly of the catalytic domain of SOS (SOScat) with KRas and three cancer-associated mutants (G12C, G13D, and Q61H), leading to the discovery of different molecular assemblies and distinct conformers of SOScat engaging KRas. We also find KRasG13D exhibits high affinity for SOScat and is a potent allosteric modulator of its activity. A structure of the KRasG13D•SOScat complex was determined using cryogenic electron microscopy providing insight into the enhanced affinity of the mutant protein. In addition, we find that KRasG13D-GTP can allosterically increase the nucleotide exchange rate of KRas at the active site more than twofold compared to KRas-GTP. Furthermore, small-molecule Ras•SOS disruptors fail to dissociate KRasG13D•SOScat complexes, underscoring the need for more potent disruptors. Taken together, a better understanding of the interaction between oncogenic Ras mutants and SOS will provide avenues for improved therapeutic interventions.

Intrinsic GTPase Activity of K-RAS Monitored by Native Mass Spectrometry.

Mutations in RAS are associated with many different cancers and have been a therapeutic target for more than three decades. RAS cycles from an active to inactive state by both intrinsic and GTPase-activating protein (GAP)-stimulated hydrolysis. The activated enzyme interacts with downstream effectors, leading to tumor proliferation. Mutations in RAS associated with cancer are insensitive to GAP, and the rate of inactivation is limited to their intrinsic hydrolysis rate. Here, we use high-resolution native mass spectrometry (MS) to determine the kinetics and transition state thermodynamics of intrinsic hydrolysis for K-RAS and its oncogenic mutants. MS data reveal heterogeneity where both 2'-deoxy and 2'-hydroxy forms of GDP (guanosine diphosphate) and GTP (guanosine triphosphate) are bound to the recombinant enzyme. Intrinsic GTPase activity is directly monitored by the loss in mass of K-RAS bound to GTP, which corresponds to the release of phosphate. The rates determined from MS are in direct agreement with those measured using an established solution-based assay. Our results show that the transition state thermodynamics for the intrinsic GTPase activity of K-RAS is both enthalpically and entropically unfavorable. The oncogenic mutants G12C, Q61H, and G13D unexpectedly exhibit a 2'-deoxy GTP intrinsic hydrolysis rate higher than that for GTP.

New insights into the metal-induced oxidative degradation pathways of transthyretin.

The amyloidogenic mechanism of transthyretin is still debated but understanding it fully could lend insight into disease progression and potential therapeutics. Transthyretin was investigated revealing a metal-induced (Cr/Cu) oxidation pathway leading to N-terminal backbone fragmentation and oligomer formation; previously hidden details were revealed only by FT-IM-Orbitrap MS and surface-induced dissociation.