The Ras gene is frequently mutated in cancer, and mutant Ras drives tumorigenesis. Although Ras is a central oncogene, small molecules that bind to Ras in a well-defined manner and exert inhibitory effects have not been uncovered to date. Through an NMR-based fragment screen, we identified a group of small molecules that all bind to a common site on Ras. High-resolution cocrystal structures delineated a unique ligand-binding pocket on the Ras protein that is adjacent to the switch I/II regions and can be expanded upon compound binding. Structure analysis predicts that compound-binding interferes with the Ras/SOS interactions. Indeed, selected compounds inhibit SOS-mediated nucleotide exchange and prevent Ras activation by blocking the formation of intermediates of the exchange reaction. The discovery of a small-molecule binding pocket on Ras with functional significance provides a new direction in the search of therapeutically effective inhibitors of the Ras oncoprotein.small G protein | guanine nucleotide exchange | nuclear magnetic resonance | crystal structure | small-molecule inhibitors R as is a small GTP-binding protein that functions as a nucleotide-dependent switch for central growth signaling pathways (1, 2). In response to extracellular signals, Ras is converted from a GDP-bound (Ras GDP ) to a GTP-bound (Ras GTP ) state, as catalyzed by guanine nucleotide exchange factors (GEFs), notably the SOS1 protein. Active Ras GTP mediates its diverse growth-stimulating functions through its direct interactions with effectors including Raf, PI3K, and Ral guanine nucleotide dissociation stimulator. The intrinsic GTPase activity of Ras then hydrolyzes GTP to GDP to terminate Ras signaling. The Ras GTPase activity can be further accelerated by its interactions with GTPase-activating proteins (GAPs), including the neurofibromin 1 tumor suppressor (2).Ras, a human oncogene identified and characterized over 30 y ago, is mutated in more than 20% of human cancers. Among the three Ras isoforms (K, N, and H), KRas is most frequently mutated (2). Mutant Ras has a reduced GTPase activity, which prolongs its activated conformation, thereby promoting Rasdependent signaling and cancer cell survival or growth (1, 2).Mutations of Ras in cancer are associated with poor prognosis (2). Inactivation of oncogenic Ras in mice results in tumor shrinkage. Thus, Ras is widely considered an oncology target of exceptional importance. However, development of small-molecule inhibitors against Ras has thus far proven unsuccessful. Given the picomolar affinity between guanine nucleotides and Ras and the high cytosolic concentration of guanine nucleotides, it is very challenging to develop a conventional inhibitor competitive against nucleotide binding (1, 2). Outside of the nucleotide-binding pocket, the Ras protein does not contain obvious cavities for small-molecule binding. A number of small molecules have been reported to bind to Ras (3-7), but their mechanisms of action and the structural basis to achieve Ras inhibition remain elusive.Fra...

The bacterial plasmid-encoded organomercurial lyase, MerB (EC 4.99.1.2), catalyzes the protonolysis of organomercury compounds yielding Hg(II) and the corresponding protonated hydrocarbon. A small, soluble protein with no known homologues, MerB is widely distributed among eubacteria in three phylogenetically distinct subfamilies whose most prominent motif includes three conserved cysteine residues. We found that the 212-residue MerB encoded by plasmid R831b is a cytosolic enzyme, consistent with its high thiol requirement in vitro. MerB is inhibited by the nonphysiological dithiol DTT but uses the physiological thiols, glutathione and cysteine, equally well. Highly conserved Cys96 and Cys159 are essential for activity, whereas weakly conserved Cys160 is not. Proteins mutant in highly conserved Cys117 are insoluble. All MerB cysteines are DTNB-reactive in native and denatured states except Cys117, which fails to react with DTNB in the native form, suggesting it is buried. Mass spectrometric analysis of trypsin fragments of reduced proteins treated with N-ethylmaleimide or iodoacetamide revealed that all cysteines form covalent adducts and remain covalently modifiable even when exposed to 1:1 PHMB prior to treatment with NEM or IAM. Stable PHMB adducts were also observed on all cysteines in mutant proteins, suggesting rapid exchange of PHMB among the remaining protein thiols. However, PHMB exposure of reduced wild-type MerB yielded only Hg adducts on the Cys159/Cys160 peptide, suggesting a trapped reaction intermediate. Using HPLC to follow release of benzoic acid from PHMB, we confirmed that fully reduced wild-type MerB and mutant C160S can carry out a single protonolysis without exogenous thiols. On the basis of the foregoing we refine the previously proposed S(E)2 mechanism for protonolysis by MerB.

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α-Synuclein (αSN) aggregation is central to the etiology of Parkinson's disease (PD). Large-scale screening of compounds to identify aggregation inhibitors is challenged by stochastic αSN aggregation and difficulties in detecting early-stage oligomers (αSOs). We developed a high-throughput screening assay combining SDS-stimulated αSN aggregation with FRET to reproducibly detect initial stages in αSN aggregation. We screened 746,000 compounds, leading to 58 hits that markedly inhibit αSN aggregation and reduce αSOs' membrane permeabilization activity. The most effective aggregation inhibitors were derivatives of (4-hydroxynaphthalen-1-yl)sulfonamide. They interacted strongly with the N-terminal part of monomeric αSN and reduced αSO-membrane interactions, possibly by affecting electrostatic interactions. Several compounds reduced αSO toxicity toward neuronal cell lines. The inhibitors introduced chemical modifications of αSN that were, however, not a prerequisite for inhibitory activity. We also identified several phenyl-benzoxazol compounds that promoted αSN aggregation (proaggregators). These compounds may be useful tools to modulate αSN aggregation in cellula.

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