Small molecule binding sites on the Ras:SOS complex can be exploited for inhibition of Ras activation.

Constitutively active mutant KRas displays a reduced rate of GTP hydrolysis via both intrinsic and GTPase-activating protein-catalyzed mechanisms, resulting in the perpetual activation of Ras pathways. We describe a fragment screening campaign using X-ray crystallography that led to the discovery of three fragment binding sites on the Ras:SOS complex. The identification of tool compounds binding at each of these sites allowed exploration of two new approaches to Ras pathway inhibition by stabilizing or covalently modifying the Ras:SOS complex to prevent the reloading of Ras with GTP. Initially, we identified ligands that bound reversibly to the Ras:SOS complex in two distinct sites, but these compounds were not sufficiently potent inhibitors to validate our stabilization hypothesis. We conclude by demonstrating that covalent modification of Cys118 on Ras leads to a novel mechanism of inhibition of the SOS-mediated interaction between Ras and Raf and is effective at inhibiting the exchange of labeled GDP in both mutant (G12C and G12V) and wild type Ras

Dynamic studies of H-Ras•GTPγS interactions with nucleotide exchange factor Sos reveal a transient ternary complex formation in solution

The cycling between GDP- and GTP- bound forms of the Ras protein is partly regulated by the binding of Sos. The structural/dynamic behavior of the complex formed between activated Sos and Ras at the point of the functional cycle where the nucleotide exchange is completed has not been described to date. Here we show that solution NMR spectra of H-Ras∙GTPγS mixed with a functional fragment of Sos (Sos(Cat)) at a 2:1 ratio are consistent with the formation of a rather dynamic assembly. H-Ras∙GTPγS binding was in fast exchange on the NMR timescale and retained a significant degree of molecular tumbling independent of Sos(Cat), while Sos(Cat) also tumbled largely independently of H-Ras. Estimates of apparent molecular weight from both NMR data and SEC-MALS revealed that, at most, only one H-Ras∙GTPγS molecule appears stably bound to Sos. The weak transient interaction between Sos and the second H-Ras∙GTPγS may provide a necessary mechanism for complex dissociation upon the completion of the native GDP → GTP exchange reaction, but also explains measurable GTP → GTP exchange activity of Sos routinely observed in in vitro assays that use fluorescently-labelled analogs of GTP. Overall, the data presents the first dynamic snapshot of Ras functional cycle as controlled by Sos

Segmental Isotope Labelling of an Individual Bromodomain of a Tandem Domain BRD4 Using Sortase A

<div><p>Bromodomain and extra-terminal (BET) family of proteins are one of the major readers of epigenetic marks and an important target class in oncology and other disease areas. The importance of the BET family of proteins is manifested by the explosion in the number of inhibitors against these targets that have successfully entered clinical trials. One important BET family member is bromodomain containing protein 4 (BRD4). Structural and biophysical studies of BRD4 are complicated by its tertiary-structure consisting of two bromodomains connected by a flexible inter-domain linker of approximately 180 amino acids. A detailed understanding of the interplay of these bromodomains will be key to rational drug design in BRD4, yet there are no reported three-dimensional structures of the multi-domain BRD4 and NMR studies of the tandem domain are hampered by the size of the protein. Here, we present a method for rapid Sortase A-mediated segmental labelling of the individual bromodomains of BRD4 that provides a powerful strategy that will enable NMR studies of ligand-bromodomain interactions with atomic detail. In our labelling strategy, we have used U-[²H,¹⁵N]-isotope labelling on the C-terminal bromodomain with selective introduction of ¹³CH₃ methyl groups on Ile (δ1), Val and Leu, whereas the N-terminal bromodomain remained unlabelled. This labelling scheme resulted in significantly simplified NMR spectra and will allow for high-resolution interaction, structure and dynamics studies in the presence of ligands.</p></div

MTH1 Substrate Recognition--An Example of Specific Promiscuity.

MTH1 (NUDT1) is an oncologic target involved in the prevention of DNA damage. We investigate the way MTH1 recognises its substrates and present substrate-bound structures of MTH1 for 8-oxo-dGTP and 8-oxo-rATP as examples of novel strong and weak binding substrate motifs. Investigation of a small set of purine-like fragments using 2D NMR resulted in identification of a fragment with weak potency. The protein-ligand X-Ray structure of this fragment provides insight into the role of water molecules in substrate selectivity. Wider fragment screening by NMR resulted in three new protein structures exhibiting alternative binding configurations to the key Asp-Asp recognition element of the protein. These inhibitor binding modes demonstrate that MTH1 employs an intricate yet promiscuous mechanism of substrate anchoring through its Asp-Asp pharmacophore. The structures suggest that water-mediated interactions convey selectivity towards oxidized substrates over their non-oxidised counterparts, in particular by stabilization of a water molecule in a hydrophobic environment through hydrogen bonding. These findings may be useful in the design of inhibitors of MTH1

Methyl-TROSY of segmentally labelled BRD4(1, 2).

<p>(A) ¹H-¹³C SOFAST TROSY HMQC of segmentally labelled BRD4(1, 2) with U-[²H,¹⁵N] and ¹³CH₃ ILV methyl labelled BRD4^C (red). (B) Spectral overlay of (A) with isolated U-[²H,¹⁵N] and ¹³CH₃ ILV methyl labelled BRD4^C (blue). (C) Spectral overlay of (A) with natural abundance BRD4^NL (purple).</p

Schematic illustration of segmental labelling on BRD4(1, 2) using SrtA.

<p>Isotopically labelled BRD4^C (orange) was ligated to unlabelled BRD4^NL (blue) to give a segmentally labelled BRD4(1, 2) fusion. TEV protease cleavage and Sortase A ligation reactions were carried out separately. The two step reaction mechanisms of SrtA is also illustrated here. Step I involves an initial nucleophilic attack of the C-terminal LPDTG recognition sequence by Cys184 of SrtA. In step II there is a subsequent nucleophilic attack by the N-terminal Gly of the BRD4^C construct.</p

Titration of I-BET762 to segmentally labelled BRD4.

<p>(A) ¹H-¹³C SOFAST TROSY HMQC of 12 μM segmentally labelled BRD4(1, 2) with U-[²H,¹⁵N] and ¹³CH₃ ILV methyl labelled BRD4^C in apo (red) and in complex with 36 μM I-BET762 (blue). Residues undergoing significant changes in chemical shifts upon binding of I-BET762 are labelled. (B) Structure of BRD4(2) (blue envelope, PDB:2YEM) with surface representation of ILV residues colour coded according to whether they have undergone a large (red) small (pink) or no change (white) in chemical shift upon binding of I-BET762. I-BET762 is shown in a bond representation in its expected binding site based on a structural alignment with the structure of the complex BRD2(1):I-BET762 (PDB:2YEK).</p

Overlay of ¹H-¹⁵N TROSY HSQC spectra of uniformly and segmentally labelled BRD4.

<p>(A) ¹H-¹⁵N TROSY HSQC of U-[²H,¹⁵N]-labelled BRD4(1, 2) (black) overlaid with the segmentally labelled BRD4(1, 2) with U-[²H,¹⁵N] and ¹³CH₃ ILV methyl labelled BRD4^C (red). The peaks corresponding to the mutated G339 and G440 are highlighted by a dashed box. (B) Segmentally labelled BRD4(1, 2) with U-[²H,¹⁵N] and ¹³CH₃ ILV methyl labelled BRD4^C (red).</p