SUMMARY MYC is an oncoprotein transcription factor that is overexpressed in the majority of malignancies. The oncogenic potential of MYC stems from its ability to bind regulatory sequences in thousands of target genes, which depends on interaction of MYC with its obligate partner, MAX. Here, we show that broad association of MYC with chromatin also depends on interaction with the WD40-repeat protein WDR5. MYC binds WDR5 via an evolutionarily conserved “MYC box IIIb” motif that engages a shallow, hydrophobic, cleft on the surface of WDR5. Structure-guided mutations in MYC that disrupt interaction with WDR5 attenuate binding of MYC to ~80% of its chromosomal locations and disable its ability to promote induced pluripotent stem cell formation and drive tumorigenesis. Our data reveal WDR5 as a key determinant for MYC recruitment to chromatin and uncover a tractable target for the discovery of anti-cancer therapies against MYC-driven tumors.

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The 3 human RAS genes, KRAS, NRAS, and HRAS, encode 4 different RAS proteins which belong to the protein family of small GTPases that function as binary molecular switches involved in cell signaling. Activating mutations in RAS are among the most common oncogenic drivers in human cancers, with KRAS being the most frequently mutated oncogene. Although KRAS is an excellent drug discovery target for many cancers, and despite decades of research, no therapeutic agent directly targeting RAS has been clinically approved. Using structure-based drug design, we have discovered BI-2852 (1), a KRAS inhibitor that binds with nanomolar affinity to a pocket, thus far perceived to be “undruggable,” between switch I and II on RAS; 1 is mechanistically distinct from covalent KRASG12C inhibitors because it binds to a different pocket present in both the active and inactive forms of KRAS. In doing so, it blocks all GEF, GAP, and effector interactions with KRAS, leading to inhibition of downstream signaling and an antiproliferative effect in the low micromolar range in KRAS mutant cells. These findings clearly demonstrate that this so-called switch I/II pocket is indeed druggable and provide the scientific community with a chemical probe that simultaneously targets the active and inactive forms of KRAS.

Fragmentsuche: Liganden, die an die GTPase K‐Ras binden und die Aktivität des Nukleotidaustauschfaktors Sos verändern, wurden mit einem fragmentbasierten Screening unter Verwendung von NMR‐Spektroskopie gefunden. Strukturdaten zeigen, wie die von den Fragmenten abgeleiteten Treffer an den K‐Ras‐Guanosindiphosphat‐Komplex binden (siehe Bild), und liefern einen Ausgangspunkt für die Entwicklung von Wirkstoffen, die K‐Ras‐Aktivierung und ‐Signalisierung beeinflussen.

Because of its stringent sequence specificity, the 3C-type protease from tobacco etch virus (TEV) is frequently used to remove affinity tags from recombinant proteins. It is unclear, however, exactly how TEV protease recognizes its substrates with such high selectivity. The crystal structures of two TEV protease mutants, inactive C151A and autolysis-resistant S219D, have now been solved at 2.2-and 1.8-Å resolution as complexes with a substrate and product peptide, respectively. The enzyme does not appear to have been perturbed by the mutations in either structure, and the modes of binding of the product and substrate are virtually identical. Analysis of the protein-ligand interactions helps to delineate the structural determinants of substrate specificity and provides guidance for reengineering the enzyme to further improve its utility for biotechnological applications.The Picornaviridae are a large superfamily of (ϩ)-strand RNA viruses that are responsible for a variety of plant and animal pathologies (1). Their RNA genomes are translated into polyprotein precursors that are co-translationally cleaved by viral proteases to generate the mature proteins (2). The majority of these processing events are mediated by the picornavirus 3C-type proteases, which are structurally similar to serine proteases like trypsin and chymotrypsin, but utilize a cysteine thiol instead of a serine hydroxyl as the active-site nucleophile (1, 3). Because they play an essential role in viral replication, 3C proteases are viewed as attractive molecular targets for antiviral therapeutics (4).The stringent sequence specificity of rhinovirus 3C protease and the 3C-like nuclear inclusion protease encoded by TEV 1 has also led to their widespread application in the biotechnology sector as reagents for endoproteolytic removal of affinity tags from recombinant proteins (5). In contrast to Factor Xa, enterokinase, and thrombin, neither of these viral proteases has ever been reported to cleave genetically engineered fusion proteins at unintended locations. All 3C-type proteases exhibit a strong preference for glutamine in the P1 position of their substrates and for small aliphatic residues in the P1Ј subsite, but these are clearly not the only specificity determinants (3, 6). Studies with oligopeptide substrates have established that the P6 and P3 subsites are also important specificity determinants for TEV protease (7), whereas it is the P4 and P2Ј positions that appear to make the greatest contribution to the unique specificity of rhinovirus 3C protease (8).Despite the fact that 3C-type proteases have been the subject of considerable interest, the structural basis of their substrate specificity remains obscure. Although the crystal structures of 3C proteases from hepatitis A virus (9), rhinovirus-14 (10), and poliovirus (11, 12) have been determined, none of them have cognate peptides in the active site. Consequently, efforts to explain the substrate specificity of these enzymes have relied on modeling or, in a few cases, on the structures of e...

Aberrant activation of the small GTPase Ras by oncogenic mutation or constitutively active upstream receptor tyrosine kinases results in the deregulation of cellular signals governing growth and survival in ∼30% of all human cancers. However, the discovery of potent inhibitors of Ras has been difficult to achieve. Here, we report the identification of small molecules that bind to a unique pocket on the Ras:Son of Sevenless (SOS):Ras complex, increase the rate of SOS-catalyzed nucleotide exchange in vitro, and modulate Ras signaling pathways in cells. X-ray crystallography of Ras:SOS:Ras in complex with these molecules reveals that the compounds bind in a hydrophobic pocket in the CDC25 domain of SOS adjacent to the Switch II region of Ras. The structureactivity relationships exhibited by these compounds can be rationalized on the basis of multiple X-ray cocrystal structures. Mutational analyses confirmed the functional relevance of this binding site and showed it to be essential for compound activity. These molecules increase Ras-GTP levels and disrupt MAPK and PI3K signaling in cells at low micromolar concentrations. These small molecules represent tools to study the acute activation of Ras and highlight a pocket on SOS that may be exploited to modulate Ras signaling.

SUMMARY The chromatin-associated protein WDR5 is a promising target for pharmacological inhibition in cancer. Drug discovery efforts center on the blockade of the “WIN site” of WDR5, a well-defined pocket that is amenable to small molecule inhibition. Various cancer contexts have been proposed to be targets for WIN site inhibitors, but a lack of understanding of WDR5 target genes and of the primary effects of WIN site inhibitors hampers their utility. Here, by the discovery of potent WIN site inhibitors, we demonstrate that the WIN site links WDR5 to chromatin at a small cohort of loci, including a specific subset of ribosome protein genes. WIN site inhibitors rapidly displace WDR5 from chromatin and decrease the expression of associated genes, causing translational inhibition, nucleolar stress, and p53 induction. Our studies define a mode by which WDR5 engages chromatin and forecast that WIN site blockade could have utility against multiple cancer types.

Thymidylate synthase (TS) is a major target in the chemotherapy of colorectal cancer and some other neoplasms. The emergence of resistance to the treatment is often related to the increased levels of TS in cancer cells, which have been linked to the elimination of TS binding to its own mRNA upon drug binding, a feedback regulatory mechanism, and/or to the increased stability to intracellular degradation of TS⅐drug complexes (versus unliganded TS). The active site loop of human TS (hTS) has a unique conformation resulted from a rotation by 180°relative to its orientation in bacterial TSs. In this conformation, the enzyme must be inactive, because the catalytic cysteine is no longer positioned in the ligandbinding pocket. The ordered solvent structure obtained from high resolution crystallographic data (2.0 Å) suggests that the inactive loop conformation promotes mRNA binding and intracellular degradation of the enzyme. This hypothesis is supported by fluorescence studies, which indicate that in solution both active and inactive forms of hTS are present. The binding of phosphate ion shifts the equilibrium toward the inactive conformation; subsequent dUMP binding reverses the equilibrium toward the active form. Thus, TS inhibition via stabilization of the inactive conformation should lead to less resistance than is observed with presently used drugs, which are analogs of its substrates, dUMP and CH 2 H 4 folate, and bind in the active site, promoting the active conformation. The presence of an extension at the N terminus of native hTS has no significant effect on kinetic properties or crystal structure. Thymidylate synthase (TS)1 catalyzes the reductive methylation of 2Ј-deoxyuridine 5Ј-monophosphate (dUMP) to thymidine 5Ј-monophosphate (dTMP), using the co-substrate, 5,10-methylenetetrahydrofolate (CH 2 H 4 folate) as a 1-carbon donor and reductant. The physical structures of bacterial TSs have been relatively well defined, and crystallographic data, in concert with data derived from kinetic, spectroscopic, and sitedirected mutagenesis studies, have led to a detailed understanding of the catalytic mechanism of these enzymes (1). In contrast, relatively few investigations of mammalian TS structure and catalysis have been conducted. The three-dimensional structure of the native human TS (hTS) has been reported previously (2). The data showed a surprising feature not observed in TSs from other sources: loop 181-197 containing the catalytic cysteine, Cys-195, was in an inactive conformation, rotated ϳ180°with respect to its orientation in bacterial TSs, with the sulfhydryl of Cys-195 over 10 Å from the location of sulfhydryls of corresponding cysteine residues in bacterial enzymes. Subsequent determination of the structure of a ternary inhibitory complex between closely related ratTS (rTS) and dUMP and Tomudex (3) has shown that the ligands bind to the enzyme in the active conformation. Recently, it was found that also in the hTS⅐dUMP⅐Tomudex complex hTS is in the active conformation (4). The inactive conformation has...