Fragment screening offers an alternative to traditional screening for discovering new leads in drug discovery programs. This paper describes a fragment screening methodology based on high throughput X-ray crystallography. The method is illustrated against five proteins (p38 MAP kinase, CDK2, thrombin, ribonuclease A, and PTP1B). The fragments identified have weak potency (>100 microM) but are efficient binders relative to their size and may therefore represent suitable starting points for evolution to good quality lead compounds. The examples illustrate that a range of molecular interactions (i.e., lipophilic, charge-charge, neutral hydrogen bonds) can drive fragment binding and also that fragments can induce protein movement. We believe that the method has great potential for the discovery of novel lead compounds against a range of targets, and the companion paper illustrates how lead compounds have been identified for p38 MAP kinase starting from fragments such as those described in this paper.

Collagenase is a zinc-dependent endoproteinase and is a member of the matrix metalloproteinase (MMP) family of enzymes. The MMPs participate in connective tissue remodeling events and aberrant regulation has been associated with several pathologies. The 2.4 angstrom resolution structure of the inhibited enzyme revealed that, in addition to the catalytic zinc, there is a second zinc ion and a calcium ion which play a major role in stabilizing the tertiary structure of collagenase. Despite scant sequence homology, collagenase shares structural homology with two other endoproteinases, bacterial thermolysin and crayfish astacin. The detailed description of protein-inhibitor interactions present in the structure will aid in the design of compounds that selectively inhibit individual members of the MMP family. Such inhibitors will be useful in examining the function of MMPs in pathological processes.

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The protein Keap1 is central to the regulation of the Nrf2-mediated cytoprotective response, and is increasingly recognized as an important target for therapeutic intervention in a range of diseases involving excessive oxidative stress and inflammation. The BTB domain of Keap1 plays key roles in sensing environmental electrophiles and in mediating interactions with the Cul3/Rbx1 E3 ubiquitin ligase system, and is believed to be the target for several small molecule covalent activators of the Nrf2 pathway. However, despite structural information being available for several BTB domains from related proteins, there have been no reported crystal structures of Keap1 BTB, and this has precluded a detailed understanding of its mechanism of action and interaction with antagonists. We report here the first structure of the BTB domain of Keap1, which is thought to contain the key cysteine residue responsible for interaction with electrophiles, as well as structures of the covalent complex with the antagonist CDDO/bardoxolone, and of the constitutively inactive C151W BTB mutant. In addition to providing the first structural confirmation of antagonist binding to Keap1 BTB, we also present biochemical evidence that adduction of Cys 151 by CDDO is capable of inhibiting the binding of Cul3 to Keap1, and discuss how this class of compound might exert Nrf2 activation through disruption of the BTB-Cul3 interface.

The cyclin D1-cyclin-dependent kinase 4 (CDK4) complex is a key regulator of the transition through the G1 phase of the cell cycle. Among the cyclin/CDKs, CDK4 and cyclin D1 are the most frequently activated by somatic genetic alterations in multiple tumor types. Thus, aberrant regulation of the CDK4/cyclin D1 pathway plays an essential role in oncogenesis; hence, CDK4 is a genetically validated therapeutic target. Although X-ray crystallographic structures have been determined for various CDK/cyclin complexes, CDK4/cyclin D1 has remained highly refractory to structure determination. Here, we report the crystal structure of CDK4 in complex with cyclin D1 at a resolution of 2.3 Å. Although CDK4 is bound to cyclin D1 and has a phosphorylated T-loop, CDK4 is in an inactive conformation and the conformation of the heterodimer diverges from the previously known CDK/cyclin binary complexes, which suggests a unique mechanism for the process of CDK4 regulation and activation. CDK4 and CDK6 associate with the D-type cyclins (D1, D2, D3) and phosphorylate and inactivate the retinoblastoma (Rb) protein family members (p107, p130, pRb). Phosphorylation of pRb by CDK4/6 then leads to the derepression and activation of E2F target genes, including the E-type cyclins, which facilitate progression through the G 1 phase of the cell cycle.Deregulation of the CDK4/cyclin D pathway has been identified in many cancers (refs. 4 and 5 and references therein and ref. 6). Notably, most genetic alterations target specifically CDK4 or cyclin D1, whereas alterations in other CDKs and cyclins are far less common. The CDK4 gene is amplified in a high percentage of liposarcomas (7), and breast cancers frequently exhibit high cyclin D1 levels, either through genetic amplification of the gene or overexpression (8). Translocation of cyclin D1 to the IgH promoter is a hallmark aberration in mantle cell lymphoma (9). Cyclin D1 translocations can also be detected in many cases of multiple myelomas (10). A mutation of CDK4 (Arg-24-Cys) that renders it refractory to inhibition by the tumor suppressor protein p16INK4a has also been identified, and, similarly, deletion or mutation of the p16INK4a gene results in defective CDK4 inhibition and dysregulated CDK4 activity (11). Finally, genetic inactivation of p16INK4 is among the most frequent tumor suppressor mutations found in human cancers. Taken together, these data indicate that an unchecked or hyperactivated CDK4/cyclin D1 pathway may be responsible for enhanced cellular proliferation in cancers and imply that CDK4 is a promising target for the development of anticancer therapies (reviewed in ref. 12).The molecular basis of CDK activation has been the focus of many studies using cellular, biochemical, and structural approaches (reviewed in ref.3). Maximal CDK activation requires both binding of a cognate cyclin and phosphorylation of residues within the CDK T-loop, and X-ray crystallographic studies of various CDKs and CDK/cyclin complexes have identified the conformational movements associated with ...

We describe the structure-guided optimization of the molecular fragments 2-amino-3-benzyloxypyridine 1 (IC(50) 1.3 mM) and 3-(2-(4-pyridyl)ethyl)indole 2 (IC(50) 35 microM) identified using X-ray crystallographic screening of p38alpha MAP kinase. Using two separate case studies, the article focuses on the key compounds synthesized, the structure-activity relationships and the binding mode observations made during this optimization process, resulting in two potent lead series that demonstrate significant increases in activity. We describe the process of compound elaboration either through the growing out from fragments into adjacent pockets or through the conjoining of overlapping fragments and demonstrate that we have exploited the mobile conserved activation loop, consisting in part of Asp168-Phe169-Gly170 (DFG), to generate significant improvements in potency and kinase selectivity.

The family of lipases (trtacylglycerol-acyl-hydrolases.EC 3.1.1.3) constitutes an interesting class of enzymes because of their ability to interact with lipid-water interfaces. their wide range of substrate specificities. and their potential industrial applications [1,2]. Here we report the first crystal structure of a bacterial lipase. from Pseudomonas glurnae. The structure is formed from three domains, the largest of which contains a subset of the a/P hydrolase fold and a calcium site. ASPIRE, the acidic residue m the catalytic tread, has previously been mutated mto an alanme with only a modest reduction m activity [3].

The first paper in this series (see previous article) described structure-activity studies of carboxamide analogues of zanamivir binding to influenza virus sialidase types A and B and showed that inhibitory activity of these compounds was much greater against influenza A enzyme. To understand the large differences in affinities, a number of protein-ligand complexes have been investigated using crystallography and molecular dynamics. The crystallographic studies show that the binding of ligands containing tertiary amide groups is accompanied by the formation of an intramolecular planar salt bridge between two amino acid residues in the active site of the enzyme. It is proposed that the unexpected strong binding of these inhibitors is a result of the burial of hydrophobic surface area and salt-bridge formation in an environment of low dielectric. In sialidase from type A virus, binding of the carboxamide moeity and salt-bridge formation have only a minor effect on the positions of the surrounding residues, whereas in type B enzyme, significant distortion of the protein is observed. The results suggest that the decreased affinity in enzyme from influenza B is directly correlated with the small changes that occur in the amino acid residue interactions accompanying ligand binding. Molecular dynamics calculations have shown that the tendency for salt-bridge formation is greater in influenza A sialidase than influenza B sialidase and that this tendency is a useful descriptor for the prediction of inhibitor potency.