Structural basis for the broad-spectrum inhibition of metallo-beta-lactamases by thiols

The development of broad-spectrum metallo-beta-lactamase (MBL) inhibitors is challenging due to structural diversity and differences in metal utilisation by these enzymes. Analysis of structural data, followed by non-denturing mass spectrometric analyses, identified thiols proposed to inhibit representative MBLs from all three sub-classes: B1, B2 and B3. Solution analyses led to the identification of broad spectrum inhibitors, including potent inhibitors of the CphA MBL (Aeromonas hydrophila). Structural studies revealed that, as observed for other B1 and B3 MBLs, inhibition of the L1 MBL thiols involves metal chelation. Evidence is reported that this is not the case for inhibition of the CphA enzyme by some thiols; the crystal structure of the CphA-Zn-inhibitor complex reveals a binding mode in which the thiol does not interact with the zinc. The structural data enabled the design and the production of further more potent inhibitors. Overall the results suggest that the development of reasonably broad-spectrum MBL inhibitors should be possible.     

Structural basis for telomeric G-quadruplex targeting by naphthalene diimide ligands

The folding of the single-stranded 3' end of the human telomere into G-quadruplex arrangements inhibits the overhang from hybridizing with the RNA template of telomerase and halts telomere maintenance in cancer cells. The ability to thermally stabilize human telomeric DNA as a four-stranded G-quadruplex structure by developing selective small molecule compounds is a therapeutic path to regulating telomerase activity and thereby selectively inhibit cancer cell growth. The development of compounds with the necessary selectivity and affinity to target parallel-stranded G-quadruplex structures has proved particularly challenging to date, relying heavily upon limited structural data. We report here on a structure-based approach to the design of quadruplex-binding ligands to enhance affinity and selectivity for human telomeric DNA. Crystal structures have been determined of complexes between a 22-mer intramolecular human telomeric quadruplex and two potent tetra-substituted naphthalene diimide compounds, functionalized with positively charged N-methyl-piperazine side-chains. These compounds promote parallel-stranded quadruplex topology, binding exclusively to the 3' surface of each quadruplex. There are significant differences between the complexes in terms of ligand mobility and in the interactions with quadruplex grooves. One of the two ligands is markedly less mobile in the crystal complex and is more quadruplex-stabilizing, forming multiple electrostatic/hydrogen bond contacts with quadruplex phosphate groups. The data presented here provides a structural rationale for the biophysical (effects on quadruplex thermal stabilization) and biological data (inhibition of proliferation in cancer cell lines and evidence of in vivo antitumor activity) on compounds in this series and, thus, for the concept of telomere targeting with DNA quadruplex-binding small molecules.     

Structural basis for bending of organic crystals

Bending is observed in organic crystals when the packing is anisotropic in such a way that strong and weak interaction patterns occur in nearly perpendicular directions.     

Structural basis for inhibition of protein tyrosine phosphatases by Keggin compounds phosphomolybdate and phosphotungstate

Protein-tyrosine phosphatases (PTPs) constitute a family of receptor-like, and cytoplasmic enzymes, which catalyze the dephosphorylation of phosphotyrosine residues in a variety of receptors and signaling molecules. Together with protein tyrosine kinases (PTKs), PTPs are critically involved in regulating many cellular signaling processes. In this study, diverse compounds were screened for PTP inhibition and selectively screened for inhibitors with the end product inhibition properties. Among phosphate analogues and their derivatives for PTP inhibition, Keggin compounds phosphomolybdate (PM) and phosphotungstate (PT) strongly inhibited both PTP-1B and SHP-1, with K(i) values of 0.06-1.2 micromM in the presence of EDTA. Unlike the vanadium compounds, inhibition potencies of PM and PT were not significantly affected by EDTA. PM and PT were potent, competitive inhibitors for PTPs, but relatively poor inhibitors of Ser/Thr phosphatase. Interestingly, PM and PT did not inhibit alkaline phosphatase at all. The crystal structure of PTP-1B in complex with PM, at 2.0 A resolution, reveals that MoO(3), derived from PM by hydrolysis, binds at the active site. The molybdenium atom of the inhibitor is coordinated with six ligands: three oxo-ligands, two apical water molecules and a S atom of the catalytic cysteine residue. In support of the crystallographic finding, we observed that molybdenium oxides (MoO(3), MoO(2), and MoO(2)Cl(2)) inhibited PTP-1B with IC(50) in the range 5-15 micromM.     

Structural basis for cyclophellitol inhibition of a beta-glucosidase

The structural basis for beta-glucosidase inhibition by cyclophellitol is demonstrated using X-ray crystallography, enzyme kinetics and mass spectrometry.     

Structural basis for a Kolbe-type decarboxylation catalyzed by a glycyl radical enzyme

4-Hydroxyphenylacetate decarboxylase is a [4Fe-4S] cluster containing glycyl radical enzyme proposed to use a glycyl/thiyl radical dyad to catalyze the last step of tyrosine fermentation in clostridia. The decarboxylation product p-cresol (4-methylphenol) is a virulence factor of the human pathogen Clostridium difficile . Here we describe the crystal structures at 1.75 and 1.81 ? resolution of substrate-free and substrate-bound 4-hydroxyphenylacetate decarboxylase from the related Clostridium scatologenes . The structures show a (βγ)(4) tetramer of heterodimers composed of a catalytic β-subunit harboring the putative glycyl/thiyl dyad and a distinct small γ-subunit with two [4Fe-4S] clusters at 40 ? distance from the active site. The γ-subunit comprises two domains displaying pseudo-2-fold symmetry that are structurally related to the [4Fe-4S] cluster-binding scaffold of high-potential iron-sulfur proteins. The N-terminal domain coordinates one cluster with one histidine and three cysteines, and the C-terminal domain coordinates the second cluster with four cysteines. Whereas the C-terminal cluster is buried in the βγ heterodimer interface, the N-terminal cluster is not part of the interface. The previously postulated decarboxylation mechanism required the substrate's hydroxyl group in the vicinity of the active cysteine residue. In contrast to expectation, the substrate-bound state shows a direct interaction between the substrate's carboxyl group and the active site Cys503, while His536 and Glu637 at the opposite side of the active site pocket anchor the hydroxyl group. This state captures a possible catalytically competent complex and suggests a Kolbe-type decarboxylation for p-cresol formation.     

Structural basis for selective inhibition of Src family kinases by PP1.

BACKGROUND: Small-molecule inhibitors that can target individual kinases are powerful tools for use in signal transduction research. It is difficult to find such compounds because of the enormous number of protein kinases and the highly conserved nature of their catalytic domains. Recently, a novel, potent, Src family selective tyrosine kinase inhibitor was reported (PP1). Here, we study the structural basis for this inhibitor's specificity for Src family kinases. RESULTS: A single residue corresponding to Ile338 (v-Src numbering; Thr338 in c-Src) in Src family tyrosine kinases largely controls PP1's ability to inhibit protein kinases. Mutation of Ile338 to a larger residue such as methionine or phenylalanine in v-Src makes this inhibitor less potent. Conversely, mutation of Ile338 to alanine or glycine increases PP1's potency. PP1 can inhibit Ser/Thr kinases if the residue corresponding to Ile338 in v-Src is mutated to glycine. We have accurately predicted several non-Src family kinases that are moderately (IC(50) approximately 1 microM) inhibited by PP1, including c-Abl and the MAP kinase p38. CONCLUSIONS: Our mutagenesis studies of the ATP-binding site in both tyrosine kinases and Ser/Thr kinases explain why PP1 is a specific inhibitor of Src family tyrosine kinases. Determination of the structural basis of inhibitor specificity will aid in the design of more potent and more selective protein kinase inhibitors. The ability to desensitize a particular kinase to PP1 inhibition of residue 338 or conversely to sensitize a kinase to PP1 inhibition by mutation should provide a useful basis for chemical genetic studies of kinase signal transduction.     

Development of pyrrole-imidazole polyamide for specific regulation of human aurora kinase-A and -B gene expression

Pyrrole-imidazole polyamide (PIP) is a nuclease-resistant novel compound that inhibits gene expression through binding to the minor groove of DNA. Human aurora kinase-A (AURKA) and -B (AURKB) are important regulators in mitosis during the cell cycle. In this study, two specific PIPs (PIP-A and PIP-B) targeting AURKA and AURKB promoter regions were designed and synthesized, and their biological effects were investigated by several in vitro assays. PIP-A and PIP-B significantly inhibited the promoter activities, mRNA expression, and protein levels of AURKA and AURKB, respectively, in a concentration-dependent manner. Moreover, 1:1 combination treatment with both PIPs demonstrated prominent antiproliferative synergy (CI value [ED(50)] = 0.256) to HeLa cells as a result of inducing apoptosis-mediated severe catastrophe of cell-cycle progression. The novel synthesized PIP-A and PIP-B are potent and specific gene-silencing agents for AURKA and AURKB.     

Structural basis for matrix metalloproteinase 1-catalyzed collagenolysis

The proteolysis of collagen triple-helical structure (collagenolysis) is a poorly understood yet critical physiological process. Presently, matrix metalloproteinase 1 (MMP-1) and collagen triple-helical peptide models have been utilized to characterize the events and calculate the energetics of collagenolysis via NMR spectroscopic analysis of 12 enzyme-substrate complexes. The triple-helix is bound initially by the MMP-1 hemopexin-like (HPX) domain via a four amino acid stretch (analogous to type I collagen residues 782-785). The triple-helix is then presented to the MMP-1 catalytic (CAT) domain in a distinct orientation. The HPX and CAT domains are rotated with respect to one another compared with the X-ray "closed" conformation of MMP-1. Back-rotation of the CAT and HPX domains to the X-ray closed conformation releases one chain out of the triple-helix, and this chain is properly positioned in the CAT domain active site for subsequent hydrolysis. The aforementioned steps provide a detailed, experimentally derived, and energetically favorable collagenolytic mechanism, as well as significant insight into the roles of distinct domains in extracellular protease function.     

Structural basis of cooperative ligand binding by the glycine riboswitch

The glycine riboswitch regulates gene expression through the cooperative recognition of its amino acid ligand by a tandem pair of aptamers. A 3.6 ? crystal structure of the tandem riboswitch from the glycine permease operon of Fusobacterium nucleatum reveals the glycine binding sites and an extensive network of interactions, largely mediated by asymmetric A-minor contacts, that serve to communicate ligand binding status between the aptamers. These interactions provide a structural basis for how the glycine riboswitch cooperatively regulates gene expression.     

Structural basis of DNA quadruplex recognition by an acridine drug

The crystal structure of a complex between the bimolecular human telomeric quadruplex d(TAGGGTTAGGGT)2 and the experimental anticancer drug BRACO-19, has been determined, to 2.5 A resolution. The binding site for the BRACO-19 molecule is at the interface of two parallel-folded quadruplexes, sandwiched between a G-tetrad surface and a TATA tetrad, and held in the site by networks of water molecules. The structure rationalizes the existing structure-activity data and provides a starting-point for the structure-based design of quadruplex-binding ligands     

Structural basis for contrasting activities of ribosome binding thiazole antibiotics

Thiostrepton and micrococcin inhibit protein synthesis by binding to the L11 binding domain (L11BD) of 23S ribosomal RNA. The two compounds are structurally related, yet they produce different effects on ribosomal RNA in footprinting experiments and on elongation factor-G (EF-G)-dependent GTP hydrolysis. Using NMR and an assay based on A1067 methylation by thiostrepton-resistance methyltransferase, we show that the related thiazoles, nosiheptide and siomycin, also bind to this region. The effect of all four antibiotics on EF-G-dependent GTP hydrolysis and EF-G-GDP-ribosome complex formation was studied. Our NMR and biochemical data demonstrate that thiostrepton, nosiheptide, and siomycin share a common profile, which differs from that of micrococcin. We have generated a three-dimensional (3D) model for the interaction of thiostrepton with L11BD RNA. The model rationalizes the differences between micrococcin and the thiostrepton-like antibiotics interacting with L11BD.     

Structural basis for the highly selective inhibition of MMP-13

Inhibitors for matrix metalloproteinases (MMPs) are under investigation for the treatment of cancer, arthritis, and cardiovascular disease. Here, we report a class of highly selective MMP-13 inhibitors (pyrimidine dicarboxamides) that exhibit no detectable activity against other MMPs. The high-resolution X-ray structures of three molecules of this series bound to MMP-13 reveal a novel binding mode characterized by the absence of interactions between the inhibitors and the catalytic zinc. The inhibitors bind in the S1' pocket and extend into an additional S1' side pocket, which is unique to MMP-13. We analyze the determinants for selectivity and describe the rational design of improved compounds with low nanomolar affinity.     

Structural basis for the exceptional stability of bisaminoacylated nucleotides and transfer RNAs

At least one bisaminoacyl-tRNA is synthesized in nature (by Thermus thermophilus phenylalanyl-tRNA synthetase), and many disubstituted tRNAs have been prepared in vitro. Such misacylated tRNAs are able to participate in protein synthesis, even though they lack the free 2'-OH group of the 3'-terminal adenosine moiety. Their ready participation in protein synthesis implies significant chemical reactivity. The basis for this reactivity has been documented previously. Surprisingly, the aminoacyl moieties of these tRNAs also exhibit exceptional chemical stability. In the present report, bisaminoacylated nucleotides are investigated computationally and experimentally to define the basis for the stability of such species. Molecular modeling of bisalanyl-AMP in the absence of solvent and in the presence of a limited number of water molecules revealed two common features among the low-energy structures. The first was the presence of H-bonding interactions between the two aminoacyl moieties. The second was the presence of a H-bonding interaction between the 2'-O-alanyl moiety and the N-3 atom of the adenine nucleobase, typically mediated through a water molecule. The prediction of an interaction between an aminoacyl moiety and the adenine nucleobase was confirmed experimentally by comparing the behavior of bisalanyl-AMP and bisalanyl-UMP in the presence of model nucleophiles. This study suggests a possible role for the adenosine moiety at the 3'-end of aminoanyl-tRNAs in controlling the stability and reactivity of the aminoacyl moiety and has important implications for the reactivity and stability of normal aminoacyl-tRNAs.     

Arresting cancer proliferation by small-molecule gene regulation

A small library of pyrrole-imidazole polyamide-DNA alkylator (chlorambucil) conjugates was screened for effects on morphology and growth characteristics of a human colon carcinoma cell line, and a compound was identified that causes cells to arrest in the G2/M stage of the cell cycle. Microarray analysis indicates that the histone H4c gene is significantly downregulated by this polyamide. RT-PCR and Western blotting experiments confirm this result, and siRNA to H4c mRNA yields the same cellular response. Strikingly, reduction of H4 protein by >50% does not lead to widespread changes in global gene expression. Sequence-specific alkylation within the coding region of the H4c gene in cell culture was confirmed by LM-PCR. The compound is active in a wide range of cancer cell lines, and treated cells do not form tumors in nude mice. The compound is also active in vivo, blocking tumor growth in mice, without obvious animal toxicity.