Interrogating the Inhibition Mechanisms of Human Aldehyde Oxidase by X-ray Crystallography and NMR Spectroscopy: The Raloxifene Case.

Human aldehyde oxidase (hAOX1) is mainly present in the liver and has an emerging role in drug metabolism, since it accepts a wide range of molecules as substrates and inhibitors. Herein, we employed an integrative approach by combining NMR, X-ray crystallography, and enzyme inhibition kinetics to understand the inhibition modes of three hAOX1 inhibitors-thioridazine, benzamidine, and raloxifene. These integrative data indicate that thioridazine is a noncompetitive inhibitor, while benzamidine presents a mixed type of inhibition. Additionally, we describe the first crystal structure of hAOX1 in complex with raloxifene. Raloxifene binds tightly at the entrance of the substrate tunnel, stabilizing the flexible entrance gates and elucidating an unusual substrate-dependent mechanism of inhibition with potential impact on drug-drug interactions. This study can be considered as a proof-of-concept for an efficient experimental screening of prospective substrates and inhibitors of hAOX1 relevant in drug discovery.

SLMP53-1 interacts with wild-type and mutant p53 DNA-binding domain and reactivates multiple hotspot mutations.

BACKGROUND: Half of human cancers harbour TP53 mutations that render p53 inactive as a tumor suppressor. As such, reactivation of mutant (mut)p53 through restoration of wild-type (wt)-like function represents one of the most promising therapeutic strategies in cancer treatment. Recently, we have reported the (S)-tryptophanol-derived oxazoloisoindolinone SLMP53-1 as a new reactivator of wt and mutp53 R280K with in vitro and in vivo p53-dependent antitumor activity. The present work aimed a mechanistic elucidation of mutp53 reactivation by SLMP53-1. METHODS AND RESULTS: By cellular thermal shift assay (CETSA), it is shown that SLMP53-1 induces wt and mutp53 R280K thermal stabilization, which is indicative of intermolecular interactions with these proteins. Accordingly, in silico studies of wt and mutp53 R280K DNA-binding domain with SLMP53-1 unveiled that the compound binds at the interface of the p53 homodimer with the DNA minor groove. Additionally, using yeast and p53-null tumor cells ectopically expressing distinct highly prevalent mutp53, the ability of SLMP53-1 to reactivate multiple mutp53 is evidenced. CONCLUSIONS: SLMP53-1 is a p53-activating agent with the ability to directly target wt and a set of hotspot mutp53. GENERAL SIGNIFICANCE: This work reinforces the encouraging application of SLMP53-1 in the personalized treatment of cancer patients harboring distinct p53 status.

The Crystal Structure of the R280K Mutant of Human p53 Explains the Loss of DNA Binding.

The p53 tumor suppressor is widely found to be mutated in human cancer. This protein is regarded as a molecular hub regulating different cell responses, namely cell death. Compelling data have demonstrated that the impairment of p53 activity correlates with tumor development and maintenance. For these reasons, the reactivation of p53 function is regarded as a promising strategy to halt cancer. In the present work, the recombinant mutant p53R280K DNA binding domain (DBD) was produced for the first time, and its crystal structure was determined in the absence of DNA to a resolution of 2.0 Å. The solved structure contains four molecules in the asymmetric unit, four zinc(II) ions, and 336 water molecules. The structure was compared with the wild-type p53 DBD structure, isolated and in complex with DNA. These comparisons contributed to a deeper understanding of the mutant p53R280K structure, as well as the loss of DNA binding related to halted transcriptional activity. The structural information derived may also contribute to the rational design of mutant p53 reactivating molecules with potential application in cancer treatment.

Molybdenum and tungsten enzymes: a crystallographic and mechanistic overview.

Molybdenum and tungsten enzymes which contain the pyranopterin cofactor are ubiquitous in Nature and perform a wide variety of biological functions. They catalyze a diversity of mostly two-electron oxidation-reduction reactions crucial in the metabolism of nitrogen, sulfur and carbon. These enzymes share common structural features, but reveal different polypeptide folding topologies and different active site coordination geometries, which, in part, dictate their function and specificity. On the basis of structural, spectroscopic and biochemical characteristics, they have been classified into three broad families named according to well-studied enzymes of each family: xanthine oxidase, sulfite oxidase and DMSO reductase. An overview of the X-ray crystallography data for representative members of the three enzyme families is given here, focusing on the mechanistic implications drawn from the structural data.

Biologically relevant O,S-donor compounds. Synthesis, molybdenum complexation and xanthine oxidase inhibition.

Two O,S-donor ligands, hydroxythiopyrone and hydroxythiopyridinone derivatives, were developed and studied, as well as the corresponding O,O-derivatives, with a view to their potential pharmacological applications as xanthine oxidase (XO) inhibitors. The biological assays revealed that the O,S-ligands present high inhibitory activity towards XO (nanomolar order, close to that of the pharmaceutical drug allopurinol), in contrast to the corresponding O,O-analogues. Due to the biomedical relevance of this molybdenum-containing enzyme, the corresponding Mo(VI) complexes were studied both in solution and in the solid state, aimed at identifying the source of the biological properties. The solution studies showed that, in comparison with the O,O-analogues, the Mo(VI) complexes with the O,S-ligands present some stabilization, which is even more pronounced for the reduced Mo(IV) species. The crystal structures of the Mo(VI) complexes with the hydroxythiopyrone revealed good flexibility of the coordination modes, with two structural isomers and two polymorphic forms for a mononuclear and a binuclear species, respectively. These results give some support to mechanistic proposals for the XO inhibition involving the interaction of the thione group with the molybdenum cofactor, thus indicating a role of the sulfur atom in the XO inhibition.

Formate-reduced E. coli formate dehydrogenase H: The reinterpretation of the crystal structure suggests a new reaction mechanism.

Re-evaluation of the crystallographic data of the molybdenum-containing E. coli formate dehydrogenase H (Boyington et al. Science 275:1305-1308, 1997), reported in two redox states, reveals important structural differences for the formate-reduced form, with large implications for the reaction mechanism proposed in that work. We have re-refined the reduced structure with revised protocols and found substantial rearrangement in some parts of it. The original model is essentially correct but an important loop close to the molybdenum active site was mistraced, and, therefore, catalytic relevant residues were located in wrong positions. In particular selenocysteine-140, a ligand of molybdenum in the original work, and essential for catalysis, is no longer bound to the metal after reduction of the enzyme with formate. These results are incompatible with the originally proposed reaction mechanism. On the basis of our new interpretation, we have revised and proposed a new reaction mechanism, which reconciles the new X-ray model with previous biochemical and extended X-ray absorption fine structure data.

Crystallization and preliminary X-ray diffraction analysis of the 16-haem cytochrome of Desulfovibrio gigas.

High-molecular-weight cytochromes (Hmcs) belong to a large family of multihaem cytochromes in sulfate-reducing bacteria. HmcA is the first cytochrome reported to have 16 c-type haems arranged in its polypeptide chain. The function of this cytochrome is still unknown, although it is clear that it belongs to a membrane-bound complex involved in electron transfer from the periplasm to the membrane. HmcA from Desulfovibrio gigas has been purified and successfully crystallized using the hanging-drop vapour-diffusion method. The crystals grew using PEG and zinc acetate as precipitants to maximum dimensions of 0.2 x 0.2 x 0.2 mm in an orthorhombic space group, with unit-cell parameters a = 88.9, b = 90.9, c = 83.7 A. The crystals diffracted to beyond 2.07 A and a MAD data set was collected.