Giulio Rastelli

Fast and accurate predictions of binding free energies using MM-PBSA and MM-GBSA

In the drug discovery process, accurate methods of computing the affinity of small molecules with a biological target are strongly needed. This is particularly true for molecular docking and virtual screening methods, which use approximated scoring functions and struggle in estimating binding energies in correlation with experimental values. Among the various methods, MM‐PBSA and MM‐GBSA are emerging as useful and effective approaches. Although these methods are typically applied to large collections of equilibrated structures of protein‐ligand complexes sampled during molecular dynamics in water, the possibility to reliably estimate ligand affinity using a single energy‐minimized structure and implicit solvation models has not been explored in sufficient detail. Herein, we thoroughly investigate this hypothesis by comparing different methods for the generation of protein‐ligand complexes and diverse methods for free energy prediction for their ability to correlate with experimental values. The methods were tested on a series of structurally diverse inhibitors of Plasmodium falciparum DHFR with known binding mode and measured affinities. The results showed that correlations between MM‐PBSA or MM‐GBSA binding free energies with experimental affinities were in most cases excellent. Importantly, we found that correlations obtained with the use of a single protein‐ligand minimized structure and with implicit solvation models were similar to those obtained after averaging over multiple MD snapshots with explicit water molecules, with consequent save of computing time without loss of accuracy. When applied to a virtual screening experiment, such an approach proved to discriminate between true binders and decoy molecules and yielded significantly better enrichment curves.

Crystal Structure and Molecular Modeling of 17-DMAG in Complex with Human Hsp90

Hsp90 is an attractive chemotherapeutic target because it chaperones the folding of proteins found in multiple signal transduction pathways. We describe the 1.75 A resolution crystal structure of human Hsp90 alpha (residues 9-236) complexed with 17-desmethoxy-17-N,N-dimethylaminoethylamino-geldanamycin (17-DMAG). The structure revealed an altered set of interactions between the 17-substituent and the protein compared to geldanamycin and the 17-dimethylaminoethyl moiety pointing into solvent, but otherwise was similar to that reported for the complex with geldanamycin. Targeted molecular dynamics simulations and energetic analysis indicate that geldanamycin undergoes two major conformational changes when it binds Hsp90, with the key step of the conversion being the trans to cis conformational change of the macrocycle amide bond. We speculate that 17-DMAG analogs constrained to a cis-amide in the ground state could provide a significant increase in affinity for Hsp90.

Diabetes complications and their potential prevention: Aldose reductase inhibition and other approaches

Despite recent advances both in the chemistry and molecular pharmacology of antidiabetic drugs, diabetes still remains a life‐threatening disease, which tends to spread all over the world. The clinical profile of diabetic subjects is often worsened by the presence of several long‐term complications, namely neuropathy, nephropathy, retinopathy, and cataract. Several attempts have been made to prevent or at least to delay them. The most relevant are reported in this review, including the development of compounds acting as aldose reductase inhibitors, anti‐advanced glycation end‐product drugs, free radical scavengers, vasoactive agents, essential fatty acid supplementation, and neurotropic growth factors.

Pharmacological approaches to the treatment of diabetic complications

Diabetes is often accompanied by several long-term complications such as neuropathy, nephropathy, retinopathy, cataract and angiopathy; their occurrence has been linked to the modification of the physiological levels of glycaemia. Several interrelated metabolic pathways have been implicated in the toxic effects of glucose; the polyol pathway was one of the first considered. However, while in diabetic animal models the inhibitors of aldose reductase (ALR2, the first enzyme of this pathway) seem to be active, 16 years of clinical trials, based mainly on neuropathy, have been inconclusive; only one drug currently being marketed. Newer potent and selective aldose reductase inhibitors have been discovered in the last few years, but the lack of commercial success has probably led to the very rapid decrease in the number of patents relating to newer aldose reductase inhibitors. Inhibition of the second enzyme of this pathway, sorbitol dehydrogenase (SDH), has been shown to be detrimental. Other approaches for the prevention and the delay of progression of diabetic complications seem to be more promising, namely, the inhibition of the formation of advanced glycated end products (AGEs) or protein kinase C (PKC) β2 inhibition; compounds acting on these two pathways have proved effective in retarding the development of diabetic complications in animal models and some products are in clinical trials at the moment. Renewed attention has been paid to vascular involvement in the pathogenesis of diabetic neuropathy; the biological activity of C-peptide and the role of endothelin-1 (ET-1) in diabetic vascular disease are emerging as a new research area for the treatment of diabetic complications.

Binding Estimation after Refinement, a New Automated Procedure for the Refinement and Rescoring of Docked Ligands in Virtual Screening

Binding estimation after refinement (BEAR) is a novel automated computational procedure suitable for correcting and overcoming limitations of docking procedures such as poor scoring function and the generation of unreasonable ligand conformations. BEAR makes use of molecular dynamics simulation followed by MM‐PBSA and MM‐GBSA binding free energy estimates as tools to refine and rescore the structures obtained from docking virtual screenings. As binding estimation after refinement relies on molecular dynamics, the entire procedure can be tailored to the needs of the end‐user in terms of computational time and the desired accuracy of the results. In a validation test, binding estimation after refinement and rescoring resulted in a significant enrichment of known ligands among top scoring compounds compared with the original docking results. Binding estimation after refinement has direct and straightforward application in virtual screening for correcting both false‐positive and false‐negative hits, and should facilitate more reliable selection of biologically active molecules from compound databases.

Histone deacetylases: structural determinants of inhibitor selectivity.

Histone deacetylases (HDACs) are epigenetic targets with an important role in cancer, neurodegeneration, inflammation, and metabolic disorders. Although clinically effective HDAC inhibitors have been developed, the design of inhibitors with the desired isoform(s) selectivity remains a challenge. Selective inhibitors could help clarify the function of each isoform, and provide therapeutic agents having potentially fewer adverse effects. Crystal structures of several HDACs have been reported, enabling structure-based drug design and providing important information to understand enzyme function. Here, we provide a comprehensive review of the structural information available on HDACs, discussing both conserved and isoform-specific structural and mechanistic features. We focus on distinctive aspects that help rationalize inhibitor selectivity, and provide structure-based recommendations for achieving the desired selectivity.

New aldose reductase inhibitors as potential agents for the prevention of long-term diabetic complications

The results of recent clinical trials emphasise the importance of an improved glycaemic control in diabetic patients in order to prevent or at least to delay long-term complications. The difficulty in obtaining normalisation of blood glucose values has underlined the importance of the search for new and effective aldose reductase inhibitors (ARIs) to control the consequences of elevated glucose levels, therefore delaying the onset and retarding the progression of diabetic complications such as neuropathy, nephropathy, retinopathy and cataract. Although the physiological role of aldose reductase (ALR2) has not been clearly elucidated yet, it has been shown that this enzyme, the first of the polyol pathway, is responsible for the production of sorbitol from glucose. There are several pieces of evidence which link this process to the occurrence of diabetic complications. Orally active aldose reductase inhibitors can be grouped into two chemical classes: cyclic imide and carboxylic acid derivatives. This revi...

Structure-Based Design of Potent Aromatase Inhibitors by High-Throughput Docking

Cytochrome P450 aromatase catalyzes the conversion of androgen substrates into estrogens. Aromatase inhibitors (AIs) have been used as first-line drugs in the treatment of estrogen-dependent breast cancer in postmenopausal women. However, the search for new, more potent, and selective AIs still remains necessary to avoid the risk of possible resistances and reduce toxicity and side effects of current available drugs. The publication of a high resolution X-ray structure of human aromatase has opened the way to structure-based virtual screening to identify new small-molecule inhibitors with structural motifs different from all known AIs. In this context, a high-throughput docking protocol was set up and led to the identification of nanomolar AIs with new core structures.

Oxidative modification of aldose reductase induced by copper ion. Definition of the metal-protein interaction mechanism.

Aldose reductase (ALR2) is susceptible to oxidative inactivation by copper ion. The mechanism underlying the reversible modification of ALR2 was studied by mass spectrometry, circular dichroism, and molecular modeling approaches on the enzyme purified from bovine lens and on wild type and mutant recombinant forms of the human placental and rat lens ALR2. Two equivalents of copper ion were required to inactivate ALR2: one remained weakly bound to the oxidized protein whereas the other was strongly retained by the inactive enzyme. Cys303 appeared to be the essential residue for enzyme inactivation, because the human C303S mutant was the only enzyme form tested that was not inactivated by copper treatment. The final products of human and bovine ALR2 oxidation contained the intramolecular disulfide bond Cys298-Cys303. However, a Cys80-Cys303 disulfide could also be formed. Evidence for an intramolecular rearrangement of the Cys80-Cys303 disulfide to the more stable product Cys298-Cys303 is provided. Molecular modeling of the holoenzyme supports the observed copper sequestration as well as the generation of the Cys80-Cys303disulfide. However, no evidence of conditions favoring the formation of the Cys298-Cys303 disulfide was observed. Our proposal is that the generation of the Cys298-Cys303 disulfide, either directly or by rearrangement of the Cys80-Cys303 disulfide, may be induced by the release of the cofactor from ALR2 undergoing oxidation. The occurrence of a less interactive site for the cofactor would also provide the rationale for the lack of activity of the disulfide enzyme forms.

Application of a post-docking procedure based on MM-PBSA and MM-GBSA on single and multiple protein conformations

In the last decades, molecular docking has emerged as an increasingly useful tool in the modern drug discovery process, but it still needs to overcome many hurdles and limitations such as how to account for protein flexibility and poor scoring function performance. For this reason, it has been recognized that in many cases docking results need to be post-processed to achieve a significant agreement with experimental activities. In this study, we have evaluated the performance of MM-PBSA and MM-GBSA scoring functions, implemented in our post-docking procedure BEAR, in rescoring docking solutions. For the first time, the performance of this post-docking procedure has been evaluated on six different biological targets (namely estrogen receptor, thymidine kinase, factor Xa, adenosine deaminase, aldose reductase, and enoyl ACP reductase) by using i) both a single and a multiple protein conformation approach, and ii) two different software, namely AutoDock and LibDock. The assessment has been based on two of the most important criteria for the evaluation of docking methods, i.e., the ability of known ligands to enrich the top positions of a ranked database with respect to molecular decoys, and the consistency of the docking poses with crystallographic binding modes. We found that, in many cases, MM-PBSA and MM-GBSA are able to yield higher enrichment factors compared to those obtained with the docking scoring functions alone. However, for only a minority of the cases, the enrichment factors obtained by using multiple protein conformations were higher than those obtained by using only one protein conformation.