Identification of FDA-approved drugs as novel allosteric inhibitors of human executioner caspases.

The regulation of apoptosis is a tightly coordinated process and caspases are its chief regulators. Of special importance are the executioner caspases, caspase-3/7, the activation of which irreversibly sets the cell on the path of death. Dysregulation of apoptosis, particularly an increased rate of cell death lies at the root of numerous human diseases. Although several peptide-based inhibitors targeting the homologous active site region of caspases have been developed, owing to their non-specific activity and poor pharmacological properties their use has largely been restricted. Thus, we sought to identify FDA-approved drugs that could be repurposed as novel allosteric inhibitors of caspase-3/7. In this study, we virtually screened a catalog of FDA-approved drugs targeting an allosteric pocket located at the dimerization interface of caspase-3/7. From among the top-scoring hits we short-listed 5 compounds for experimental validation. Our enzymatic assays using recombinant caspase-3 suggested that 4 out of the 5 drugs effectively inhibited caspase-3 enzymatic activity in vitro with IC50 values ranging ~10-55 µM. Structural analysis of the docking poses show the 4 compounds forming specific non-covalent interactions at the allosteric pocket suggesting that these molecules could disrupt the adjacently-located active site. In summary, we report the identification of 4 novel non-peptide allosteric inhibitors of caspase-3/7 from among FDA-approved drugs.

N-H···N Hydrogen Bonds Involving Histidine Imidazole Nitrogen Atoms: A New Structural Role for Histidine Residues in Proteins.

The amino acid histidine can play a significant role in the structure and function of proteins. Its various functions include enzyme catalysis, metal binding activity, and involvement in cation-π, π-π, salt-bridge, and other types of noncovalent interactions. Although histidine's imidazole nitrogens (Nδ and Nε) are known to participate in hydrogen bond (HB) interactions as an acceptor or a donor, a systematic study of N-H···N HBs with the Nδ/Nε atom as the acceptor has not been conducted. In this study, we have examined two data sets of ultra-high-resolution (data set I) and very high-resolution (data set II) protein structures and identified 28 and 4017 examples of HBs of the N-H···Nδ/Nε type from both data sets involving histidine imidazole nitrogen as the acceptor. In nearly 70% of them, the main-chain N-H bond is the HB donor, and a majority of the examples are from the N-H group separated by two residues (Ni+2-Hi+2) from histidine. Quantum chemical calculations using model compounds were performed with imidazole and N-methylacetamide, and they assumed conformations from 19 examples from data set I with N-H···Nδ/Nε HBs. Basis set superposition error-corrected interaction energies varied from -5.0 to -6.78 kcal/mol. We also found that the imidazole nitrogen of 9% of histidine residues forming N-H···Nδ/Nε interactions in data set II participate in bifurcated HBs. Natural bond orbital analyses of model compounds indicate that the strength of each HB is mutually influenced by the other. Histidine residues involved in Ni+2-Hi+2···Nδi/Nεi HBs are frequently observed in a specific N-terminal capping position giving rise to a novel helix-capping motif. Along with their predominant occurrence in loop segments, we propose a new structural role for histidines in protein structures.

Oxygen-aromatic contacts in intra-strand base pairs: analysis of high-resolution DNA crystal structures and quantum chemical calculations.

Three-dimensional structures of biomolecules are stabilized by a large number of non-covalent interactions and some of them such as van der Waals, electrostatic and hydrogen bond interactions are well characterized. Delocalized π-electron clouds of aromatic residues are known to be involved in cation-π, CH-π, OH-π and π-π interactions. In proteins, many examples have been found in which the backbone carbonyl oxygen of one residue makes close contact with the aromatic center of aromatic residues. Quantum chemical calculations suggest that such contacts may provide stability to the protein secondary structures. In this study, we have systematically analyzed the experimentally determined high-resolution DNA crystal structures and identified 91 examples in which the aromatic center of one base is in close contact (<3.5Ǻ) with the oxygen atom of preceding (Group-I) or succeeding base (Group-II). Examples from Group-I are overwhelmingly observed and cytosine or thymine is the preferred base contributing oxygen atom in Group-I base pairs. A similar analysis of high-resolution RNA structures surprisingly did not yield many examples of oxygen-aromatic contact of similar type between bases. Ab initio quantum chemical calculations on compounds based on DNA crystal structures and model compounds show that interactions between the bases in base pairs with oxygen-aromatic contacts are energetically favorable. Decomposition of interaction energies indicates that dispersion forces are the major cause for energetically stable interaction in these base pairs. We speculate that oxygen-aromatic contacts in intra-strand base pairs in a DNA structure may have biological significance.

Recent Advances in Structure, Function, and Pharmacology of Class A Lipid GPCRs: Opportunities and Challenges for Drug Discovery.

Great progress has been made over the past decade in understanding the structural, functional, and pharmacological diversity of lipid GPCRs. From the first determination of the crystal structure of bovine rhodopsin in 2000, much progress has been made in the field of GPCR structural biology. The extraordinary progress in structural biology and pharmacology of GPCRs, coupled with rapid advances in computational approaches to study receptor dynamics and receptor-ligand interactions, has broadened our comprehension of the structural and functional facets of the receptor family members and has helped usher in a modern age of structure-based drug design and development. First, we provide a primer on lipid mediators and lipid GPCRs and their role in physiology and diseases as well as their value as drug targets. Second, we summarize the current advancements in the understanding of structural features of lipid GPCRs, such as the structural variation of their extracellular domains, diversity of their orthosteric and allosteric ligand binding sites, and molecular mechanisms of ligand binding. Third, we close by collating the emerging paradigms and opportunities in targeting lipid GPCRs, including a brief discussion on current strategies, challenges, and the future outlook.

Imidazole Nitrogens of Two Histidine Residues Participating in N-H···N Hydrogen Bonds in Protein Structures: Structural Bioinformatics Approach Combined with Quantum Chemical Calculations.

Protein structures are stabilized by different types of hydrogen bonds. However, unlike the DNA double helical structure, the N-H···N type of hydrogen bonds is relatively rare in proteins. N-H···N hydrogen bonds formed by imidazole groups of two histidine residues have not been investigated. We have systematically analyzed 5333 high-resolution protein structures with resolution 1.8 Å or better and identified 285 histidine pairs in which the nitrogen atoms of the imidazole side chains can potentially participate in N-H···N hydrogen bonds. The histidine pairs were further divided into two groups, neutral-neutral and protonated-neutral, depending on the protonation state of the donor histidine. Quantum chemical calculations were performed on imidazole groups adopting the same geometry observed in the protein structures. Average interaction energies between the interacting imidazole groups are -6.45 and -22.5 kcal/mol for neutral-neutral and protonated-neutral, respectively. Hydrogen bond interaction between the imidazole moieties is further confirmed by natural bond orbital analyses of the model compounds. Histidine residues involved in N-H···N hydrogen bonds are relatively more buried and have low B-factor values in the protein structures. N-H···N hydrogen bond formed by a pair of buried histidine residues can significantly contribute to the structural stability of proteins.

Comparison of metal-binding strength between methionine and cysteine residues: Implications for the design of metal-binding motifs in proteins.

Metals play vital role in various physiological processes and are bound to biomolecules. Although cysteine sulfur is more frequently found as metal-binding ligand, methionine prefers to occur in copper-binding motifs of some proteins. To address methionine's lower preference in copper-binding sites in comparison to cysteine, we have considered copper-binding motifs (His-Cys-His-Met) from seven different high-resolution protein structures. We performed quantum chemical calculations to find out the strength of interactions between sulfur and metal ion in both Met and Cys residues. In the case of Cys, both neutral (CysH) and the deprotonated form (Cys-) were considered. We used two different levels of theory (B3LYP and M06-2X) and the model compounds methyl propyl sulfide, ethanethiol and ethanethiolate were used to represent Met, CysH and Cys- respectively. To compare the metal-binding strength, we mutated Met in silico to CysH/Cys- and performed the calculations. We also carried out calculations with wild-type Cys present in the same metal-binding motif. On average, interactions of Met with copper ion are stronger by 13-35kcal/mol compared to CysH. However, Cys- interactions with copper is stronger than that of Met by ~250kcal/mol. We then considered the entire metal-binding motif with four residues and calculated the interaction energies with the copper ion. We also considered Met→Cys- mutation in the motif and repeated the calculations. Interaction of the wild-type motif with the copper ion is ~160kcal/mol weaker than that of mutated motif. Our studies suggest the factors that could explain why Met is not as frequently observed as Cys in the metal-binding motifs. Results of these studies will help in designing metal-binding motifs in proteins with varying interaction strengths.

Controlling in Vitro Insulin Amyloidosis with Stable Peptide Conjugates: A Combined Experimental and Computational Study.

Insulin aggregation, to afford amyloidogenic polypeptide fibrils, is an energetically driven, well-studied phenomenon, which presents interesting biological ramifications. These aggregates are also known to form around insulin injection sites and in diabetic patients suffering from Parkinson's disease. Such occurrences force considerable reduction in hormone activity and are often responsible for necrotic deposits in diabetic patients. Changes in physicochemical environment, such as pH, temperature, ionic strength, and mechanical agitation, affect insulin fibrillation, which also presents intrigue from the structural viewpoint. Several reports have tried to unravel underlying mechanisms concerning the aggregation process taking into account a three aromatic amino acid patch Phe(B24)-Phe(B25)-Tyr(B26) located in the C-terminal part of the B chain, identified as a key site for human insulin-receptor interaction. The present study describes design and inhibitory effects of novel peptide conjugates toward fibrillation of insulin as investigated by thioflavin T assay, circular dichroism, and AFM. Possible interaction of insulin with peptide-based fibrillation inhibitors reveals an important role of hydrophobic interactions in the inhibition process. Molecular dynamics simulation studies demonstrate that inhibitor D4 interacts with insulin residues from the helix and the C-terminal extended segment of chain B. These studies present a novel approach for the discovery of stable, peptide-based ligands as novel antiamyloidogenic agents for insulin aggregation.