Arnold T. Hagler

Chemoinformatics and Drug Discovery

This article reviews current achievements in the field of chemoinformatics and their impact on modern drug discovery processes. The main data mining approaches used in cheminformatics, such as descriptor computations, structural similarity matrices, and classification algorithms, are outlined. The applications of cheminformatics in drug discovery, such as compound selection, virtual library generation, virtual high throughput screening, HTS data mining, and in silico ADMET are discussed. At the conclusion, future directions of chemoinformatics are suggested.

Keeping it in the family: folding studies of related proteins

Investigators have recently turned to studies of protein families to shed light on the mechanism of protein folding. In small proteins for which detailed analysis has been performed, recent studies show that transition-state structure is generally conserved. The number and structures of populated folding intermediates have been found to vary in homologous families of larger (greater than 100-residue) proteins, reflecting a balance of local and global interactions.

Generation of receptor structural ensembles for virtual screening using binding site shape analysis and clustering.

Accounting for protein flexibility is an essential yet challenging component of structure‐based virtual screening. Whereas an ideal approach would account for full protein and ligand flexibility during the virtual screening process, this is currently intractable using available computational resources. An alternative is ensemble docking, where calculations are performed on a set of individual rigid receptor conformations and the results combined. The primary challenge associated with this approach is the choice of receptor structures to use for the docking calculations. In this work, we show that selection of a small set of structures based on clustering on binding site volume overlaps provides an efficient and effective way to account for protein flexibility in virtual screening. We first apply the method to crystal structures of cyclin‐dependent kinase 2 and HIV protease and show that virtual screening for ensembles of four cluster representative structures yields consistently high enrichments and diverse actives. We then apply the method to a structural ensemble of the androgen receptor generated with molecular dynamics and obtain results that are in agreement with those from the crystal structures of cyclin‐dependent kinase 2 and HIV protease. This work provides a step forward in the incorporation of protein flexibility into structure‐based virtual screening.

Sequence and structural analysis of cellular retinoic acid‐binding proteins reveals a network of conserved hydrophobic interactions

Abstract Proteins in the intracellular lipid‐binding protein (iLBP) family show remarkably high structural conservation despite their low‐sequence identity. A multiple‐sequence alignment using 52 sequences of iLBP family members revealed 15 fully conserved positions, with a disproportionately high number of these (n=7) located in the relatively small helical region. The conserved positions displayed high structural conservation based on comparisons of known iLBP crystal structures. It is striking that the β‐sheet domain had few conserved positions, despite its high structural conservation. This observation prompted us to analyze pair‐wise interactions within the β‐sheet region to ask whether structural information was encoded in interacting amino acid pairs. We conducted this analysis on the iLBP family member, cellular retinoic acid‐binding protein I (CRABP I), whose folding mechanism is under study in our laboratory. Indeed, an analysis based on a simple classification of hydrophobic and polar amino acids revealed a network of conserved interactions in CRABP I that cluster spatially, suggesting a possible nucleation site for folding. Significantly, a small number of residues participated in multiple conserved interactions, suggesting a key role for these sites in the structure and folding of CRABP I. The results presented here correlate well with available experimental evidence on folding of CRABPs and their family members and suggest future experiments. The analysis also shows the usefulness of considering pair‐wise conservation based on a simple classification of amino acids, in analyzing sequences and structures to find common core regions among homologues. Proteins 2003.

Discovery of New Selective Human Aldose Reductase Inhibitors through Virtual Screening Multiple Binding Pocket Conformations

Aldose reductase reduces glucose to sorbitol. It plays a key role in many of the complications arising from diabetes. Thus, aldose reductase inhibitors (ARI) have been identified as promising therapeutic agents for treating such complications of diabetes, as neuropathy, nephropathy, retinopathy, and cataracts. In this paper, a virtual screening protocol applied to a library of compounds in house has been utilized to discover novel ARIs. IC50s were determined for 15 hits that inhibited ALR2 to greater than 50% at 50 μM, and ten of these have an IC50 of 10 μM or less, corresponding to a rather substantial hit rate of 14% at this level. The specificity of these compounds relative to their cross-reactivity with human ALR1 was also assessed by inhibition assays. This resulted in identification of novel inhibitors with IC50s comparable to the commercially available drug, epalrestat, and greater than an order of magnitude better selectivity.

Allosteric mechanisms of nuclear receptors: insights from computational simulations.

The traditional structural view of allostery defines this key regulatory mechanism as the ability of one conformational event (allosteric site) to initiate another in a separate location (active site). In recent years computational simulations conducted to understand how this phenomenon occurs in nuclear receptors (NRs) has gained significant traction. These results have yield insights into allosteric changes and communication mechanisms that underpin ligand binding, coactivator binding site formation, post-translational modifications, and oncogenic mutations. Moreover, substantial efforts have been made in understanding the dynamic processes involved in ligand binding and coregulator recruitment to different NR conformations in order to predict cell/tissue-selective pharmacological outcomes of drugs. They also have improved the accuracy of in silico screening protocols so that nowadays they are becoming part of optimisation protocols for novel therapeutics. Here we summarise the important contributions that computational simulations have made towards understanding the structure/function relationships of NRs and how these can be exploited for rational drug design.

Diversity of Quaternary Structures Regulates Nuclear Receptor Activities

Nuclear receptors (NRs) form homo- and/or heterodimers as central scaffolds of multiprotein complexes, which activate or repress gene transcription to regulate development, homeostasis, and metabolism. Recent studies on NR quaternary structure reveal novel mechanisms of receptor dimerization, the existence of tetrameric chromatin-bound NRs, and previously unanticipated protein-protein/protein-DNA interactions.

Local and Long-Range Sequence Contributions to the Folding of a Predominantly β-Sheet Protein

Understanding how a linear sequence of amino acids guides acquisition of the native state of a protein — the protein folding problem — is key to interpreting information present in the human genome and to developing therapeutic approaches to diseases caused by protein misfolding. While considerable progress has been made in elucidating the interplay of local and long-range forces in the folding of α-helical proteins, such understanding has lagged for primarily β-sheet proteins [1]. We have undertaken studies aimed at determining the roles of local and global sequence information in the folding of the predominantly β-sheet protein, cellular retinoic acid binding protein I (CRABP I). CRABP I is a 136 residue β-clamshell protein (Figure 1) whose physiological role is the sequestration and transport of the hydrophobic ligand retinoic acid. CRABP I is a member of the large family of intracellular lipid binding proteins (iLBPs) [2], which contains 52 members with sequence identity greater than 30% with respect to CRABP I. Past work in our laboratory using stopped-flow (SF) mixing to follow the fluorescence of CRABP I has allowed us to define a series of kinetic phases from the urea-denatured conformational ensemble to the native fold [3]. Within the ≈10 ms dead time of the SF instrument, the unfolded ensemble forms a hydro-phobically collapsed state with considerable secondary structure. The presence of significant secondary structure along with hydrophobic collapse suggests that both global and local forces are acting in the earliest folding events.