Characterization of the NPC1L1 gene and proteome from an exceptional responder to ezetimibe.

BACKGROUND: Strategies to reduce LDL-cholesterol involve reductions in cholesterol synthesis or absorption. We identified a familial hypercholesterolemia patient with an exceptional response to the cholesterol absorption inhibitor, ezetimibe. Niemann-Pick C 1-like 1 (NPC1L1) is the molecular target of ezetimibe. METHODS AND RESULTS: Sequencing identified nucleotide changes predicted to change amino acids 52 (L52P), 300 (I300T) and 489 (S489G) in exceptional NPC1L1. In silico analyses identified increased stability and cholesterol binding affinity in L52P-NPC1L1 versus WT-NPC1L1. HEK293 cells overexpressing WT-NPC1L1 or NPC1L1 harboring amino acid changes singly or in combination (Comb-NPC1L1) had reduced cholesterol uptake in Comb-NPC1L1 when ezetimibe was present. Cholesterol uptake was reduced by ezetimibe in L52P-NPC1L1, I300T-NPC1L1, but increased in S489G-NPC1L1 overexpressing cells. Immunolocalization studies found preferential plasma membrane localization of mutant NPC1L1 independent of ezetimibe. Flotillin 1 and 2 expression was reduced and binding to Comb-NPC1L1 was reduced independent of ezetimibe exposure. Proteomic analyses identified increased association with proteins that modulate intermediate filament proteins in Comb-NPC1L1 versus WT-NPC1L1 treated with ezetimibe. CONCLUSION: This is the first detailed analysis of the role of NPC1L1 mutations in an exceptional responder to ezetimibe. The results point to a complex set of events in which the combined mutations were shown to affect cholesterol uptake in the presence of ezetimibe. Proteomic analysis suggests that the exceptional response may also lie in the nature of interactions with cytosolic proteins.

Handheld impedance biosensor system using engineered proteinaceous receptors.

We put forward an impedometric protein-based biosensor platform suitable for point-of-care diagnostics. A hand-held scale impedance reader system is described for the detection of corresponding physiochemical changes as the immobilized proteins bind to the analyte molecules in the proximity of the microfabricated electrodes. Specifically, we study the viability of this approach for glucose biosensing purposes using genetically engineered glucokinase as receptor proteins. The proposed reagent-less biosensor offers a high sensitivity of 0.5 mM glucose concentration level in the physiologically relevant range of 0.5 mM to 7.5 mM with less than 10 s response time.

A combined crystallographic and molecular dynamics study of cathepsin L retrobinding inhibitors.

We report the crystal structures of three noncovalent retrobinding inhibitors in complex with mature cathepsin L up to resolutions of 2.5, 1.8, and 2.5 A, respectively. These inhibitors were Bpa-(Nepsilon-Bpa)Lys-DArg-Tyr-Npe, Bpa-(Nepsilon-Bpa)Lys-DArg-Phe-Npe, and Bpa-MCys-DArg-Phe-Npe, where Bpa = biphenylacetyl and Pea = N-phenylethyl. These were selected to clarify the binding mode of the biphenyl groups in the S' subsites because the addition of a second biphenyl does not improve potency. Examination of the symmetry-related monomers in the crystal structures revealed inhibitor-inhibitor crystal packing interactions. Molecular dynamics simulations were then used to explore the structure and dynamical behavior of the isolated protein-ligand complexes in solution. In the simulations, the backbone biphenyl groups for all three inhibitors ended up in the same location despite having started out in different orientations in the initial crystal structure conformations. The lack of improved potency of the larger inhibitors over the smaller one is attributed to a correspondingly greater entropic cost of binding.

An investigation into CAG repeat length variation and N/C terminal interactions in the T877A mutant androgen receptor found in prostate cancer.

Prostate cancer may progress by circumventing ablation therapy due to mutations in the androgen receptor (AR) gene. The most intensively studied is the T877A mutation in the ligand binding domain (LBD), which causes the AR to become promiscuous, i.e., respond to a number of different ligands. Our investigations have shown that the T877A mutation alters the inverse relationship between CAG repeat length and transactivation in a noticeable albeit minor manner, while increasing N/C terminal interactions. In the presence of beta-catenin, a coactivator over-expressed in prostate cancer, the inverse relationship between CAG repeat length and transactivation is reversed for the wild type (wt) AR as well. We have also used molecular modeling with the AR and FXXLF and LXXLL peptides to investigate N/C terminal and coactivator interactions. In T877A, this approach revealed an increase in the flexibility of amino acid residues in the activation function 2 (AF-2) domain in the LBD, and a larger solvent accessible surface in T877A compared to the wt AR AF-2 domain. Thus, the improved induced fit of the AR N-terminal domain FXXLF-containing peptide into the T877A LBD could be due to the increased flexibility and solvent accessibility of the AF-2 domain. These new observations suggest that the AR CAG effect can be overridden by prostate cancer mutations, and also further our understanding of hormone-refractory prostate cancer by helping to explain the promiscuity of the T877A mutation.

Exploring inhibitor binding at the S' subsites of cathepsin L.

We report a series of noncovalent, reversible inhibitors of cathepsin L that have been designed to explore additional binding interactions with the S' subsites. The design was based on our previously reported crystal structure that suggested the possibility of engineering increased interactions with the S' subsites ( Chowdhury et al. J. Med. Chem. 2002, 45, 5321-5329 ). A representative of these new inhibitors has been co-crystallized with mature cathepsin L, and the structure has been solved and refined at 2.2 A. The inhibitors described in this work extend farther into the S' subsites of cathepsins than any inhibitors reported in the literature thus far. These interactions appear to make use of a S3' subsite that can potentially be exploited for enhanced specificity and/or affinity.

Solvated interaction energy (SIE) for scoring protein-ligand binding affinities. 1. Exploring the parameter space.

We present a binding free energy function that consists of force field terms supplemented by solvation terms. We used this function to calibrate the solvation model along with the binding interaction terms in a self-consistent manner. The motivation for this approach was that the solute dielectric-constant dependence of calculated hydration gas-to-water transfer free energies is markedly different from that of binding free energies (J. Comput. Chem. 2003, 24, 954). Hence, we sought to calibrate directly the solvation terms in the context of a binding calculation. The five parameters of the model were systematically scanned to best reproduce the absolute binding free energies for a set of 99 protein-ligand complexes. We obtained a mean unsigned error of 1.29 kcal/mol for the predicted absolute binding affinity in a parameter space that was fairly shallow near the optimum. The lowest errors were obtained with solute dielectric values of Din = 20 or higher and scaling of the intermolecular van der Waals interaction energy by factors ranging from 0.03 to 0.15. The high apparent Din and strong van der Waals scaling may reflect the anticorrelation of the change in solvated potential energy and configurational entropy, that is, enthalpy-entropy compensation in ligand binding (Biophys. J. 2004, 87, 3035-3049). Five variations of preparing the protein-ligand data set were explored in order to examine the effect of energy refinement and the presence of bound water on the calculated results. We find that retaining water in the final protein structure used for calculating the binding free energy is not necessary to obtain good results; that is the continuum solvation model is sufficient. Virtual screening enrichment studies on estrogen receptor and thymidine kinase showed a good ability of the binding free energy function to recover true hits in a collection of decoys.

Effect of limited solid-state glycation on the conformation of lysozyme by ESI-MSMS peptide mapping and molecular modeling.

Although protein glycation has been implicated in the alteration of protein functionality, both in vivo (in biological systems) and in vitro (in food systems), the effect of the protein-bound glycan moiety on the structure/conformation of proteins that result in the modification of functionality is not clear. In this article, we report a study of the conformational changes of glycated lysozyme using LC-ESI-MSMS peptide mapping, and molecular modeling. A comparison of the RP-HPLC of the tryptic digests of unglycated and glycated lysozyme showed markedly different chromatographic profiles. Analysis of the peptide composition of the chromatographic fractions of the tryptic digests revealed that glycation of lysozyme resulted in the modification of its conformation. Glycation-induced changes in the conformation of lysozyme resulted in the exposure of its active site region to increased proteolytic activity of trypsin. Molecular simulation of triglycated lysozyme also showed that limited glycation of lysozyme caused reorientation of the active site residues (Arg 45, Arg 68, Asn 44, and Trp 62) and increased solvent accessibility into the active site region of the protein. The results of the modeling experiment corroborated the results of the RP-HPLC and ESI-MSMS peptide mapping.

Design of noncovalent inhibitors of human cathepsin L. From the 96-residue proregion to optimized tripeptides.

A novel series of noncovalent inhibitors of cathepsin L have been designed to mimic the mode of autoinhibition of procathepsin L. Just like the propeptide, these peptide-based inhibitors have a reverse-binding mode relative to a substrate and span both the S' and S subsites of the enzyme active site. In contrast to previous studies in which even moderate truncation of the full-length propeptide led to rapid reduction in potency, these blocked tripeptide-sized inhibitors maintain nanomolar potency. Moreover, these short peptides show higher selectivity (up to 310-fold) for inhibiting cathepsin L over K versus only 2-fold selectivity of the 96-residue propeptide of cathepsin L. A 1.9 A X-ray crystallographic structure of the complex of cathepsin L with one of the inhibitors confirms the designed reverse-binding mode of the inhibitor as well as its noncovalent nature. Enzymatic analysis also shows the inhibitors to be resistant to hydrolysis at elevated concentrations of the enzyme. The mode of inhibition of these molecules provides a general strategy for inhibiting other cathepsins as well as other proteases.

Synthesis and testing of 5-benzyl-2,4-diaminopyrimidines as potential inhibitors of leishmanial and trypanosomal dihydrofolate reductase.

Dihydrofolate reductase is a drug target that has not been thoroughly investigated in leishmania and trypanosomes. Work has previously shown that 5-benzyl-2,4-diaminopyrimidines are selective inhibitors of the leishmanial and trypanosome enzymes. Modelling predicted that alkyl/aryl substitution on the 6-position of the pyrimidine ring should increase enzyme activity of 5-benzyl-2,4-diaminopyrimidines as inhibitors of leishmanial and trypanosomal dihydrofolate reductase. Various compounds were prepared and evaluated against both the recombinant enzymes and the intact organisms. The presence of a substituent had a small or negative effect on activity against the enzyme or intact parasites compared to unsubstituted compounds.