Gregory V. Nikiforovich

Community-wide assessment of GPCR structure modelling and ligand docking

Recent breakthroughs in the determination of the crystal structures of G protein-coupled receptors (GPCRs) have provided new opportunities for structure-based drug design strategies targeting this protein family. With the aim of evaluating the current status of GPCR structure prediction and ligand docking, a community-wide, blind prediction assessment — GPCR Dock 2008 — was conducted in coordination with the publication of the crystal structure of the human adenosine A2A receptor bound to the ligand ZM241385. Twenty-nine groups submitted 206 structural models before the release of the experimental structure, which were evaluated for the accuracy of the ligand binding mode and the overall receptor model compared with the crystal structure. This analysis highlights important aspects for success and future development, such as accurate modelling of structurally divergent regions and use of additional biochemical insight such as disulphide bridges in the extracellular loops.

Genetic Analysis of the First and Third Extracellular Loops of the C5a Receptor Reveals an Essential WXFG Motif in the First Loop

The extracellular loops of G protein-coupled receptors (GPCRs) frequently contain binding sites for peptide ligands. However, the mechanism of receptor activation following ligand binding and the influence of the extracellular loops in other aspects of receptor function are poorly understood. Here we report a structure-function analysis of the first and third extracellular loops of the human C5a receptor, a GPCR that binds a 74-amino acid peptide ligand. Amino acid substitutions were randomly incorporated into each loop, and functional receptors were identified in yeast. The first extracellular loop contains a large number of positions that cannot tolerate amino acid substitutions, especially residues within the WXFG motif found in many rhodopsin-like GPCRs, yet disruption of these residues does not alter C5a binding affinity. These results demonstrate an unanticipated role for the first extracellular loop, and the WXFG motif in particular, in ligand-mediated activation of the C5a receptor. This motif likely serves a similar role in other GPCRs. The third extracellular loop, in contrast, contains far fewer preserved residues and appears to play a less essential role in receptor activation.

Ratiometric Analysis of Fluorescence Lifetime for Probing Binding Sites in Albumin with Near‐Infrared Fluorescent Molecular Probes

A number of diseases have been linked to abnormal conformation of albumin, a major extracellular protein in blood. Current protein structural analysis requires pure isolated samples, thereby limiting their use for albumin analysis in blood. In this study, we report a new approach for high‐throughput structure‐related analysis of albumin by using the fluorescence lifetime properties of near‐infrared (NIR) polymethine dyes. Based on molecular modeling, polymethine dyes are bound to two binding sites with different polarities on albumin. As a result, an NIR molecular probe exhibits two distinct lifetimes with two corresponding fluorescent fractional contributions. The distribution of fractional contributions along with individual fluorescence lifetimes represents unique parameters for characterizing albumin architecture by ratiometric analysis. After screening a small library of NIR polymethine dyes, we identified and used a polymethine dye with optimal fluorescence lifetime properties to assess structure‐related differences in commercially available bovine serum albumin as model systems. The results show that changes in the lifetime of NIR dyes reflect the perturbation of the tertiary structures of albumin and that albumin prepared by different methods has slightly altered tertiary structures. Because of the reduced absorption of light by blood in the NIR region, the method developed can be used to determine structural changes in albumin in whole blood without prior isolation of the pure protein.

Examination of the conformational meaning of “δ-address” in the dermenkephalin sequence

Summary Comprehensive energy calculations were applied to four opioid-related peptides with different receptor selectivities, namely the δ-selective dermenkephalin (Tyr-D-Met-Phe-His-Leu-Met-Asp-NH 2 , DRE), the μ-selective dermorphin (Tyr-D-Ala-Phe-Gly-Tyr-Pro-Ser-NH 2 , DRM) and their “hybrid” peptides DRM/DRE (Tyr-D-Ala-Phe-Gly-Leu-Met-Asp-NH 2 ) and DRE/DRM (Tyr-D-Met-Phe-His-Tyr-Pro-Ser-NH 2 ). It was shown that the N-terminal tripeptide “μ-messages” in the δ-selective ligands DRE and DRM/DRE can possess similar low energy space arrangements of their functionally important elements (the N-terminal α-amino group and the aromatic moieties of Tyr and Phe), but that these are different from the space arrangement of these moieties in μ-selective DRM and DRE/DRM. These results suggest that the C-terminal tripeptide “δ-address” in DRE may influence the conformation of the “μ-message” in DRM. A refined model for the δ-receptor-bound conformation of DRE is proposed based on these calculations which is similar to that previously suggested for the cyclic δ-selective peptide [D-Pen 2 , D-Pen 5 ]enkephalin (DPDPE). This model also has partial correspondence with the structure of the δ-selective alkaloid naltrindole.

Insight into the Binding Mode for Cyclopentapeptide Antagonists of the CXCR4 Receptor

The finding that the chemokine receptor CXCR4 is involved in T‐cell HIV entry has encouraged the development of antiretroviral drugs targeting this receptor. Additional evidence that CXCR4 plays a crucial role in both angiogenesis and metastasis provides further motivation for the development of a CXCR4 inhibitor for therapeutic applications in oncology. To facilitate the design of such ligands, we have investigated the possible binding modes for cyclopentapeptide CXCR4 antagonists by docking 11 high/medium affinity cyclopentapeptides to a developed three‐dimensional model of the CXCR4 G‐protein‐coupled receptors transmembrane region. These ligands, expected to bind in the same mode to the receptor, were docked in the previously deduced receptor‐bound conformation [Våbenøet al., in press; doi 10.1002/bip.20508]. Ligand–receptor complexes were generated using an automated docking procedure that allowed ligand flexibility. By comparing the resulting ligand poses, only two binding modes common for all 11 compounds were identified. Inspection of these two ligand–receptor complexes identified several CXCR4 contact residues shown by mutation to be interaction sites for ligands and important for HIV gp120 binding. Thus, the results provide further insight into the mechanism by which these cyclopentapeptides block HIV entry as well as a basis for rational design of CXCR4 mutants to map potential contacts with small peptide ligands.

Modeling the possible conformations of the extracellular loops in G-protein-coupled receptors.

This study presents the results of a de novo approach modeling possible conformational dynamics of the extracellular (EC) loops in G‐protein‐coupled receptors (GPCRs), specifically in bovine rhodopsin (bRh), squid rhodopsin (sRh), human β‐2 adrenergic receptor (β2AR), turkey β‐1 adrenergic receptor (β1AR), and human A2 adenosine receptor (A2AR). The approach deliberately sacrificed a detailed description of any particular 3D structure of the loops in GPCRs in favor of a less precise description of many possible structures. Despite this, the approach found ensembles of the low‐energy conformers of the EC loops that contained structures close to the available X‐ray snapshots. For the smaller EC1 and EC3 loops (6–11 residues), our results were comparable with the best recent results obtained by other authors using much more sophisticated techniques. For the larger EC2 loops (25–34 residues), our results consistently yielded structures significantly closer to the X‐ray snapshots than the results of the other authors for loops of similar size. The results suggested possible large‐scale movements of the EC loops in GPCRs that might determine their conformational dynamics. The approach was also validated by accurately reproducing the docking poses for low‐molecular‐weight ligands in β2AR, β1AR, and A2AR, demonstrating the possible influence of the conformations of the EC loops on the binding sites of ligands. The approach correctly predicted the system of disulfide bridges between the EC loops in A2AR and elucidated the probable pathways for forming this system. Proteins 2010.

A minimalistic 3D pharmacophore model for cyclopentapeptide CXCR4 antagonists.

Because of its involvement in HIV entry, the chemokine receptor CXCR4 is an attractive target for antiretroviral drugs. Despite the large number of CXCR4 inhibitors studied, the 3D pharmacophore for binding to CXCR4 remains elusive, mainly as a result of conformational flexibility inherent in the identified ligands. In the present study, an exhaustive systematic exploration of the conformational space for a series of analogs of FC131, a cyclopentapeptide CXCR4 antagonist, has been performed. By comparing the resulting low‐energy conformations using different sets of atoms, specific conformational features common only to the high/medium affinity compounds were identified. These features included the spatial arrangement of three pharmacophoric side chains as well as the orientation of a specific backbone amide bond. Together these features represent a minimalistic 3D pharmacophore model for binding of the cyclopentapeptide antagonists to CXCR4. The model enables rationalization of the experimental affinity data for this class of compounds as well as for the peptidomimetic KRH‐1636.

Structure of the complement factor 5a receptor-ligand complex studied by disulfide trapping and molecular modeling.

Complement factor 5a (C5a) is an anaphylatoxin that acts by binding to a G protein-coupled receptor, the C5aR. The relative orientation of this ligand-receptor pair is investigated here using the novel technique of disulfide trapping by random mutagenesis (DTRM) and molecular modeling. In the DTRM technique, an unpaired cysteine is introduced in the ligand, and a library of randomly mutagenized receptors is screened to identify mutants that introduce a cysteine at a position in the receptor that allows functional interactions with the ligand. By repeating this analysis at six positions of C5a, we identify six unique sets of intermolecular interactions for the C5a-C5aR complex, which are then compared with an independently developed computational three-dimensional model of the complex. This analysis reveals that the interface of the receptor N terminus with the cysteine-containing ligand molecules is selected from a variety of possible receptor conformations that exist in dynamic equilibrium. In contrast, DTRM identifies a single position in the second extracellular loop of the receptor that interacts specifically with a cysteine probe placed in the C-terminal tail of the C5a ligand.

Possible locally driven folding pathways of TC5b, a 20-residue protein

A novel computational procedure for modeling possible locally driven folding pathways by stepwise elongations of the peptide chain was successfully applied to TC5b, a 20‐residue miniprotein. Systematic exploration of the possible locally driven pathways showed that the Trp‐cage structure of TC5b could be obtained by stepwise elongation starting from the noncentral local nucleation centers preexisting in the unfolded state of TC5b. The probable locally driven folding pathway starts with folding of α‐helical fragment 4‐9, followed by formation of the proper three‐dimensional structure of fragment 4‐12, and then 4‐18. Accordingly, the Trp‐cage‐forming interactions emerge successively, first Trp6–Pro12, then Trp6–Pro18, and then Trp6–Tyr3. The Trp‐cage‐like structures of TC5b found in this study by independent energy calculations are in excellent agreement with the NMR experimental data. The same procedure rationalizes the incomplete Trp‐cage formation observed for two analogs of TC5b. Generally, the success of this novel approach is encouraging and provides some justification for the use of computational simulations of locally driven protein folding. Proteins 2003;52:292–302.

Conformational analysis of β-methyl-para-nitrophenylalanine stereoisomers of cyclo[D-Pen2, D-Pen5]enkephalin by NMR spectroscopy and conformational energy calculations

Solution conformations of beta-methyl-para-nitrophenylalanine4 analogues of the potent delta-opioid peptide cyclo[D-Pen2, D-Pen5]enkephalin (DPDPE) were studied by combined use of nmr and conformational energy calculations. Nuclear Overhauser effect connectivities and 3JHNC alpha H coupling constants measured for the (2S, 3S)-, (2S, 3R)-, and (2R, 3R)-stereoisomers of [beta-Me-p-NO2Phe4]DPDPE in DMSO were compared with low energy conformers obtained by energy minimization in the Empirical Conformational Energy Program for Peptides (ECEPP/2) force field. The conformers that satisfied all available nmr data were selected as probable solution conformations of these peptides. Side-chain rotamer populations, established using homonuclear (3JH alpha H beta) and heteronuclear (3JH alpha C gamma) coupling constants and 13C chemical shifts, show that the beta-methyl substituent eliminates one of the three staggered rotamers of the torsion angle chi 1 for each stereoisomer of the beta-Me-p-NO2Phe4. Similar solution conformations were suggested for the L-Phe4-containing (2S, 3S)- and (2S, 3R)-stereoisomers. Despite some local differences, solution conformations of L- and D-Phe4-containing analogues have a common shape of the peptide backbone and allow similar orientations of the main delta-opioid pharmacophores. This type of structure differs from several models of the solution conformations of DPDPE, and from the model of biologically active conformations of DPDPE suggested earlier. The latter model is allowed for the potent (2S, 3S)- and (2S, 3R)-stereoisomers of [beta-Me-p-NO2Phe4]DPDPE, but it is forbidden for the less active (2R, 3R)- and (2R, 3S)-stereoisomers. It was concluded that the biologically active stereoisomers of [beta-Me-p-NO2Phe4]DPDPE in the delta-receptor-bound state may assume a conformation different from their favorable conformations in DMSO.