Physicochemical prediction of a brain-blood distribution profile in polycyclic amines

Recent investigation into the pharmacological character of the pentacyclo[5.4.0.0(2,6).0(3,10).0(5,9)]undecyl and related polycyclic amines has revealed interesting facts regarding their possible use as neuroprotective agents. At this stage however, a clear shortcoming in the quest for further development of this novel class of compounds is the lack of concrete data on their ability to cross the blood-brain barrier (BBB). Working towards the aim of predicting BBB permeability, a series of related N-substituted 8-amino-8,11-oxapentacyclo[5.4.0.0(2,6).0(3,10).0(5,9)]undecanes were synthesised. Compounds were characterised by both experimental and calculative methods, followed by biological assessment and statistical manipulation of the results obtained. In doing so, a simple biological model was established for the comparative evaluation of brain-blood distribution properties within the class. A highly sensitive ESI-MS.MS analytical procedure was developed for the detection of these compounds in biological tissues, indicating significant drug concentrations in the brain after intraperitoneal administration to C57Bl/6 mice. Stepwise multiple linear regression analysis of all data yielded two meaningful models (R(2)=0.9996 and R(2)=0.7749) depicting lipophilicity (log P(oct)), solvent accessible molecular volume (SV), molar refractivity (MR) and system energy as the prime determinants of the brain-blood profile for these amines. The inherently high lipophilicity potential within the series is attributed to strong hydrophobic influences dominating hydrogen bonding effects. A possible conformational and energy dependent preference at the site of permeation is also suggested. The proposed estimations allow for the expedient and reliable prediction of brain partitioning behaviour for related polycyclic amines, facilitating the early rejection of unsuitable candidates and enabling research to focus on neuroprotective activity.     

An automatic method for predicting transmembrane protein structures using cryo-EM and evolutionary data

The transmembrane (TM) domains of many integral membrane proteins are composed of alpha-helix bundles. Structure determination at high resolution (<4 A) of TM domains is still exceedingly difficult experimentally. Hence, some TM-protein structures have only been solved at intermediate (5-10 A) or low (>10 A) resolutions using, for example, cryo-electron microscopy (cryo-EM). These structures reveal the packing arrangement of the TM domain, but cannot be used to determine the positions of individual amino acids. The observation that typically, the lipid-exposed faces of TM proteins are evolutionarily more variable and less charged than their core provides a simple rule for orienting their constituent helices. Based on this rule, we developed score functions and automated methods for orienting TM helices, for which locations and tilt angles have been determined using, e.g., cryo-EM data. The method was parameterized with the aim of retrieving the native structure of bacteriorhodopsin among near- and far-from-native templates. It was then tested on proteins that differ from bacteriorhodopsin in their sequences, architectures, and functions, such as the acetylcholine receptor and rhodopsin. The predicted structures were within 1.5-3.5 A from the native state in all cases. We conclude that the computational method can be used in conjunction with cryo-EM data to obtain approximate model structures of TM domains of proteins for which a sufficiently heterogeneous set of homologs is available. We also show that in those proteins in which relatively short loops connect neighboring helices, the scoring functions can discriminate between near- and far-from-native conformations even without the constraints imposed on helix locations and tilt angles that are derived from cryo-EM.     

The structural context of disease-causing mutations in gap junctions

Gap junctions form intercellular channels that mediate metabolic and electrical signaling between neighboring cells in a tissue. Lack of an atomic resolution structure of the gap junction has made it difficult to identify interactions that stabilize its transmembrane domain. Using a recently computed model of this domain, which specifies the locations of each amino acid, we postulated the existence of several interactions and tested them experimentally. We introduced mutations within the transmembrane domain of the gap junction-forming protein connexin that were previously implicated in genetic diseases and that apparently destabilized the gap junction, as evidenced here by the absence of the protein from the sites of cell-cell apposition. The model structure helped identify positions on adjacent helices where second-site mutations restored membrane localization, revealing possible interactions between residue pairs. We thus identified two putative salt bridges and one pair involved in packing interactions in which one disease-causing mutation suppressed the effects of another. These results seem to reveal some of the physical forces that underlie the structural stability of the gap junction transmembrane domain and suggest that abrogation of such interactions bring about some of the effects of disease-causing mutations.     

RosettaDock in CAPRI rounds 6-12

A challenge in protein-protein docking is to account for the conformational changes in the monomers that occur upon binding. The RosettaDock method, which incorporates sidechain flexibility but keeps the backbone fixed, was found in previous CAPRI rounds (4 and 5) to generate docking models with atomic accuracy, provided that conformational changes were mainly restricted to protein sidechains. In the recent rounds of CAPRI (6-12), large backbone conformational changes occur upon binding for several target complexes. To address these challenges, we explicitly introduced backbone flexibility in our modeling procedures by combining rigid-body docking with protein structure prediction techniques such as modeling variable loops and building homology models. Encouragingly, using this approach we were able to correctly predict a significant backbone conformational change of an interface loop for Target 20 (12 A rmsd between those in the unbound monomer and complex structures), but accounting for backbone flexibility in protein-protein docking is still very challenging because of the significantly larger conformational space, which must be surveyed. Motivated by these CAPRI challenges, we have made progress in reformulating RosettaDock using a "fold-tree" representation, which provides a general framework for treating a wide variety of flexible-backbone docking problems.     

Growth Inhibition of Plasmodium Falciparum Involving Carbon Centered Iron-Chelate Radical (L., X-)-Fe(III) Based on Pyridoxal-Betaine. A Novel Type of Antimalarials Active Against Chloroquine-Resistant Parasites

Malaria parasites have been shown to be more susceptible to oxidative stress than their host erythro- cytes. In the present work, a chloroquine resistant malaria parasite, Plasmodium falciparum (FCR-3) was found to be susceptible in vitro to a pyridoxal based iron chelator — (l-[N-ethoxycarbonylmethyl- pyridoxlidenium]-2-[2'-pyridyl] hydrazine bromide — (code named L2-9). 2h exposure to 20μM L2-9 was sufficient to irreversibly inhibit parasite growth. Desferrioxamine blocked the drug effect, indicating the requirement for iron. Oxygen however, was not essential. Spectrophotometric analysis showed that under anoxic conditions, L2-9-Fe(II) chelate undergoes an intramolecular redox reaction which presumably involves a one electron transfer and is expected to result in the formation of free radical. Spin trapping coupled to electron spin resonance (ESR) studies of L2-9-iron chelate showed that L2-9-Fe(II) produced free radicals both in the presence and absence of cells, while L2-9-Fe(III) produced free radicals only in the presence of actively metabolising cells.     

Electrical and mechanical response in biventricular mechanical alternans.

Mechanical alternans of various degrees is produced by rapid heart rates, slower rates in failing hearts and can be brought about by a single extra systole. It has also been shown that the two ventricles may exhibit different degrees of mechanical alternation. The present study was planned to clarify the possible mechanism inducing this latter phenomenon. For this reason myocardial tension was recorded simultaneously from the two ventricles as well as through a miniature strain gage capable of measuring electrogram and myocardial tension of a small area -- just adjacent to a stimulating electrode. The heart was driven at a steady heart rate through one electrode and very late premature beats were applied at various coupling times at another site through an electrode attached to the miniature strain gage. It was found that the degree of mechanical alternans is markedly different at the sites of measurements in either ventricle. These changes could be related to the time interval elapsed between the application of the electrical stimulus and the occurrence of the mechanical response.