Thursday, November 14, 2013

Using water for drug design

Alfonso García-Sosa at the University of Tartu in Estonia has published a paper getting to grips with precisely the question that I have long wanted to see addressed: how to employ water molecules in design strategies for drug binding (J. Chem. Inform. Model. 53, 1388; 2013 – paper here). He has analysed over 2,000 crystal structures of hydrated and non-hydrated ligand-receptor complexes (including many drugs), and finds that bridging water molecules are an effective strategy for tight binding. He concludes that such a binding mechanism could be beneficially targeted, and that “if a tightly bound, bridging water molecule is observed in the binding site, attempts to replace it [in a designed drug that binds competitively] should only be made if the subsequent ligand modification would improve also its ligand efficiency, enthalpy, specificity, and pharmacokinetic properties.” This is just the kind of large-scale study that is needed to extract the advantages or otherwise, and the effective modes, of using water molecules in ligand design.

It seems increasingly likely that the mechanism of hydrophobic association, once considered in terms of a static picture involving expulsion of ‘bound’ water, is in fact associated with dynamical effects, in particular the fluctuations in the hydrogen-bonded network around the hydrophobes. That view is emphatically supported in a paper by Aljaz Godec and Franci Merzel of the National Institute of Chemistry in Ljubljana, working with Jeremy Smith at Oak Ridge (Phys. Rev. Lett. 111, 127801; 2013 – paper here). Their MC simulations of two hydrophobic particles (of about the size of a methane molecule) coming together shows that crossing over the desolvation barrier to the associated state involves a large collective fluctuation in hydration water in which the intervening hydrogen-bonded clusters are mostly displaced from the inner to the outer hydration shell. As the authors conclude, “a complete description of hydrophobic association can be obtained only by explicitly considering collective fluctuations involving many-body correlations between water molecules.”

The observation by Amish Patel, Shekhar Garde and their colleagues that hydrophobic association is faster near a hydrophobic surface, which I have mentioned before in this blog, is fleshed out in detail by these researchers in a new paper (S. Vembanur et al., J. Phys. Chem. B 117, 10261; 2013 – paper here). The key issue is that the interfacial water, like that at the air-liquid interface, is more easily displaced, lowering the desolvation barrier to association. Naturally, this means that hydrophobic surfaces might be used to catalyse aggregation processes. I wonder if it might also help to explain Barry Sharpless’s observations some years back of an increase in organic reaction rates at the air-water interface?

Water diffusion close to lipid membranes is not like that close to solid surfaces, Roland Netz at the Free University of Berlin and colleagues say (Y. von Hansen et al., Phys. Rev. Lett. 111, 118103; 2013 – paper here). Their MD simulations reveal that, whereas at solid surfaces water diffuses faster laterally than perpendicularly, at lipid membranes the opposite is true. This seems to be because the lipids present a rough surfaces, with some head groups protruding from the leaflets, so that lateral diffusion takes place amidst a rough free-energy landscape and is correspondingly hindered. As a result, lateral motion in the fluid phase at the membrane surface involves a distinct perpendicular component to the trajectories: water molecules wander in local energy traps before moving up and away an then “descending” elsewhere.

The dynamics of interfacial water at lipid membranes is also probed by Kevin Kubarych and colleagues at the University of Michigan using IR spectroscopy (D. G. Osborne et al., J. Phys. Chem. B jp4049428 – paper here). They infer this from the spectral diffusion of a labelled cholesterol derivative in the membrane, which reflects the dynamics of its hydration water, and find that these dynamics are three times slower than those of the same molecule in bulk water. The paper largely establishes this as a nice tool that might be used to look at variations in water dynamics at different locations in a membrane.

One explanation for the apparent long-ranged hydrophobic interaction measured between adsorbed monolayers in the surface force apparatus invokes electrostatic interactions between the restructured monolayers themselves (see, for example, Jacob Klein’s work: Phys. Rev. Lett. 96, 038301; 2006 and 109, 168305; 2012). Max Berkowitz and colleagues at UNC investigate this idea using a lattice model to look at how a surfactant coated charged (mica) surface might become reconfigured into heterogeneous structures (C. Eun et al., J. Phys. Chem. B jp405979n – paper here). They find that indeed an initially uniform surfactant monolayer can become transformed into a non-uniform surface covered with patches of surfactant bilayer separated by patches of bare (water-covered) mica. This simple model system might help to understand not just Klein’s studies but also the somewhat related issue of formation of lipid rafts in mixed-lipid membranes.

Another ongoing discussion is the mechanism by which osmolytes stabilize proteins. Piotr Bruzdziak and colleagues at Gdansk University of Technology in Poland examine that issue by using IR spectroscopy and DSC to study the effects of various osmolyes (TMAO, Gly, NMG, DMG) on the stability of lysozyme (P. Bruzdziak et al., J. Phys. Chem. B 117, 11502; 2013 – paper here). In contrast to many recent simulation studies of this problem, the authors here conclude that the osmolyte-protein interaction is indirect, the osmolytes “enhancing” water structure with a consequent “tightening” of protein folding.

OK, why not touch on another controversial topic too: the molecular basis for the dormancy of bacterial spores, and in particular whether this involves an ‘altered state’ of water. That’s addressed in a preprint ( by Bertil Halle at Lund and colleagues, using deuteron magnetic relaxation dispersion to look at the state of water in spores of B. subtilis. Two models for the structure of the core aqueous phase have been proposed: a gel, in which mobile water permeates a macromolecular network, and a glass, in which everything inside the cells (including water) forms a solid amorphous phase. The NMR results clearly support the former picture – the water remains mobile, albeit with rotational motion about 15 times slower than the bulk.

Calcium release-activated calcium (CRAC) channels play an important role in cell signalling, and their dysfunction is associated with cardiac arrythmia and immunodeficiency problems. Thanks to a recent crystal structure of the pore region of a CRAC channel, Michael Klein and colleagues at Temple University have been able to investigate the mechanism of ion permeation through simulations (H. Dong et al., PNAS 110, 17332; 2013 – paper here). They say that a central hydrophobic region of the channel is the crucial region for switching on and off, and that it is the hydration of this region that controls ion transport: a small change in the number and orientation of water molecules here significantly alters the local electrostatic field. In other words, this is another channel that is water-regulated.

Moving onto another channel: the high water permeability of aquaporins may be a result of an optimization of shape at the pore entrance to minimize viscous dissipation, according to a study by Laurent Joly of the University of Lyon 1 and colleagues (S. Gravelle et al., PNAS 110, 16367; 2013 – paper here). Their finite-element calculations suggest that the hour-glass profile of the pore is particularly efficient at reducing dissipation, and that the optimal opening angle of 5-20 degrees is in the range of those observed in the proteins.

Aquaporins have of course a well resolved crystal structure, but many membrane proteins are intrinsically disordered, especially in the regions that extend beyond the lipid membrane and into the extracellular matrix. Songi Han of UCSB and colleagues suggest that hydration dynamics – in their case as measured by Overhauser dynamic nuclear polarization NMR – can provide a proxy for at least locating these extended protein segments (C.-Y. Cheng et al., PNAS 110, 16838; 2013 – paper here). The technique relies on the apparent existence of a gradient of diffusion dynamics up to 3 nm away from the membrane surface (really?). The authors can seemingly also use the technique to look at protein structure parallel to the membrane surface, as in the case of an amyloid-forming protein called alpha-synuclein associated with Parkinson’s disease. They report a sinusoidal variation in water retardation close to the lipid surface which they interpret as the result of the alpha-S coiled into an alpha helix embedded laterally in the membrane with its axis about 1-3 Å below the phosphate head groups, while the C-terminus end floats freely and disordered above the membrane surface.

Water dynamics generally found to be retarded in the vicinity of proteins. But NMR measurements on crystals of the protein Crh (protein catabolite repression Hpr) have indicated that water in the crystals may remain mobile down to at least -30 C – in other words, it seems more mobile than bulk water at these temperatures (Böckmann et al., J. Biomol. NMR 45, 319; 2009). How can this be? To answer that question, Anja Böckmann of the University of Lyon, who did the NMR wirk, has collaborated with Wilfred van Gunsteren at ETH Zurich and colleagues to conduct MD simulations of the crystalline form of Crh (D. Wang et al., J. Phys. Chem. B 117, 11433; 2013 – paper here). They find that indeed while retarded dynamics of hydration water are seen at 291 K, at 200 K (close to the glass transition) the rotational and translational mobility of this water is greater than in bulk. But while introducing artificial perturbations such as rigidifying the protein or switching off protein-solvent electrostatics does induce some enhanced mobility of either translation or rotation, the cause of the overall water mobility at this temperature remains obscure.

While on the subject of glassy low-temperature behaviour, Thomas Loerting of the University of Innsbruck and colleagues have reported a second glass transition in amorphous ice at 116 K, which they say is related to the coexistence of two highly viscous metastable liquid-like phases: a low-density phase with a glass transition of 136 K and a high-density phase which undergoes this second glass transition at 116 K (K. Amann-Winkel et al., PNAS 110, 17720; 2013 – paper here). Thus, they say, the putative HDL and LDL “can both be observed experimentally” – although the results don’t directly address the question of whether the two can be interconverted by a first-order liquid-liquid transition ending at a critical point.

A stark and rather remarkable reminder of how much still remains to be understood about the hydrogen bond is provided by Michele Ceriotti at Oxford and colleagues (PNAS 110, 15591; 2013 – paper here). Their ab initio simulations that incorporate nuclear quantum effects show that the protons in water’s hydrogen bonds undergo fluctuations that take them towards the oxygen acceptor atoms for significant fractions of time, sometimes leading to spontaneous proteolysis. What’s more, these events are strongly correlated among neighbouring hydrogen bonds, so that perturbations to the hydrogen-bonded network seem likely to modulate this distinctly non-classical effect in significant ways.

The discussion continues of how ions alter the dynamics of water in their hydration shells. MD simulations by Ana Vila Verde and Reinhard Lipowsky at the MPI for Colloids and Interfaces in Potsdam look at the effect of ion pairs (here magnesium sulphate and caesium chloride) to see to what extent the slowdown of water dynamics in cooperative between the anion and cation (J. Phys. Chem. B 117, 10556; 2013 – paper here). They find that the cooperative slowdown can be intense (especially for ions of high charge density), but only in the first hydration shell. I note their comment “our results do not support the notion that the Hofmeister series is due primarily to long-range effects of ions on water properties. Instead, they point to the possibility that models of ion solvation may focus primarily on the first hydration layer without excessive loss of accuracy.”

Directional transport of water through a carbon nanotube connecting two reservoirs, driven by electric fields, has been reported in simulations by several teams over the past few years. Hangjun Wu of Zhejiang Normal University in Jinhua and colleagues now add another instance (X. Zhou et al., J. Phys. Chem. B 117, 11681; 2013 – paper here). They show that a vibrating charge that deforms the nanotube away from its halfway point (thus introducing the asymmetry that underpins the ratchet-like effect) will create a directional flux of water. But the direction of that flux is dependent on the displacement of the nanotube wall, and can even change sign when the displacement gets very large.

Daryl Eggers at San José State University continues to develop his picture of solvation water as a reactant in solution equilibria. With Brian Castellano he considers what happens when one includes in the thermodynamic treatment of binding equilibria a representation of how water is perturbed next to solutes (so that it is not ‘equivalent’ on both sides of the equilibrium) (B. M. Castellano & D. K. Eggers, J. Phys. Chem. B 117, 8180; 2013) – paper here). The spirit of the exercise is nicely encapsulated thus: “When water is viewed as a coreactant, it becomes apparent that the traditional equation for the standard-state Gibbs free energy of binding represents an unbalanced equation.” In fact, this means that the equilibria and binding constants become concentration-dependent. Perhaps this might account for discrepancies between different ways of experimentally measuring the enthalpy changes of some reactions.

To what extent can the vibrations of liquid water be decomposed into essentially single-molecule bending and stretching OH modes, plus intermolecular contributions? Less than was thought, according to Andrei Tokmakoff, currently at the University of Chicago, and colleagues (K. Ramasesha et al., Nat. Chem. 5, 935; 2013 – paper here). Their studies using ultrafast 2D IR spectroscopy show that there is strong mixing between all of these modes, so that all the vibrations have some collective component. This will require some significant rethinking of relaxation and energy dissipation in water.


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