Drug design for ever, from hype to hope.

In its first 25 years JCAMD has been disseminating a large number of techniques aimed at finding better medicines faster. These include genetic algorithms, COMFA, QSAR, structure based techniques, homology modelling, high throughput screening, combichem, and dozens more that were a hype in their time and that now are just a useful addition to the drug-designers toolbox. Despite massive efforts throughout academic and industrial drug design research departments, the number of FDA-approved new molecular entities per year stagnates, and the pharmaceutical industry is reorganising accordingly. The recent spate of industrial consolidations and the concomitant move towards outsourcing of research activities requires better integration of all activities along the chain from bench to bedside. The next 25 years will undoubtedly show a series of translational science activities that are aimed at a better communication between all parties involved, from quantum chemistry to bedside and from academia to industry. This will above all include understanding the underlying biological problem and optimal use of all available data.

Modeling GPCRs.

Many GPCR models have been built over the years for many different purposes, of which drug-design undoubtedly has been the most frequent one. The release of the structure of bovine rhodopsin in August 2000 enabled us to analyze models built before that period to learn things for the models we build today. We conclude that the GPCR modeling field is riddled with "common knowledge". Several characteristics of the bovine rhodopsin structure came as a big surprise, and had obviously not been predicted, which led to large errors in the models. Some of these surprises, however, could have been predicted if the modelers had more rigidly stuck to the rule that holds for all models, namely that a model should explain all experimental facts, and not just those facts that agree with the modeler's preconceptions.

Privileged structures in GPCRs.

Certain kinds of ligand substructures recur frequently in pharmacologically successful synthetic compounds. For this reason they are called privileged structures. In seeking an explanation for this phenomenon, it is observed that the privileged structure represents a generic substructure that matches commonly recurring conserved structural motifs in the target proteins, which may otherwise be quite diverse in sequence and function. Using sequence-handling tools, it is possible to identify which other receptors may respond to the ligand, as dictated on the one hand by the nature of the privileged substructure itself or by the rest of the ligand in which a more specific message resides. It is suggested that privileged structures interact with the partially exposed receptor machinery responsible for the switch between the active and inactive states. Depending on how they have been designed to interact, one can predispose these substructures to favour either one state or the other; thus privileged structures can be used to create either agonists or antagonists. In terms of the mechanism of recognition, the region that the privileged structures bind to are rich in aromatic residues, which explains the prevalence of aromatic groups and atoms such as sulphur or halogens in many of the ligands. Finally, the approach described here can be used to design drugs for orphan receptors whose function has not yet been established experimentally.

Identification and surveillance of antimicrobial resistance dissemination in animal production.

Antimicrobial resistance is a growing problem in human medicine, and concern has been expressed that use of antimicrobials in animals may be a contributing factor. Although the majority of human pathogens showing antibiotic resistance have no link with animals, the issue of animal use of antimicrobials remains controversial, particularly with respect to antibiotic growth promoters (AGP). The European Union (EU) has withdrawn as AGP some compounds that remain in use in the United Sates. This difference in availability allows comparisons to be made of antimicrobial resistance outcomes with and without use of an AGP. Such comparisons so far show little apparent measurable benefit to human health resulting from the EU removal of AGP, and there is evidence of increased use of therapeutic antibiotics in animals to treat an apparent increased incidence of clinical disease. Microbial risk assessments are important in judging quantitatively or qualitatively whether the risk of using a particular AGP is acceptable in terms of potential hazard to human health. Resistance surveillance is an essential part of such microbial risk assessments, but such surveillance should be carefully planned to avoid confounding factors that could invalidate any conclusions.

Veterinary use of antimicrobials and emergence of resistance in zoonotic and sentinel bacteria in the EU.

Antimicrobials are essential for treatment of sick animals, but even if used correctly, may eventually lead to antimicrobial resistance. While this represents a potential hazard to humans, the great majority of resistant human pathogens, especially the more important ones, are unrelated to animal sources. A survey of informed medical opinion suggested that of the human antimicrobial resistance problem, <4% was seen as potentially linked to animal sources. This proportion related largely to zoonotic bacteria which by definition have the capacity to carry resistance between species, although the evidence for resulting harm remains limited. A recent study compared resistance among chicken, pig and cattle isolates of Salmonella spp., Campylobacter spp. and Escherichia coli from a series of EU countries. When tested against antimicrobial agents, this survey showed variation of resistance between countries, between hosts and between organisms. Such variation may give insight into preferred methods of antimicrobial administration or disease control, but it is clear that the epidemiology of antimicrobial resistance induction and dissemination in animals remains complex and is yet to be fully understood.

An analysis of inhibitory junction potentials in the guinea-pig proximal colon.

Intracellular recordings were made from either sheets or isolated bundles of the circular muscle layer of guinea-pig proximal colon and the responses evoked by stimulating inhibitory nerve fibres were analysed. Inhibitory junction potentials (IJPs), evoked by single stimuli, had two components which could be separated on their pharmacological and temporal characteristics and their voltage sensitivities. The initial component, which was abolished by apamin and reduced in amplitude by pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid (PPADS), had a brief time course: its amplitude was changed when the external concentration of potassium ions ([K+](o)) was changed. The second component of the IJP had a slower onset than the first component, was abolished by l-nitroarginine (NOLA) and oxadiazolo quinoxalin-1-one (ODQ), an inhibitor of soluble guanylate cyclase: its amplitude was little affected by changing [K+](o) and was increased when the membrane potential of the circular layer was hyperpolarized. The observations suggest that the initial component of the IJP results from the release of ATP which triggers an increase in membrane conductance to K+ and that the second component results from the release of nitric oxide which suppresses a background inward current.

Murine intestinal migrating motor complexes: longitudinal components.

Spontaneous migrating contractions have been described in the circular muscle of the isolated mouse colon and terminal ileum, however, spontaneous events equivalent to these have not been reported in the longitudinal muscle. The longitudinal muscle shortenings in the colon and ileum, which are of similar form, frequency and pharmacology to the circular muscle colonic and ileal migrating motor complexes (CMMCs and IMMCs), are recorded in the present study. The spontaneous ileal and colonic longitudinal muscle shortenings appear to be neurally organized as they are abolished by tetrodotoxin (1 micro mol L-1), hexamethonium (500 micro mol L-1) and morphine (1 micro mol L-1). Endogenously released nitric oxide slowed the frequency of spontaneous ileal and colonic longitudinal muscle shortenings and 5-hydroxytryptamine increased their frequency. Hyoscine (1 micro mol L-1) abolished longitudinal shortenings in the ileum and reduced the amplitude of longitudinal shortening by approximately 44% in the colon. Shortenings were effectively abolished by nifedipine (1 micro mol L-1). Surgical sectioning of the colon identified that each region of the colon contracted longitudinally in an independent fashion; the distal colon contracted to the greatest amplitude and lowest frequency. The longitudinal preparation is suitable to initially assess the actions of novel pharmacological agents on spontaneous, neurally coordinated, CMMCs and IMMCs in emptied isolated murine intestines.

Motility in the isolated mouse colon: migrating motor complexes, myoelectric complexes and pressure waves.

This study has used mechanical, together with pressure/volume recordings or electrophysiological recordings, to investigate the spontaneous activity in isolated preparations of mouse colon. In the former preparations, when not distended with fluid, spontaneous colonic migrating motor complexes (CMMCs) were observed using isotonic transducers. When the colons were distended with fluid, CMMCs continued at an increased frequency and in addition were associated temporally, with rises in intraluminal pressure and pulses of distally ejected fluid. 5-Hydroxytryptamine (1 micro mol L-1) or NG-nitro-l-arginine (100 micro mol L-1) increased the frequency of propulsive activity and this activity was abolished by hexamethonium (500 micro mol L-1). In a second preparation, myoelectric complexes recorded from circular muscle cells in colons using intracellular microelectrodes, were found to correlate in frequency and phase with CMMCs. The experiments indicate that CMMCs are intimately related to pressure waves in the fluid-filled viscus and the muscle membrane potential changes that have been recorded during myoelectric complexes are likely to be analogous to those occurring during fluid-filled propulsive activity.

Enteric nerve stimulation evokes a premature colonic migrating motor complex in mouse.

The effects of enteric nerve stimuli were investigated on spontaneously occurring colonic migrating motor complexes (CMMCs) in isolated mouse colon. Changes in circular smooth muscle tension were recorded simultaneously from the proximal, mid and distal regions of an in vitro preparation of whole mouse colon at 36 +/- 1 degrees C. The CMMCs were recorded from all preparations with a mean interval between contractions ranging from 135.2 +/- 9.3 to 163.3 +/- 22.4 s. The CMMCs migrated spontaneously from the proximal to distal colon and were abolished by tetrodotoxin (1 micromol L-1). In approximately half of all trials (57 of 103, n = 31), trains of stimuli (20 Hz, 2-5 s, 1 ms, 40-70 V) delivered to the mid or distal regions of colon, during the intervals between CMMCs, elicited a premature CMMC. However, similar trains of stimuli delivered to the proximal colon were without similar effects (33 trials, n = 13). It is suggested that in isolated whole mouse colon, CMMCs can be evoked prematurely by trains of electrical stimuli applied to the enteric nerves. The observation that nerve stimuli failed to evoke a premature CMMC from the proximal colon suggests that selective activation of functional ascending pathways may be required to initiate a premature CMMC.

Construction of the simplest model to explain complex receptor activation kinetics.

We study the mathematical solutions to the kinetic equations arising from various simple ligand-receptor [corrected] models. Focusing on the prediction of the various models for the activity vs. concentration curve, we find that solutions to the kinetic equations arising from the so-called dimer model exhibit features observed in some experiments, most noticeably a distinct maximum in the activity curve.

Neural integrity is essential for the propagation of colonic migrating motor complexes in the mouse.

The mechanisms that underlie the propagation of contractions along the colon are uncertain. We have examined whether spontaneous colonic migrating motor complexes (CMMCs) migrate through a region of muscle paralysis, or through a region where neural transmission was disrupted in the isolated mouse colon. Mouse colon was mounted in a separately perfused three-compartment organ bath and recordings of circular muscle tension were made. Drug application was restricted to the middle compartment. Application of nifedipine (1 micromol L(-1)), an l-type calcium channel antagonist, reduced the contraction amplitude by approximately 94%, without affecting the form of contractions in the proximal and distal compartments. Moreover, CMMCs appeared to remain temporally related in all compartments. In contrast, interruption of neural transmission in the middle compartment by either tetrodotoxin (1.6 micromol L(-1)), hexamethonium (500 micromol L(-1)) or a low-calcium, high-magnesium solution abolished CMMCs in this compartment; contractions recorded in the proximal and distal compartments became slower in frequency and were no longer synchronized. The experiments suggest that there may be more than one 'pacemaker' generating spontaneous CMMCs and that CMMCs can migrate through a region where there is minimal tension generation, but not through a region where neural integrity has been compromised.

Lipid-facing correlated mutations and dimerization in G-protein coupled receptors.

A correlated mutation analysis has been performed on the aligned protein sequences of a number of class A G-protein coupled receptor families, including the chemokine, neurokinin, opioid, somatostatin, thyrotrophin and the whole biogenic amine family. Many of the correlated mutations are observed flanking or neighbouring conserved residues. The correlated residues have been plotted onto the transmembrane portion of the rhodopsin crystal structure. The structure shows that a significant proportion of the correlated mutations are located on the external (lipid-facing) region of the helices. The occurrence of these highly correlated patterns of change amongst the external residues suggest that they are sites for protein-protein interactions. In particular, it is suggested that the correlated residues may be involved in either large conformational changes, the formation of heterodimers or homodimers (which may be domain swapped) or oligomers required for activation or internalization. The results are discussed in the light of the subtype-specific heterodimerization observed for the chemokine, opioid and somatostatin receptors.

Dimerization of G-protein-coupled receptors.

The evolutionary trace (ET) method, a data mining approach for determining significant levels of amino acid conservation, has been applied to over 700 aligned G-protein-coupled receptor (GPCR) sequences. The method predicted the occurrence of functionally important clusters of residues on the external faces of helices 5 and 6 for each family or subfamily of receptors; similar clusters were observed on helices 2 and 3. The probability that these clusters are not random was determined using Monte Carlo techniques. The cluster on helices 5 and 6 is consistent with both 5,6-contact and 5,6-domain swapped dimer formation; the possible equivalence of these two types of dimer is discussed because this relates to activation by homo- and heterodimers. The observation of a functionally important cluster of residues on helices 2 and 3 is novel, and some possible interpretations are given, including heterodimerization and oligomerization. The application of the evolutionary trace method to 113 aligned G-protein sequences resulted in the identification of two functional sites. One large, well-defined site is clearly identified with adenyl cyclase, beta/gamma and regulator of G-protein signaling (RGS) binding. The other G-protein functional site, which extends from the ras-like domain onto the helical domain, has the correct size and electrostatic properties for GPCR dimer binding. The implications of these results are discussed in terms of the conformational changes required in the G-protein for activation by a receptor dimer. Further, the implications of GPCR dimerization for medicinal chemistry are discussed in the context of these ET results.

Influence of a lipid interface on protein dynamics in a fungal lipase.

Lipases catalyze lipolytic reactions and for optimal activity they require a lipid interface. To study the effect of a lipid aggregate on the behavior of the enzyme at the interfacial plane and how the aggregate influences an attached substrate or product molecule in time and space, we have performed molecular dynamics simulations. The simulations were performed over 1 to 2 ns using explicit SPC water. The interaction energies between protein and lipid are mainly due to van der Waals contributions reflecting the hydrophobic nature of the lipid molecules. Estimations of the protonation state of titratable residues indicated that the negative charge on the fatty acid is stabilized by interactions with the titratable residues Tyr-28, His-143, and His-257. In the presence of a lipid patch, the active site lid opens wider than observed in the corresponding simulations in an aqueous environment. In that lid conformation, the hydrophobic residues Ile-85, Ile-89, and Leu-92 are embedded in the lipid patch. The behavior of the substrate or product molecule is sensitive to the environment. Entering and leaving of substrate molecules could be observed in presence of the lipid patch, whereas the product forms strong hydrogen bonds with Ser-82, Ser-144, and Trp-88, suggesting that the formation of hydrogen bonds may be an important contribution to the mechanism by which product inhibition might take place.

Dynamics of the substrate binding pocket in the presence of an inhibitor covalently attached to a fungal lipase.

To gain insight into the mobility of the occupied ligand-binding pocket of the Rhizomucor miehei lipase we have conducted a rigorous molecular dynamics analysis. The covalently attached inhibitor, ethylhexylphosphonate, was employed as a mimic of the putative tetrahedral intermediate in the esterolytic reaction. Our results show that in this lipase, ligand recognition is influenced by the flexibility of the binding pocket, a feature that is common to many other enzymes. Several regions around the active site were found to move significantly to adapt to the inhibitor. These motions are correlated to the flexibility of the inhibitor. In particular, the hexyl chain of the inhibitor shows considerable mobility, and adjacent residues in the binding cleft accommodate to this flexibility. Pronounced fluctuations in the binding pocket induced by the flexibility of the inhibitor are observed in the hinge region F79-S82, the active site loop region W88-V95 and the protein regions P209-F215/H257-Y260. The flexibility in the regions F79-S82 and H257-Y260, where the shorter ethyl chain is located, indicates that additional space in this binding cleft region is available for accommodating a larger moiety. Fluctuations in the region W88-V95 and P209-F215 are due to the relatively short flexible hexyl carbon chain. This part of the binding pocket could be stiffened by the presence of a longer carbon chain. Though the inhibitor is covalently attached through the phosphonate moiety, interaction of the remainder of the molecule and the enzyme are determined by hydrophobic interactions, where the Van der Waals energies are approximately 25% lower than the electrostatic contributions.

Ongoing nicotinic and non-nicotinic inputs to inhibitory neurons in the mouse colon.

1. Intracellular microelectrodes were used to record spontaneous and evoked inhibitory junction potentials (IJP) from the circular muscle layer of the mid-distal region of the mouse isolated colon in the presence of nifedipine (1 micromol/L) and hyoscine (1 micromol/L). 2. The length of the tissue preparation (> 1 cm) or the presence of the mucosa had no effect on the frequency of spontaneous IJP. 3. Hexamethonium (500 micromol/L) reduced the frequency of spontaneous IJP to approximately 70% of the control frequency, whereas D-tubocurarine (280 micromol/L) reduced the frequency to approximately 17% of control. Apamin (250 nmol/L) abolished all spontaneous IJP activity. 4. The greater inhibition of spontaneous IJP in the presence of D-tubocurarine compared with hexamethonium is discussed as a possible 'apamin-like' effect. 5. Although electrically evoked IJP (single pulse at 15 V, 0.6 msec) were not significantly affected by hexamethonium, D-tubocurarine and apamin reduced the amplitude of evoked IJP to approximately 65 and 50% of control, respectively. 6. These results suggest that the properties of spontaneous IJP cannot be inferred by a study of evoked IJP alone.

A sequence and structural study of transmembrane helices.

A comparison is made between the distribution of residue preferences, three dimensional nearest neighbour contacts, preferred rotamers, helix-helix crossover angles and peptide bond angles in three sets of proteins: a non-redundant set of accurately determined globular protein structures, a set of four-helix bundle structures and a set of membrane protein structures. Residue preferences for the latter two sets may reflect overall helix stabilising propensities but may also highlight differences arising out of the contrasting nature of the solvent environments in these two cases. The results bear out the expectation that there may be differences between residue type preferences in membrane proteins and in water soluble globular proteins. For example, the beta-branched residue types valine and isoleucine are considerably more frequently encountered in membrane helices. Likewise, glycine and proline. residue types normally associated with 'helix-breaking' propensity are found to be relatively more common in membrane helices. Three dimensional nearest neighbour contacts along the helix, preferred rotamers, and peptide bond angles are very similar in the three sets of proteins as far as can be ascertained within the limits of the relatively low resolution of the membrane proteins dataset. Crossing angles for helices in the membrane protein set resemble the four helix bundle set more than the general non-redundant set, but in contrast to both sets they have smaller crossing angles consistent with the dual requirements for the helices to form a compact structure while having to span the membrane. In addition to the pairwise packing of helices we investigate their global packing and consider the question of helix supercoiling in helix bundle proteins.