PRO_LIGAND: an approach to de novo molecular design. 2. Design of novel molecules from molecular field analysis (MFA) models and pharmacophores.

A computational approach for molecular design, PRO_LIGAND, has been developed within the PROMETHEUS molecular design and simulation system in order to provide a unified framework for the de novo generation of diverse molecules which are either similar or complementary to a specified target. In this instance, the target is a pharmacophore derived from a series of active structures either by a novel interpretation of molecular field analysis data or by a pharmacophore-mapping procedure based on clique detection. After a brief introduction to PRO_LIGAND, a detailed description is given of the two pharmacophore generation procedures and their abilities are demonstrated by the elucidation of pharmacophores for steroid binding and ACE inhibition, respectively. As a further indication of its efficacy in aiding the rational drug design process, PRO_LIGAND is then employed to build novel organic molecules to satisfy the physicochemical constraints implied by the pharmacophores.

[D-Pen2, D-Pen5]enkephalin, the standard delta opioid agonist, induces morphine-like behaviors in mice.

[D-Pen2, D-Pen5]enkephalin (DPDPE; 3-30 micrograms) and morphine (10 micrograms) both caused Straub tails, increased locomotion, and circling after ICV administration to ICR mice. DPDPE-induced tail stiffening was reduced when mice were pretreated with naloxone (0.5 mg/kg SC) or beta-funaltrexamine (10 micrograms ICV), but not with ICI 174864 (2 mg/kg SC), the selective antagonist at delta opioid receptors. These results point to (a) mu receptors mediating the tail stiffening and (b) the loss of delta receptor selectivity after 10 and 30 micrograms DPDPE.

Formalin nociception in the mouse does not lead to increased spinal serotonin turnover.

The mouse formalin test is a model of tonic (continuous), chemical/inflammatory nociception. To test the hypothesis that bulbospinal serotonergic pathways modulate such nociception, whole spinal cords from mice pretreated with probenecid and sacrificed at 15, 30, 45 and 60 min after injection of 5% formalin or 0.9% saline in the hindpaw were assayed by high performance liquid chromatography with electrochemical detection for the serotonin metabolite, 5-hydroxyindoleacetic acid, as an index of turnover. No difference in serotonin turnover was found between formalin and saline groups, indicating that increased spinal serotonin release is not a normal response to formalin nociception in the mouse.

Facility planning and development in Latin America.

Since the mid-1980s, the author has been involved in the development of healthcare facilities in the Caribbean and Latin America. This article highlights his experiences to date and provides insights relative to the many challenges U.S. developers and contractors might encounter when exporting U.S. healthcare expertise to Caribbean and Latin American countries.

Here's some good medicine to supplement hospitals' efforts to stimulate pharmacies.

Hospitals started retail pharmacies with visions of revenues that would offset prospective payment shortages, but they soon found out that some key assumptions about profitability were off key. There are ways to tune the pharmacy operation, however, so it reaches out to its market and comes through for the hospital. Charles W. Murray explains some problems and solutions.

Tonic pain perception in the mouse: differential modulation by three receptor-selective opioid agonists.

The proposition that tonic nociception models are more analogous to clinical pain than traditional acute models prompted our previous development of a modified mouse paw formalin test. To discern possible modulatory roles and site(s) of action of endogenous opioid systems, the receptor-preferring agonists sufentanil (mu), U-50,488H (kappa) and [D-Pen2,5]enkephalin (DPDPE, delta) were evaluated for antinociceptive activity in the formalin paradigm by systemic (except DPDPE), spinal and supraspinal routes. All observations were done under blind conditions. Doses causing overt behaviors that indicated a breach of receptor specificity (during the observation period) were rejected. Higher doses of centrally administered DPDPE (greater than 0.3 micrograms/mouse, intrathecal; greater than 3 micrograms/mouse, intracerebroventricular) induced a behavioral syndrome traditionally associated with mu agonism, and thus were not considered for this study. A50 values from behaviorally acceptable dose ranges for mean percent analgesia (reduction of paw licking compared to controls) were: trans-(+/- )-3,4-dichloro-N-methyl-N-[U-50,488H 2-pyrrolidinyl)cyclohexyl]-benzeneacetamide methanesulfonate,U-50,488H--3200 nmol/kg, subcutaneous, 1100 nmol/kg, intrathecal and 314 nmol/kg, intracerebroventricular; sufentanil--11.1 nmol/kg, subcutaneous, 8.6 nmol/kg, intrathecal; and DPDPE--inactive. On the basis of our dose-response data, we suggest that, in mice, kappa and mu, but not delta, opioid receptors modulate tonic pain perception at both spinal and supraspinal loci. The results also support inclusion of the modified formalin test in preclinical evaluations of potential kappa agonists.

Methodological refinements to the mouse paw formalin test. An animal model of tonic pain.

The formalin tonic pain test has recently been adapted for use in mice. We have observed two problems with this procedure. First, compared to a vehicle control group, the majority of mice injected in a hind paw with dilute formalin exhibited a significant nociceptive response (paw licking), including peaks of activity, well beyond the end of the recommended observation period; and, second, an unacceptably wide variation in response occurred. We report two modifications that eliminate these difficulties: a) extending the observation period to 20-45 min postformalin injection, and b) lightly anesthetizing the mice with ether before injection of formalin into the paw. The modified protocol was tested by evaluating morphine, pentazocine and zomepirac (analgesic A50 values were 2.1, 23.8, and greater than 100 mg/kg, s.c., respectively). An intrathecal or intracerebroventricular injection of saline, given to the lightly anesthetized mouse, did not interfere with the nociceptive behavior; this finding widens the applicability and usefulness of the test. Collectively, these refinements significantly improve the mouse formalin model of tonic pain.

Empirical scoring functions. II. The testing of an empirical scoring function for the prediction of ligand-receptor binding affinities and the use of Bayesian regression to improve the quality of the model.

This paper tests the performance of a simple empirical scoring function on a set of candidate designs produced by a de novo design package. The scoring function calculates approximate ligand-receptor binding affinities given a putative binding geometry. To our knowledge this is the first substantial test of an empirical scoring function of this type on a set of molecular designs which were then subsequently synthesised and assayed. The performance illustrates that the methods used to construct the scoring function and the reliance on plausible, yet potentially false, binding modes can lead to significant over-prediction of binding affinity in bad cases. This is anticipated on theoretical grounds and provides caveats on the reliance which can be placed when using the scoring function as a screen in the choice of molecular designs. To improve the predictability of the scoring function and to understand experimental results, it is important to perform subsequent Quantitative Structure-Activity Relationship (QSAR) studies. In this paper, Bayesian regression is performed to improve the predictability of the scoring function in the light of the assay results. Bayesian regression provides a rigorous mathematical framework for the incorporation of prior information, in this case information from the original training set, into a regression on the assay results of the candidate molecular designs. The results indicate that Bayesian regression is a useful and practical technique when relevant prior knowledge is available and that the constraints embodied in the prior information can be used to improve the robustness and accuracy of regression models. We believe this to be the first application of Bayesian regression to QSAR analysis in chemistry.

A comparison of heuristic search algorithms for molecular docking.

This paper describes the implementation and comparison of four heuristic search algorithms (genetic algorithm, evolutionary programming, simulated annealing and tabu search) and a random search procedure for flexible molecular docking. To our knowledge, this is the first application of the tabu search algorithm in this area. The algorithms are compared using a recently described fast molecular recognition potential function and a diverse set of five protein-ligand systems. Statistical analysis of the results indicates that overall the genetic algorithm performs best in terms of the median energy of the solutions located. However, tabu search shows a better performance in terms of locating solutions close to the crystallographic ligand conformation. These results suggest that a hybrid search algorithm may give superior results to any of the algorithms alone.

PRO_LIGAND: an approach to de novo molecular design. 6. Flexible fitting in the design of peptides.

This paper describes the further development of the functionality of our in-house de novo design program, PRO_LIGAND. In particular, attention is focused on the implementation and validation of the 'direct tweak' method for the construction of conformationally flexible molecules, such as peptides, from molecular fragments. This flexible fitting method is compared to the original method based on libraries of prestored conformations for each fragment. It is shown that the directed tweak method produces results of comparable quality, with significant time savings. By removing the need to generate a set of representative conformers for any new library fragment, the flexible fitting method increases the speed and simplicity with which new fragments can be included in a fragment library and also reduces the disk space required for library storage. A further improvement to the molecular construction process within PRO_LIGAND is the inclusion of a constrained minimisation procedure which relaxes fragments onto the design model and can be used to reject highly strained structures during the structure generation phase. This relaxation is shown to be very useful in simple test cases, but restricts diversity for more realistic examples. The advantages and disadvantages of these additions to the PRO_LIGAND methodology are illustrated by three examples: similar design to an alpha helix region of dihydrofolate reductase, complementary design to the active site of HIV-1 protease and similar design to an epitope region of lysozyme.

MOLMAKER: de novo generation of 3D databases for use in drug design.

A program, MOLMAKER, is described which, in conjunction with a 2D-3D conversion program and 3D database software, can generate de novo 3D databases to aid in drug design. MOLMAKER is based upon graph-theoretical techniques for vertex degree set generation and constructive enumeration of molecular graphs. The generated molecular graphs are then functionalised in a probabilistic manner but in accordance with various constraints specified by the user. The resulting connection tables can be converted into 3D structures by commercial software and loaded into a 3D database for pharmacophore searching. The utility of MOLMAKER is illustrated by two examples of interest from the recent scientific literature: the design of novel protein kinase C agonists and of a bridging ligand for cyclophilin-calcineurin.

The sensitivity of the results of molecular docking to induced fit effects: application to thrombin, thermolysin and neuraminidase.

This paper describes the application of PRO_LEADS to the flexible docking of ligands into crystallographically derived enzyme structures that are assumed to be rigid. PRO_LEADS uses a Tabu search methodology to perform the flexible search and an empirically derived estimate of the binding affinity to drive the docking process. The paper tests the extent to which the assumption of a rigid enzyme compromises the accuracy of the results. All-pairs docking experiments are performed for three enzymes (thrombin, thermolysin and influenza virus neuraminidase) based on six or more ligand-enzyme crystal structures for each enzyme. In 76% of the cases, PRO_LEADS can successfully identify the correct ligand conformation as the lowest energy configuration when the enzyme structure is derived from that ligand's crystal structure, but the methodology only docks 49% of the cases successfully when the ligand is docked against enzyme crystal structures derived from other ligands. Small movements in the enzyme structure lead to an under-prediction in the energy of the correct binding mode by up to 14 kJ/mol and in some cases this under-prediction can lead to the native mode not being recognised as the lowest energy solution. The type of movements responsible for mis-docking are: the movement of sidechains as a result of changes in C alpha position; the movement of sidechains without changes in C alpha position; the movement of flexible portions of main chains to facilitate the formation of hydrogen bonds; and the movement of metal atoms bound to the enzyme active site. The work illustrates that the assumption of a rigid active site can lead to errors in identification of the correct binding mode and the assessment of binding affinity, even for enzymes which show relatively small shift in atomic positions from one ligand to the next. A good docking code, such as PRO_LEADS, can usually dock successfully if there is induced fit in relatively rigid enzymes but there remains the need to develop improved strategies for dealing with enzyme flexibility. The work implies that treatments of enzyme flexibility which focus only on sidechain rotations will not deal with the critical shifts responsible for mis-docking of ligands in thrombin, thermolysin and neuraminidase. The paper demonstrates the utility of all pairs docking experiments as a method of assessing the effectiveness of docking methodologies in dealing with enzyme flexibility.

Neurokinin-induced salivation in the anesthetized rat: a three receptor hypothesis.

Substance P (3 micrograms/kg), neurokinin A (20 micrograms/kg), neurokinin B (6 micrograms/kg) and acetylcholine (875 micrograms/kg) all produced salivation upon i.v. infusion in the anesthetized rat. Against single equivalent agonist doses, atropine (135 micrograms/kg i.v.) blocked both acetylcholine- and neurokinin B-, but not substance P- or neurokinin A-induced salivation. [D-Pro2,D-Trp7,9]-substance P (1 mg/kg i.v.), a putative substance P antagonist, reduced responses to mammalian neurokinins but caused a 2-fold potentiation of acetylcholine-induced salivation. [D-Pro2,D-Trp6,8,Nle10]-Neurokinin B (1 mg/kg i.v.), a novel putative neurokinin B antagonist, significantly reduced substance P- and neurokinin B- but not acetylcholine- or neurokinin A-induced salivation. The three agonists (at doses that produced salivation) and [D-Pro2,D-Trp6,8,Nle10]-neurokinin B (1 mg/kg i.v.) lowered blood pressure in anesthetized rats by 35 to 40%. [D-Pro2,D-Trp7,9]-Substance P (1 mg/kg i.v.) had no significant effect on blood pressure. Hydralazine at 0.60 mg/kg (i.v.), a dose which lowered blood pressure by 47%, did not reduce substance P-induced salivation. Thus, blockade of neurokinin-induced salivation by [D-Pro2,D-Trp6,8,Nle10]-neurokinin B was probably not due to hypotension. Based on the differential effects of the three antagonists on neurokinin- and acetylcholine-induced salivation, we hypothesize the existence of three distinct neurokinin receptors in rat salivary gland, and suggest that neurokinin B receptors reside presynaptically.

Active-site-directed 3D database searching: pharmacophore extraction and validation of hits.

Two new computational tools, PRO_PHARMEX and PRO_SCOPE, for use in active-site-directed searching of 3D databases are described. PRO_PHARMEX is a flexible, graphics-based program facilitating the extraction of pharmacophores from the active site of a target macromolecule. These pharmacophores can then be used to search a variety of databases for novel lead compounds. Such searches can often generate many 'hits' of varying quality. To aid the user in setting priorities for purchase, synthesis or testing, PRO_SCOPE can be used to dock molecules rapidly back into the active site and to assign them a score using an empirical scoring function correlated to the free energy of binding. To illustrate how these tools can add value to existing 3D database software, their use in the design of novel thrombin inhibitors is described.

Empirical scoring functions: I. The development of a fast empirical scoring function to estimate the binding affinity of ligands in receptor complexes.

This paper describes the development of a simple empirical scoring function designed to estimate the free energy of binding for a protein-ligand complex when the 3D structure of the complex is known or can be approximated. The function uses simple contact terms to estimate lipophilic and metal-ligand binding contributions, a simple explicit form for hydrogen bonds and a term which penalises flexibility. The coefficients of each term are obtained using a regression based on 82 ligand-receptor complexes for which the binding affinity is known. The function reproduces the binding affinity of the complexes with a cross-validated error of 8.68 kJ/mol. Tests on internal consistency indicate that the coefficients obtained are stable to changes in the composition of the training set. The function is also tested on two test sets containing a further 20 and 10 complexes, respectively. The deficiencies of this type of function are discussed and it is compared to approaches by other workers.

PRO-LIGAND: an approach to de novo molecular design. 3. A genetic algorithm for structure refinement.

Recently, the development of computer programs which permit the de novo design of molecular structures satisfying a set of steric and chemical constraints has become a burgeoning area of research and many operational systems have been reported in the literature. Experience with PRO-LIGAND-the de novo design methodology embodied in our in-house molecular design and simulation system PRO-METHEUS-has suggested that the addition of a genetic algorithm (GA) structure refinement procedure can 'add value' to an already useful tool. Starting with the set of designed molecules as an initial population, the GA can combine features from both high- and low-scoring structures and, over a number of generations, produce individuals of better score than any of the starting structures. This paper describes how we have implemented such a procedure and demonstrates its efficacy in improving two sets of molecules generated by different de novo design projects.

Flexible docking using Tabu search and an empirical estimate of binding affinity.

This article describes the implementation of a new docking approach. The method uses a Tabu search methodology to dock flexibly ligand molecules into rigid receptor structures. It uses an empirical objective function with a small number of physically based terms derived from fitting experimental binding affinities for crystallographic complexes. This means that docking energies produced by the searching algorithm provide direct estimates of the binding affinities of the ligands. The method has been tested on 50 ligand-receptor complexes for which the experimental binding affinity and binding geometry are known. All water molecules are removed from the structures and ligand molecules are minimized in vacuo before docking. The lowest energy geometry produced by the docking protocol is within 1.5 A root-mean square of the experimental binding mode for 86% of the complexes. The lowest energies produced by the docking are in fair agreement with the known free energies of binding for the ligands.

PRO_LIGAND: an approach to de novo molecular design. 4. Application to the design of peptides.

In some instances, peptides can play an important role in the discovery of lead compounds. This paper describes the peptide design facility of the de novo drug design package, PRO_LIGAND. The package provides a unified framework for the design of peptides that are similar or complementary to a specified target. The approach uses single amino acid residues, selected from preconstructed libraries of different residues and conformations, and places them on top of predefined target interaction sites. This approach is a well-tested methodology for the design of organics but has not been used for peptides before. Peptides represent a difficulty because of their great conformational flexibility and a study of the advantages and disadvantages of this simple approach is an important step in the development of design tools. After a description of our general approach, a more detailed discussion of its adaptation to peptides is given. The method is then applied to the design of peptide-based inhibitors to HIV-1 protease and the design of structural mimics of the surface region of lysozyme. The results are encouraging and point the way towards further development of interaction site-based approaches for peptide design.

Protein fold recognition by threading: comparison of algorithms and analysis of results.

Optimal sequence threading can be used to recognize members of a library of protein folds which are closely related in 3-D structure to the native fold of an input test sequence, even when the test sequence is not significantly homologous to the sequence of any member of the fold library. The methods provide an alignment between the residues of the test sequence and the residue positions in a template fold. This alignment optimizes a score function, and the predicted fold is the highest scoring member of the library of folds. Most score functions contain a pairwise interaction energy term. This, coupled with the need to introduce gaps into the alignment, means that the optimization problem is NP hard. We report a comparison between two heuristic optimization algorithms used in the literature, double dynamic programming and an iterative algorithm based on the so-called frozen approximation. These are compared in terms of both the ranking of likely folds and the quality of the alignment produced.

PRO-LIGAND: an approach to de novo molecular design. 1. Application to the design of organic molecules.

An approach to de novo molecular design, PRO-LIGAND, has been developed that, in the environment of a large, integrated molecular design and simulation system, provides a unified framework for the generation of novel molecules which are either similar or complementary to a specified target. The approach is based on a methodology that has proved to be effective in other studies--placing molecular fragments upon target interaction sites-but incorporates many novel features such as the use of a rapid graph-theoretical algorithm for fragment placing, a generalised driver for structure generation which offers a large variety of fragment assembly strategies to the user and the pre-screening of library fragments. After a detailed description of the relevant modules of the package, PRO-LIGAND's efficacy in aiding rational drug design is demonstrated by its ability to design mimics of methotrexate and potential inhibitors for dihydrofolate reductase and HIV-1 protease.