Design, docking, and evaluation of multiple libraries against multiple targets.

We present a general approach to the design, docking, and virtual screening of multiple combinatorial libraries against a family of proteins. The method consists of three main stages: docking the scaffold, selecting the best substituents at each site of diversity, and comparing the resultant molecules within and between the libraries. The core "divide-and-conquer" algorithm for side-chain selection, developed from an earlier version (Sun et al., J Comp Aided Mol Design 1998;12:597-604), provides a way to explore large lists of substituents with linear rather than combinatorial time dependence. We have applied our method to three combinatorial libraries and three serine proteases: trypsin, chymotrypsin, and elastase. We show that the scaffold docking procedure, in conjunction with a novel vector-based orientation filter, reproduces crystallographic binding modes. In addition, the free-energy-based scoring procedure (Zou et al., J Am Chem Soc 1999;121:8033-8043) is able to reproduce experimental binding data for P1 mutants of macromolecular protease inhibitors. Finally, we show that our method discriminates between a peptide library and virtual libraries built on benzodiazepine and tetrahydroisoquinolinone scaffolds. Implications of the docking results for library design are explored.

Monte Carlo calculations on HIV-1 reverse transcriptase complexed with the non-nucleoside inhibitor 8-Cl TIBO: contribution of the L100I and Y181C variants to protein stability and biological activity.

A computational model of the non-nucleoside inhibitor 8-Cl TIBO complexed with HIV-1 reverse transcriptase (RT) was constructed in order to determine the binding free energies. Using Monte Carlo simulations, both free energy perturbation and linear response calculations were carried out for the transformation of wild-type RT to two key mutants, Y181C and L100I. The newer linear response method estimates binding free energies based on changes in electrostatic and van der Waals energies and solvent-accessible surface areas. In addition, the change in stability of the protein between the folded and unfolded states was estimated for each of these mutations, which are known to emerge upon treatment with the inhibitor. Results from the calculations revealed that there is a large hydrophobic contribution to protein stability in the native, folded state. The calculated absolute free energies of binding from both the linear response, and also the more rigorous free energy perturbation method, gave excellent agreement with the experimental differences in activity. The success of the relatively rapid linear response method in predicting experimental activities holds promise for estimating the activity of the inhibitors not only against the wild-type RT, but also against key protein variants whose emergence undermines the efficacy of the drugs.

Estimation of the binding affinities of FKBP12 inhibitors using a linear response method.

A series of non-immunosuppressive inhibitors of FK506 binding protein (FKBP12) are investigated using Monte Carlo statistical mechanics simulations. These small molecules may serve as scaffolds for chemical inducers of protein dimerization, and have recently been found to have FKBP12-dependent neurotrophic activity. A linear response model was developed for estimation of absolute binding free energies based on changes in electrostatic and van der Waals energies and solvent-accessible surface areas, which are accumulated during simulations of bound and unbound ligands. With average errors of 0.5 kcal/mol, this method provides a relatively rapid way to screen the binding of ligands while retaining the structural information content of more rigorous free energy calculations.

Prognostic significance of K-ras codon 12 mutations in patients with resected stage I and II non-small-cell lung cancer.

PURPOSE: The aim of this study was to investigate the prognostic importance of codon 12 K-ras mutations in patients with early-stage non-small-cell lung cancer (NSCLC). PATIENTS AND METHODS: We identified 260 patients with surgically resected stage I (n = 193) and stage II (n = 67) NSCLC with at least a 5-year follow-up. We performed polymerase chain reaction analysis of DNA obtained from paraffin-embedded NSCLC tissue, using mutation-specific probes for codon 12 K-ras. RESULTS: K-ras mutations were detected in 35 of 213 assessable specimens (16.4%). K-ras mutations were detected in 27 of 93 adenocarcinomas (29.0%), one of 61 squamous cell carcinomas (1.6%), five of 39 large-cell carcinomas (12.8%), and two of 20 adenosquamous carcinomas (10%) (P = .001). G to T transversions accounted for 71% of the mutations. There was no statistically significant difference in overall survival for all patients with K-ras mutations (median survival, 39 months) compared with patients without K-ras mutations (median survival, 53 months; P = .33). There was no statistically significant difference in overall or disease-free survival for subgroups with stage I disease, adenocarcinoma, or non-squamous cell carcinoma or for specific amino acid substitutions. The median survival time for stage II patients with K-ras mutations was 13 months, compared with 38 months for patients without K-ras mutations (P = .03). CONCLUSION: Codon 12 K-ras mutations were more common in adenocarcinomas than in squamous cell carcinomas. For the subgroup with stage II NSCLC, there was a statistically significant adverse effect on survival for the presence of K-ras mutations. However, when the entire group was considered, the presence of K-ras mutations was not of prognostic significance in this cohort of patients with resected early-stage NSCLC.

Prediction of binding affinities for TIBO inhibitors of HIV-1 reverse transcriptase using Monte Carlo simulations in a linear response method.

Monte Carlo (MC) simulations in combination with a linear response approach were used to estimate the free energies of binding for a series of 12 TIBO nonnucleoside inhibitors of HIV-1 reverse transcriptase. Separate correlations were made for the R6 and S6 absolute conformations of the inhibitors, as well as for the analogous N6-monoprotonated species. Models based upon the neutral unbound inhibitors produced overall better fits to experimental values than did those using the protonated unbound inhibitors, with only slight differences between the neutral R6 and S6 cases. The best results were obtained with a three-parameter linear response equation containing van der Waals (alpha), electrostatic (beta), and solvent accessible surface area (SASA, gamma) terms. The averaged (R6 and S6) rms error was approximately 0.88 kcal/mol for the observed range of 4.06 kcal/mol in inhibitor activities. The averaged values of alpha, beta, and gamma were -0.150, 0.114, and 0. 0286, respectively. Omission of the alpha term gave beta 0.152 and gamma 0.022 with a rms of 0.92. The unweighted van der Waals components were found to be highly attractive but failed to correlate well across the series of inhibitors. Contrastingly, while the electrostatic components are all repulsive, they show a direct correlation with inhibitor activity as measured by DeltaGbinding. The role of gamma is primarily to produce an overall negative binding energy, and it can effectively be replaced with a negative constant. During the MC simulations of the unbound solvated inhibitors, the R6 and S6 absolute conformations do not interconvert due to the formation of a favorable hydrogen bond to solvent. In the complex, however, interconversion of these conformations of the inhibitor is observed during the course of the simulations, a phenomenon which is apparently not observed in the crystalline state of the complex. Hydrogen bonding of the inhibitor to the backbone NH of K101 and the lack of such an interaction with the C=O of K101 or with solvent correlate with enhanced activity, as does the ability to assume a number of different orientations of the inhibitor dimethylallyl moiety with respect to residues Y181 and Y188 while retaining contact with W229. Overall, the use of a combination of MC simulation with a linear response method shows promise as a relatively rapid means of estimating inhibitor activities. This approach should be useful in the preliminary evaluation of potential modifications to known inhibitors to enhance activity.

Investigations of neurotrophic inhibitors of FK506 binding protein via Monte Carlo simulations.

The binding and solution-phase properties of six inhibitors of FK506 binding protein (FKBP12) were investigated using free energy perturbation techniques in Monte Carlo statistical mechanics simulations. These nonimmunosuppressive molecules are of current interest for their neurotrophic activity when bound to FKBP12 as well as for their potential as building blocks for chemical inducers of protein dimerization. Relative binding affinities were computed and analyzed for ligands differing by a phenyl ring, an external phenyl or pyridyl substituent, and a pipecolyl or prolyl ring. Such results are, in general, valuable for inhibitor optimization and, in the present case, bring into question some of the previously reported binding data.

Computational approaches to molecular recognition.

Recent advances in the computation of free energies have facilitated the understanding of host-guest and protein-ligand recognition. Rigorous perturbation methods have been assessed and expanded, and more approximate techniques have been developed that allow faster treatment of diverse systems.

Pseudocleavage is dispensable for polarity and development in C. elegans embryos.

The first cleavage of the Caenorhabditis elegans embryo is asymmetrical, producing daughters with different cell fates. During the first cell cycle, P granules, cytoplasmic components that are segregated to the germ-line, are localized to the posterior of the embryo. It has been hypothesized that the asymmetrical behavior of the daughters of the first division results from a similar localization of developmental determinants. A process called pseudocleavage also occurs during the first cell cycle: Anterior cortical contractions culminate in a single partial constriction of the embryo called the pseudocleavage furrow. Coincident with pseudocleavage, there is an anteriorly directed flow of cortical cytoplasm and a posteriorly directed flow of internal cytoplasm. Foci of filamentous cortical actin become asymmetrically distributed into an anterior cap. Roles for these various first cell cycle events in cytoplasmic localization and development have been suggested but remain unclear. We have isolated a maternal effect mutation, nop-1(it142), which abolishes the anterior cortical contractions and the pseudocleavage furrow. In addition, cortical actin foci remain uniformly distributed in most embryos. Despite these defects, cytoplasmic and cortical streaming is present and P granules are localized to the posterior of early embryos. In most embryos from mutant mothers, development proceeds normally and the embryos hatch and grow into fertile adults. We conclude that the pseudocleavage contractions and furrow are dispensable for the development of C. elegans.

Novel functional M1 selective muscarinic agonists. 2. Synthesis and structure-activity relationships of 3-pyrazinyl-1,2,5,6-tetrahydro-1-methylpyridines. Construction of a molecular model for the M1 pharmacophore.

A series of 3-(3-substituted-pyrazinyl)-1,2,5,6-tetrahydro-1-methylpyridines were synthesized and found to have high affinity for central muscarinic receptors. The ability of some of these compounds to inhibit the electrically stimulated twitch of the guinea pig vas deferens indicated that the compounds were M1 agonists. M1 agonist activity was related to the length of the side chain attached to the pyrazine ring, with maximal activity being obtained with the hexyloxy side chain. The (hexyloxy)pyrazine 3f lacked M2 agonist activity as it failed to affect the guinea pig atria and was also relatively devoid of M3 agonist activity as determined by its lack of tremorogenic and sialogogic effects in mice. A comparison of the M1 agonist efficacy of these pyrazines and related 1,2,5-thiadiazoles and 1,2,5-oxadiazoles suggested that M1 efficacy was related to the magnitude of electrostatic potential located over the nitrogens of the respective heterocycles. The heteroatom directly attached to the 3 position of the pyrazine or 1,2,5-thiadiazole heterocycle markedly influenced the M1 efficacy of the compounds by determining the energetically favorably conformers for rotation about the bond connecting the tetrahydropyridyl ring and the heterocycle. A three-dimensional model for the M1-activating pharmacophore was proposed based on computational studies and the model of the muscarinic pharmacophore proposed by Schulman.

Detection and follow-up of moderate and severe hypertensive subjects in the Australia community.

A national screening project has detected 989 subjects aged 30 to 69 years with untreated moderate or severe hypertension. The 1-2% incidence of this condition indicates a total of about 100,000 people in the Australian community potentially requiring therapy. The sample showed that half the subjects were previously unaware of a blood pressure problem. Those so detected showed a very high rate (90-7%) of acceptance of advice to see their own doctor. The local doctors confirmed the elevated pressure level in nearly two-thirds of the patients referred, and initiated treatment in a similar proportion.

Report on progress in the Australian National Blood Pressure Study (NBPS).

1. The National Blood Pressure Study (NBPS) is a single blind trial designed to test the efficacy of active drug treatment in reducing complications from mild hypertension (mean diastolic pressure = 95-109 mmHg). 2. Between 1973 and 1975, four centres screened about 104 000 subjects aged 30-69 years, yielding an estimated prevalence of hypertension (greater than or equal to 95 mmHg diastolic) of 16% and of moderate-to-severe hypertension (greater than of equal to 110 mmHg diastolic) of 1-3%. 3. Some 4000 subjects selected for untreated uncomplicated mild hypertension were randomized to either active treatment (cholorothiazide +alpha-methyldopa and/or a beta-adrenoreceptor antagonist as required) or to matching placebos. 4. At 1 year mean pressures had fallen significantly below entry pressures in both groups but in the active group the fall was greater by a margin of 14-4+/-1-3 (SEM) mmHg systolic and 7-1+/-0-7 mmHg diastolic. At 1 year 5% of subjects in the placebo group had been placed on active treatment on the ethical grounds that pressure had exceeded the mild hypertension limit. 5. Trial end-points (death, morbidity from stroke, hypertensive heart and renal disease, and ischaemic heart disease) number 106 (nine deaths) thus far, of which ischaemic heart disease accounts for 71% and stroke 19%. 6. The duration of trial may need to be extended beyond the original estimate of 5 years.

Borderline hypertension discovered during screening: effect of twelve months' treatment.

The frequency of borderline hypertension (diastolic BP 95 to 109 mm Hg) is described in 5,877 men and 1,125 women attending a screening clinic. Borderline hypertension is more frequent in each 5-year age group in men than women. The results of the initial 12 months treatment show a greater fall in mean systolic and diastolic pressures in those on active treatment in comparison with placebo. Over the same 12 months the sum of selected ECG precordial lead voltages shows a significant fall in those on active treatment as compared with those on placebo.