Pharmacogenomic analysis: correlating molecular substructure classes with microarray gene expression data.

Genomic studies are producing large databases of molecular information on cancers and other cell and tissue types. Hence, we have the opportunity to link these accumulating data to the drug discovery processes. Our previous efforts at 'information-intensive' molecular pharmacology have focused on the relationship between patterns of gene expression and patterns of drug activity. In the present study, we take the process a step further-relating gene expression patterns, not just to the drugs as entities, but to approximately 27,000 substructures and other chemical features within the drugs. This coupling of genomic information with structure-based data mining can be used to identify classes of compounds for which detailed experimental structure-activity studies may be fruitful. Using a systematic substructure analysis coupled with statistical correlations of compound activity with differential gene expression, we have identified two subclasses of quinones whose patterns of activity in the National Cancer Institute's 60-cell line screening panel (NCI-60) correlate strongly with the expression patterns of particular genes: (i) The growth inhibitory patterns of an electron-withdrawing subclass of benzodithiophenedione-containing compounds over the NCI-60 are highly correlated with the expression patterns of Rab7 and other melanoma-specific genes; (ii) the inhibitory patterns of indolonaphthoquinone-containing compounds are highly correlated with the expression patterns of the hematopoietic lineage-specific gene HS1 and other leukemia genes. As illustrated by these proof-of-principle examples, we introduce here a set of conceptual tools and fluent computational methods for projecting directly from gene expression patterns to drug substructures and vice versa. The analysis is presented in terms of the NCI-60 cell lines and microarray-based gene expression patterns, but the concept and methods are broadly applicable to other large-scale pharmacogenomic database sets as well. The approach (SAT for Structure-Activity-Target) provides a systematic way to mine databases for the design of further structure-activity studies, particularly to aid in target and lead identification.

Anomeric effects in non-carbohydrate compounds: conformational differences between the oxazolidine rings of a cis-fused bicyclic system.

Tris(hydroxymethyl)aminomethane (Tris) can react with benzaldehyde (1:2 molar ratio) to produce cis-2,8-diphenyl-5-hydroxymethyl-1-aza-3,7-dioxabicyclo[3.3.0]octa ne, the structure of which has been confirmed by nuclear magnetic resonance spectroscopy and X-ray crystallography. The crystal structure showed that both oxazolidine rings A and B are puckered in opposite directions. Ring A exists in an E3 envelope form with 0-3 noticeably down (0.65 A) the plane of the remaining atoms, whereas ring B adopts the 7E envelope conformation with the 0-7 atom displaced up from the mean reference plane by 0.70 A. Comparison of bond angles and bond distances showed that both oxazolidine rings A and B exhibit cross endo-anomeric effects resulting from electron delocalization over the bond sequence O-3-C-2-N-1-C-8-O-7.

All-L-Leu-Pro-Leu-Pro: a challenging cyclization.

In this paper, we report the difficult synthesis of cyclo(Leu-Pro-Leu-Pro). While the cyclization of Leu-Pro-Leu-D-Pro did not cause problems, the all-L-peptide afforded cyclodimer rather than cyclotetrapeptide (cyclomonomer). A first attempt using our reversible backbone substitution methodology failed. However, we were successful in obtaining the desired cyclo(Leu-Pro-Leu-Pro) by decreasing the concentration. The ratio of cyclomonomer to cyclodimer was raised to 1:1.1 using BOP and 1:0.6 using HATU under our high dilution condition. The structures of the cyclopeptides were confidently assigned by electrospray ionization mass spectrometry and NMR.

Comparison of indirect calorimetry, the Fick method, and prediction equations in estimating the energy requirements of critically ill patients.

BACKGROUND: Accurate measurement of resting energy expenditure (REE) is helpful in determining the energy needs of critically ill patients requiring nutritional support. Currently, the most accurate clinical tool used to measure REE is indirect calorimetry, which is expensive, requires trained personnel, and has significant error at higher inspired oxygen concentrations. OBJECTIVE: The purpose of this study was to compare REE measured by indirect calorimetry with REE calculated by using the Fick method and prediction equations by Harris-Benedict, Ireton-Jones, Fusco, and Frankenfield. DESIGN: REEs of 36 patients [12 men and 24 women, mean age 58+/-22 y and mean Acute Physiology and Chronic Health Evaluation II score 22+/-8] in a hospital intensive care unit and receiving mechanical ventilation and total parenteral nutrition (TPN) were measured for > or = 15 min by using indirect calorimetry and compared with REEs calculated from a mean of 2 sets of hemodynamic measurements taken during the metabolic testing period with an oximetric pulmonary artery catheter. RESULTS: Mean REE by indirect calorimetry was 8381+/-1940 kJ/d and correlated poorly with the other methods tested (r = 0.057-0.154). This correlation did not improve after adjusting for changes in respiratory quotient (r2 = 0.28). CONCLUSIONS: These data do not support previous findings showing a strong correlation between REE determined by the Fick method and other prediction equations and indirect calorimetry. In critically ill patients receiving TPN, indirect calorimetry, if available, remains the most appropriate clinical tool for accurate measurement of REE.

Changes in measured resting energy expenditure after Roux-en-Y gastric bypass for clinically severe obesity are not related to bypass limb-length.

BACKGROUND: Roux-en-Y gastric bypass (RYGB) for clinically severe obesity (CSO) results in a 'paradoxical' response of the measured resting energy expenditure (MREE) in which the MREE remains within the predicted range based upon the Harris-Benedict (HB) equation, despite a significant decrease in caloric intake to 500-1000 kcal/day. The mechanism for this response is unknown. A study was undertaken to determine whether the changes in MREE after RYGB are related to limb-length of the gastric bypass. METHODS: A prospective clinical trial of varying limb-lengths based on body mass index (BMI) in patients having RYGB for CSO. The records of patients who underwent RYGB for CSO and had MREE measured at baseline, 6 months and 12 months postoperation were reviewed. MREE was performed using a Med Graphics CCM system after an overnight fast or at least 4 hours after a light meal, and a 30 minute rest in a supine position in a neutral environment, on the same day of the week between the hours of 10 a.m. and 4 p.m. Patients were selected for RYGB in accordance with NIH recommendations. RYGB was performed in a standardized fashion with the Roux limb-length varied as follows: (A) BMI < or = 51 kg/m2 - 75 cm limb (n = 20); (B) BMI < or = 51 kg/m2 - 150 cm limb (n = 16); (C) BMI > or = 51 kg/m2 - 150 cm limb (n = 18); or (D) BMI > or = 51 kg/m2 - 250 cm limb (n = 6). RESULTS: Data from 60 patients (nine male, 51 female; mean age 39 years; mean baseline BMI 51.5 +/- 10 kg/m2; mean baseline weight 145 +/- 32 kg) were analyzed. There were no significant differences in MREE or percentage HB-predicted energy expenditure between the groups. CONCLUSIONS: These data suggest that the observed changes in MREE following RYGB for CSO are not related to the limb-length of the bypass.

Study IV: concurrent validity of the DSM-IV revised Children's Interview for Psychiatric Syndromes (ChIPS).

OBJECTIVE: To determine validity of the Diagnostic and Statistical Manual of Mental Disorders (4th ed.) (DSM-IV) revised Children's Interview for Psychiatric Syndromes (ChIPS) in inpatient children and adolescents. METHOD: Participants were 47 psychiatric inpatients 6-12 (n = 23) and 12 to 18 years of age (n = 24). ChIPS was administered to all participants. The Diagnostic Interview for Children and Adolescents-Revised-Child Version (DICA-R-C) was administered to 40 participants. Discharge diagnoses were recorded for all participants. Kappas, low base rate kappas, and percentage agreement were used to assess diagnostic agreement between sources for 18 disorders. RESULTS: ChIPS/DICA-R-C kappas could not be calculated for two disorders because of 100% agreement on their absence. Fourteen of 16 kappas were significant (p < 0.05). The remaining 2 of 16 disorders had 98% agreement (kappax = 0.494, p < 0.157). When ChIPS results were compared with discharge diagnoses, sensitivity for each disorder averaged 70%, whereas specificity averaged 84%. When disagreements occurred between all three sources, ChIPS was somewhat more likely than DICA-R-C to agree with discharge diagnoses (27% versus 22%). Analysis were repeated for children and adolescents, then for boys and girls. Boys and children had fewer significant ChIPS and DICA-R-C kappa coefficients compared with girls and adolescents; this appeared to be related to the fewer number of diagnoses they endorsed. ChIPS/clinician agreement was similar for boys and girls as well as for children and adolescents. Administration time was less for ChIPS than for DICA-R-C (p < 0.08). CONCLUSION: Psychometric properties of the DSM-IV revised ChIPS compare favorably with that of other structured interviews. ChIPS appears to work well for adolescents as well as children.

Study V: Children's Interview for Psychiatric Syndromes (ChIPS): psychometrics in two community samples.

OBJECTIVE: To determine sensitivity and specificity of the Diagnostic and Statistical Manual of Mental Disorders (4th ed.) (DSM-IV) revised Children's Interview for Psychiatric Syndromes (ChIPS) in nonclinical samples. METHOD: Participants were 40 children 6 to 18 years of age from a community sample (n = 22) or a bereaved sample 1 to 2 years following the death of a parent (n = 18). ChIPS and the Diagnostic Interview for Children and Adolescents (DICA-R-C) were administered in a Latin Square design. A consensus conference (CC) of child psychopathology experts determined presence or absence of syndromes or symptoms after reviewing assessment materials not including ChIPS. RESULTS: Sensitivity is commensurate with epidemiologic base rates (17.5% of participants endorsed at least one syndrome). Low base rate kappas and percentage agreement were calculated to determine agreement on symptom or syndrome endorsement for 20 disorders. For syndrome analyses, over half the kappas could not be calculated due to 100% agreement on absence. For symptom analyses, 3 of 20 kappas could not be calculated (100% agreement on absence). Eleven of ChIPS/DICA-R-C symptom kappas were significant (p < 0.04), 2 of 17 had 95% agreement (kappas, p < 0.08), and 4 of 17 had 97.5% agreement (kappas, p < 0.16). Thirteen of 17 ChIPS/CC symptom kappas were significant (p < 0.04), and 4 of 17 had 97.5% agreement (kappas, p < 0.16). CONCLUSION: ChIPS' psychometrics in nonclinical samples compares favorably with that of other structured interviews.

Destruxin analogs: variations of the alpha-hydroxy acid side chain.

This work describes the synthesis of three destruxin E cyclodepsipeptidic analogs. These compounds have an identical amino acid sequence but differ by the nature of the hydroxy acid residue with is 2-hydroxy-3-phenylpropionic (Hpp), 2-hydroxy-5-trimethylsilyl-4-pentynoic (Hpy-TMS) and 2-hydroxy-4-pentynoic (Hpy) acid, respectively. The insecticidal properties on the Galleria mellonella larvae (paralysis and lethal effect) of these analogs are presented in comparison with the natural destruxin E. All these compounds have toxic effects, the most potent being Hpy that induces the same effect as destruxin E.

Metabolism of tentoxin by hepatic cytochrome P-450 3A isozymes.

The interaction between rat and human liver cytochrome P-450 with tentoxin, a natural phytotoxic cyclotetrapeptide having chlorotic properties, was studied by difference ultraviolet visible spectroscopy. Tentoxin interacted with rat liver microsomes and the difference spectrum was characteristic of binding to a protein site close to the heme. The intensity of this spectrum was clearly dependent on the amounts of P-450 3A in the microsomes and was optimal in dexamethasone-treated rat microsomes. Tentoxin exhibited a high affinity for P-450 3A (Ks approximately 10 microM). Similar results were observed with human P-450 isozymes expressed in yeast. Only P-450 3A4 and 3A5 were able to give spectral interactions with tentoxin. Liver microsomes from rats pretreated with dexamethasone, a specific inducer of P-450 3A, were found to be particularly active for the oxidation of tentoxin, which occurs mainly on its Ala(Me) function leading to demethylation. Yeast-expressed P-450 3A also exhibited high activity to metabolize tentoxin. The metabolites were identified by their ultraviolet and mass spectra in fast atom bombardment and collision-activated dissociation modes. In addition to the major N-demethylated metabolite, other hydroxylated metabolites were formed. Preliminary analysis showed that as tentoxin, some metabolites were still efficient chloroplast ATPase inhibitors, while at least one of them exhibited even at low concentration stimulatory effects.

Synthesis, structure, and properties of MeSer1-tentoxin, a new cyclic tetrapeptide which interacts specifically with chloroplast F1 H(+)-ATPase differentiation of inhibitory and stimulating effects.

A new tentoxin analogue, in which the L-methyl alanine residue is substituted by L-methylserine, has been prepared following the synthetic pathway recently described for the synthesis of tentoxin [Cavelier, F., & Verducci, J. (1995) Tetrahedron Lett. 36, 4425-4428]. Using two-dimensional homonuclear proton nuclear magnetic resonance and structural analysis, we observed that MeSer1-tentoxin, like tentoxin, adopts several conformations in aqueous solution and presents self-aggregative properties. This analogue was found to be conformationally similar to the natural toxin. It showed the same efficiency as tentoxin in inhibition of ATPase activity of the isolated chloroplast F1 proton ATPase (CF1) as well as in inhibition of the ATP synthase activity of the membrane-bound enzyme (CF0CF1) in thylakoids and proteoliposomes. At concentrations above 10 microM, MeSer1-tentoxin did not reactivate CF1 to a high extent, contrary to tentoxin. It appeared, however, to bind in the same way, since the reactivating effect of tentoxin was inhibited by MeSer1-tentoxin. These results show that it is possible, using tentoxin analogues, to separate inhibitory and activating effects on the chloroplast ATPase, despite the limited chemical difference between the two toxins.

Tentoxin has at least two binding sites on CF1 and epsilon-depleted CF1 ATPases isolated from spinach chloroplast.

A new procedure for synthesis of 14C-labeled tentoxin [14C-MePhe[(Z)delta]3-tentoxin], with a high specific activity, is described. Binding experiments with CF1 or CF1-epsilon isolated from spinach chloroplast have been carried out using equilibrium dialysis technique. The results show the presence of two classes of binding sites. The association constants of the two major binding sites were derived from non-linear fitting of the binding curves. At 4 degrees C, the first binding site has a value of Ka1 = 8.2 x 10(5) M(-1) in CF1 and 8.7 x 10(5) M(-1) in CF1-epsilon, while the second binding site has lower affinity with Ka2 = 1.5 x 10(4) M(-1) in CF1 and 2.3 x 10(3) M(-1) in CF1-epsilon.

A symmetric analysis of paired rankings with application to temporal patterns of hormonal concentration.

Mallows (1957, Biometrika 44, 114-130) introduced a simple model to describe the distribution of subjects' rankings of the items from some fixed set. Both Feigin (1992, in Probability Models and Statistical Analyses for Ranking Data, 75-91. New York: Springer-Verlag) and Critchlow and Verducci (1992, Journal of the Royal Statistical Society, Series C 41, 17-29) have considered an asymmetric extension of this model, designed to analyze the distribution of subjects' post-treatment rankings, conditional on their pretreatment rankings. Here a symmetric extension is introduced and is used to analyze the joint distribution of paired rankings associated with the patterns of prolactin hormonal response of wife-husband pairs during the course of a marital conflict. Locally most powerful tests are developed to check for independence of the wife and husband responses, and for differences in their divergence from a theoretical pattern of response. Additionally, conditional on the rankings of one group (e.g., the wives), the locally most powerful test of an affinity for the theoretical pattern in the other group (e.g., the husbands) turns out to be closely related to the test given by Critchlow and Verducci.

Synthesis and biological activity of a destruxin analogue: D-Lac-6 destruxin E.

A general strategy for the synthesis of destruxin analogues is described and applied to a particular example, D-Lac-6 destruxin E. The tetrapeptide Boc-Ile-N-MeVal-N-MeAla-beta-Ala-OMe (2) was chosen as the basic starting compound, and its preparation was optimized. This fragment was then coupled with the compound (D)Lac-Pro, and the resulting peptide was cyclized by the DEPC or DPPA/HOBt/DMAP methods at 21 and 30% yield, respectively. The biological activity of the analogue obtained was established by injection to an insect host.

Ionic pores formed by cyclic peptides.

It is shown that 2 cyclic tetrapeptides, namely tentoxin and HC toxin, are able to induce the formation of transmembrane ionic channels, although a carrier mechanism could be expected on the basis of their chemical structure (presence of proline or N-methylated residues). Since other cyclic peptides but of larger size, i.e., tyrocidines, gramicidin S (decapeptides) and an octapeptide with a sequence similar to that of HC toxin, are also able to form pores, it appears that this property can be extended to a large number of cyclic peptides. A pore structure based on aggregates is proposed.

Aggregation and ion transfer induced by tentoxin.

It is shown that tentoxin, a cyclic tetrapeptide with two N-methylated residues, is able, when added to lipid bilayers, to increase the transmembrane current through discrete events. Conformational investigations involving 1H-NMR, infrared and circular dichroism studies show that, at concentrations above 7 X 10(-5) M, the cyclic tetrapeptide aggregates in chloroform. We suggest that the aggregates could form a pore through a stacking of cycles.