Studies on 3-deoxy-D-manno-octulosonic acid 8-phosphate synthase using chorismate mutase inhibitors.

The proposed cyclic mechanism of 3-deoxy-D-manno-octulosonic acid 8-phosphate synthase and the mechanism of chorismate mutase share certain structural and electronic similarities. In this report, we examine several inhibitors of chorismate mutase for their efficacy against KDO 8-P synthase.

A selective inhibitor of Escherichia coli prephenate dehydratase.

To identify selective prephenate dehydratase (PDT) inhibitors, a series of substituted biphenic acid derivatives was synthesized using the Ullmann reaction. Screening experiments identified 18 as a promising new PDT inhibitor.

Asymmetric total synthesis of (-)-alpha-kainic acid using an enantioselective, metal-promoted ene cyclization.

[figure: see text] A short and efficient asymmetric total synthesis of the title compound 1, which is an important neurotransmitter, has been achieved. The synthesis features a metal-promoted, enantioselective ene reaction that provides entry into the kainic acid ring system from very simple precursors. Moreover, the zirconium-mediated Strecker reaction, which represents an outgrowth of earlier amide-to-imine methodology developed in our laboratory, demonstrates remarkable chemoselectivity and stereoselectivity.

Selective fowler reductions: asymmetric total syntheses of isofagomine and other 1-azasugars from methyl nicotinate.

[figure: see text] An efficient, high-yielding strategy has been developed for the asymmetric synthesis of 1-N-iminosugars (1-azasugars), a new class of glycosidase inhibitors with promising biomedical applications. A highly regioselective procedure for the 1,2-reduction of substituted pyridines was employed to transform methyl nicotinate into several representative 1-azasugars.

Polyamines with N-(3-phenylpropyl) substituents are effective competitive inhibitors of trypanothione reductase and trypanocidal agents.

Several N-(3-phenylpropyl)-substituted spermidine and spermine derivatives were prepared and found to be potent competitive inhibitors of Trypanosoma cruzi trypanothione reductase (seven compounds with Ki values < 5 microM are described). The most effective inhibitor studied was compound 12 with a Ki value of 0.151 microM. Six of the compounds described are also effective trypanocides with IC50 values < 1 microM.

A theoretical study of the chorismate synthase reaction.

Density functional calculations (B3LYP/6-31+G(d,p)) were carried out to investigate the mechanism of the anti-1,4-elimination of phosphate from 5-enolpyruvylshikimate-3-phosphate 1 that is catalyzed by chorismate synthase. Of particular interest was the functional role of the reduced flavin cofactor. [reaction: see text]

Total synthesis of mololipids: a new series of anti-HIV Moloka'iamine derivatives.

A new family of bioactive bromotyrosine derivatives, termed mololipids, was recently isolated from a Hawaiian sponge, but could not be resolved into individual components by chromatography. To complete their structural characterization and better understand structure-activity relationships, the first pure samples of dimyristoyl, distearoyl, dioleoyl, and stearoyl/oleoyl mololipids have now been prepared by total synthesis, and their anti-HIV activity investigated.

Reaction of COTC with glutathione: structure of the putative glyoxalase I inhibitor.

The structure of the active glyoxalase I inhibitor derived from the Streptomyces griseosporeus metabolite COTC 1 has been conclusively identified by means of total synthesis as 2c. Human glyoxalase I is competitively inhibited by 2c (K(i)() = 183 +/- 6 microM) but is not inhibited by 1 itself.

Synthesis of glycolipid analogues that disrupt binding of HIV-1 gp120 to galactosylceramide.

HIV-1 has been shown to infect CD4 negative cells by the binding of HIV gp120 to the glycolipid galactosylceramide (1) (GalCer). Several analogues of 1 were prepared to investigate the specific orientation of 1 in the membrane bilayer that is involved in gp120 binding. Interestingly, N-stearyl-1-deoxynojirimycin (8) displayed potent and specific affinity for gp120 equal to that of 1, a finding that may shed light on the antiviral activity of N-butyl-1-deoxynojirimycin.

Stereoselective synthetic approaches to highly substituted cyclopentanes via electrophilic additions to mono-, di-, and trisubstituted cyclopentenes.

Electrophilic additions to allylically substituted alkenes are of broad synthetic utility. The control of stereoselectivity in such reactions has attracted considerable interest. However, the effect of allylic and homoallylic substituents in cyclopentenyl systems has not been investigated systematically. Studies on a series of mono, di-, and trisubstituted cyclopentenes are reported in which trans-vicinal-additions favor a syn-selective approach of electrophiles to the cyclopentene system. The formal addition of HOBr, HOCl, CH(3)SCl, and dimethyl(methylthio)sulfonium tetrafluoroborate (DMTSF)/NaN(3) with a variety of cyclopentene substrates has been carried out, and the effects of various allylic substituents on these selectivities have been examined. Additions of HOBr, HOCl, and DMTSF to highly functionalized substrates proceed predictably with syn selectivity, giving predominantly or exclusively one product. Methanesulfenyl chloride additions are less predictable, but can be tuned by suitable alteration of solvent and substrate. Results have proven useful in total syntheses of (+)-trehazolin and (+)-allosamidin.

Probing the catalytic mechanism of prephenate dehydratase by site-directed mutagenesis of the Escherichia coli P-protein dehydratase domain.

The Escherichia coli bifunctional P-protein, which plays a central role in L-phenylalanine (Phe) biosynthesis, contains distinct chorismate mutase (CM) and prephenate dehydratase (PDT) domains as well as a regulatory (R) domain for feedback control by Phe. To elucidate the catalytic mechanism of PDT in the P-protein, 24 mutations of 15 conserved residues in the PDT domain were created, expressed in the pheA(-)E. coli strain NK6024, and studied for their effect on PDT activity. Fourteen mutant enzymes were purified to homogeneity, tested for feedback inhibition by Phe, and characterized by kinetic analysis and circular dichroism spectroscopy. Selected mutant enzymes were further studied by gel filtration, fluorescence emission, and microcalorimetry. In addition, a monofunctional PDT domain (PDT20, residues 101-285) was cloned and overexpressed in plasmid pET with expression levels up to 200-250 mg/L. PDT20 retained full PDT activity, lacked CM activity, and was insensitive to feedback inhibition by Phe. Four residues (T278, N160, Q215, and S208) were shown to be important for PDT catalysis. The values of k(cat)/K(m) for the S208A/C and T278S mutant enzymes were 100-fold lower, and 500-fold lower for the N160A and Q215A mutant enzymes than the wild-type (WT) protein. The T278A and T278V mutant enzymes displayed no measurable catalytic activity, yet bound both prephenate and a competitive inhibitor (S-DNBA) comparably to the WT protein. These data, taken together with the normal CD spectra of the mutant enzymes, strongly suggested that T278 was involved in the catalytic mechanism. To establish whether acidic residues were involved in catalysis, all the conserved Glu and Asp residues in the PDT domain were mutated to Ala. None of these mutations significantly reduced PDT activity, indicating that the acidic residues of the PDT domain are not directly involved in catalysis. However, two mutant enzymes (E159A and E232A) displayed higher levels of PDT activity (2.2- and 3.5-fold, respectively), which was due to enhanced substrate binding. For the double mutant enzyme (E159A-E232A), k(cat)/K(m) was ca. 7-fold higher than for the WT enzyme, while its K(m) was 4.6-fold lower.

A pharmacokinetic-pharmacodynamic modelling of the antihistaminic (H1) effects of cetirizine.

AIM: The pharmacokinetic-pharmacodynamic modelling developed here characterizes the time course of cetirizine effect on histamine-induced skin reactions (wheal and flare). METHOD: The model incorporated data from the study of Simons et al. [1993] in which the cetirizine plasma concentrations and the wheal and flare areas were recorded in a group of 6 patients after a 10 mg oral administration. RESULTS: The peak plasma concentration (>500 ng/ml) was rapidly reached in 1 h and the maximal effects were observed later at approximately 6 h. The cetirizine effect was ascribed to a physiologic indirect response model in which the drug concentration in the central compartment is linked to a response function that describes the inhibition or stimulation of the factors affected, input or output of response control. Cetirizine was characterized by two-compartmental kinetics with a rapid absorption phase (Ka = 1.0-1.4 h(-1)), a rapid distribution phase (alpha = 0.33-0.69 h(-1)) and a slower terminal half-life, 13.2-13.6 h (beta = 0.051-0.052 h(-1)). The total clearance was 1.4-1.5 l/h. Cetirizine effects on flare and wheal were characterized by the inhibition of the input factor (k(in)), the concentrations producing 50% of maximal effect (EC50) were 13 and 40 ng/ml and k(in) were 0.99 and 0.96 h(-1), respectively. These results were then used to simulate repeated daily oral administration of 10 mg cetirizine. CONCLUSION: At this dosage the histamine-induced flare was at least 80% inhibited at the start of the second administration Thereafter, on successive administrations, the inhibition was even more pronounced and the response control was nearly total.

Regulation of phenylalanine biosynthesis. Studies on the mechanism of phenylalanine binding and feedback inhibition in the Escherichia coli P-protein.

Isothermal titration calorimetry (ITC) and site-directed mutagenesis were used to study the interaction of Phe with (a) the Escherichia coli P-protein, a bifunctional chorismate mutase/prephenate dehydratase that is feedback inhibited by Phe, (b) PDT32, a 32 kDa P-protein fragment (residues 101-386) containing the prephenate dehydratase and regulatory domains, and (c) R12, a C-terminal 12 kDa P-protein fragment (residues 286-386) containing the regulatory domain. DeltaH(total) values for PDT32, which included the heats of Phe binding, conformational change, and dimerization, established that in developing a mechanism for end product feedback inhibition, the P-protein has evolved a ligand recognition domain that exhibits Phe-binding enthalpies comparable to those reported for other full-fledged amino acid receptor proteins. Sequence alignments of R12 with other Phe-binding enzymes identified two highly conserved regions, GALV (residues 309-312) and ESRP (residues 329-332). Site-directed mutagenesis and ITC established that changes in the GALV and ESRP regions affected Phe binding and feedback inhibition to different extents. Mutagenesis further showed that C374 was essential for feedback inhibition, but not for Phe binding, while W338 was involved in Phe binding, but not in the Phe-induced conformational change required for feedback inhibition.

Thermodynamics of a transition state analogue inhibitor binding to Escherichia coli chorismate mutase: probing the charge state of an active site residue and its role in inhibitor binding and catalysis.

Electrostatic interactions play important roles in the catalysis of chorismate to prephenate by chorismate mutase. Mutation of Gln88 to glutamate in the monofunctional chorismate mutase from Escherichia coli results in an enzyme with a pH profile of activity significantly different from that of the wild type protein. To investigate whether the mutation alters the substrate binding process or the catalysis, we have directly determined the thermodynamic parameters of a transition state analogue inhibitor binding to the wild-type chorismate mutase and its Q88E mutant using isothermal titration calorimetry. The results demonstrate that solvent reorganization and hydrophobic interactions contribute the predominant free energy to inhibitor binding. The charge state of Glu88 in the Q88E mutant was experimentally determined and was shown to be protonated at pH 4.5 and ionized at pH 7.8, consistent with earlier hypotheses. Most surprisingly, inhibitor binding energetics do not exhibit significant pH dependency for both enzymes. Our findings indicate that the charge state of Glu88 has a small impact on inhibitor binding but plays an important role in the catalytic process.

Chorismate mutase-prephenate dehydratase from Escherichia coli. Study of catalytic and regulatory domains using genetically engineered proteins.

The bifunctional P-protein, which plays a central role in Escherichia coli phenylalanine biosynthesis, contains two catalytic domains (chorismate mutase and prephenate dehydratase activities) as well as one R-domain (for feedback inhibition by phenylalanine). Six genes coding for P-protein domains or subdomains were constructed and successfully expressed. Proteins containing residues 1-285 and residues 1-300 retained full mutase and dehydratase activity, but exhibited no feedback inhibition. Proteins containing residues 101-386 and residues 101-300 retained full dehydratase activity, but lacked mutase activity. Fluorescence emission spectra and binding assays indicated that residues 286-386 were crucial for phenylalanine binding. The mutase (residues 1-109), dehydratase (residues 101-285), and regulatory (residues 286-386) activities were thus shown to reside in discrete domains of the P-protein. Both the mutase domain and the native P-protein formed dimers. Deletion of the mutase domain diminished phenylalanine binding to the regulatory site as well as prephenate binding to the dehydratase domain, both through cooperative effects. Besides eliminating feedback inhibition, removal of the R-domain decreased the affinity of chorismate mutase for chorismate.

Characterization of benzodiazepine "combinatorial" chemical libraries by on-line immunoaffinity extraction, coupled column HPLC-ion spray mass spectrometry-tandem mass spectrometry.

To characterize combinatorial chemical libraries of small drug compounds, an automated column switching system incorporating an immunoaffinity extraction (IAE) column and two reversed-phase HPLC columns was coupled to a triple-quadrupole mass spectrometer. A Protein G column and antibodies to benzodiazepines were used to screen library components. A pH change in the mobile phase eluted the benzodiazepine-antibody complexes onto a C-18 restricted access media (RAM) column, thereby separating the selected benzodiazepines from the antibody. In a final step, backflushing the RAM column eluted the benzodiazepines onto a C-8 analytical reversed-phase column for separation before detection and preliminary structural characterization using ion spray mass spectrometry (MS) and tandem mass spectrometry (MS/ MS). A known 19-component library and an unknown 20-component library were analyzed. Full-scan IAE/LC/ LC/MS and IAE/LC/LC/MS/MS chromatograms suggested the feasibility of this combination of techniques, although the antibodies used were not highly specific. Inspection of MS/MS spectra of components in the unknown library compared to the MS/MS spectrum of a known standard (chlordiazepoxide) identified a subclass of benzodiazepines. Productions of the known standard and an unknown benzodiazepine were successively captured and fragmented (MSn experiments) using an iontrap mass spectrometer off-line, which confirmed that the unknown was an analogue of chlordiazepoxide.

Charge is the major discriminating factor for glutathione reductase versus trypanothione reductase inhibitors.

Benson et al. (Biochem. J. 1992, 286, 9) reported three novel competitive inhibitors of trypanothione reductase (TR), which were selected to complement a hydrophobic region identified on the TR structure which was not present on human glutathione reductase (hGR). Benson et al. also noted that chlorpromazine, a tricyclic antidepressant known to have trypanocidal activity, was an inhibitor of TR. Here we show that chlorpromazine is a competitive inhibitor of TRs from Crithidia fasciculata (Ki = 14 microM) and Trypanosoma cruzi (Ki = 10 microM), but the drug binds > 50-fold more weakly (Ki = 762 microM) to hGR. Analogues of chlorpromazine differing in the length of the side chain carrying the positively charged R-group are also selective TR inhibitors whereas, a tricyclic structure carrying a negatively charged side chain is a competitive inhibitor with selectivity for hGR (K(hGR)i = 165 microM vs. K(TR)i = 1400 microM). This finding suggests that simple charge characteristics, rather than differences in hydrophobicity, may account for a significant portion of the selectivity of this series of inhibitors for these two enzymes. Electrostatic analysis of the structures of TR and hGR thus provides a rationale for these results, and offers a new principle for inhibitor design. The principle gains further support from the observation that all known tricyclic competitive inhibitors of TR are positively charged. In order to investigate the in vivo relevance of our findings we have examined the effect of chlorpromazine and its negatively charged analogue on the growth of C. fasciculata parasites. Consistent with our kinetics, chlorpromazine (50 microM) inhibited the growth of parasites by 50%, while no measurable decrease in parasite growth rate was noted in the presence of the negatively charged inhibitor (400 microM). Furthermore, the highly similar inhibitory profiles of C. fasciculata TR and T. cruzi TR suggest that drug-design studies using the structurally better-studied C. fasciculata TR are also relevant to the human pathogen T. cruzi.

Site-directed mutagenesis of monofunctional chorismate mutase engineered from the E. coli P-protein.

Analysis of the active-site residues of a fully functional chorismate mutase representing the N-terminal 113 amino acids of the Escherichia coli P-protein suggests that Lys39 and Gln88 play critical roles in catalyzing the rearrangement of chorismate to prephenate. Five site-directed mutants at these positions have been constructed in which Lys39 was replaced with Arg, Asn, and Gln, and Gln88 was replaced with Arg and Glu. Although the Gln88Arg plasmid failed to produce detectable cross-reacting proteins in E. coli, the other four plasmids were expressed, and the mutant proteins purified to homogeneity. Their structures were similar to wild type enzyme, as indicated by circular dichroism spectra, with Lys39Gln showing a small deviation. In accordance with predictions, all mutations result in major loss of catalytic activity at pH 7.8. However, activity of the Gln88Glu mutant at pH 4.5 exceeded wild-type EcCM. Implications for the mechanism of mutase catalysis are discussed.

Trypanosomal nucleoside hydrolase. Resonance Raman spectroscopy of a transition-state inhibitor complex.

The transition state for hydrolysis of the N-ribosidic bond of inosine by nucleoside hydrolase has oxocarbenium character and a protonated leaving group hypoxanthine with an sp2-hybridized C1' of the ribosyl [Horenstein, B. A., Parkin, D. W., Estupinan, B., & Schramm, V. L. (1991) Biochemistry 30, 10788-10795]. These features are incorporated into N-(p-nitrophenyl)-D-riboamidrazone, a transition state analogue which binds with a dissociation constant of 2 nM [Boutellier, M., Horenstein, B. A., Semenyaka, A., Schramm, V. L., & Ganem, B. (1994) Biochemistry 33, 3994-4000]. Resonance Raman and ultraviolet-visible absorbance spectroscopy has established that the inhibitor binds as the neutral, zwitterionic species. The enzyme stabilizes a specific resonance state characterized by the quinonoid form of the p-nitrophenyl group with evidence for ion pairing at the nitro group. Incorporation of 15N into a specific position of the amidrazone reveals that the exo-ribosyl nitrogen bonded to the C1' position carries the proton while that bonded to the p-nitrophenyl carbon is unprotonated. This tautomer carries a distributed positive charge centered at the position analogous to C1' of the ribosyl group at the transition state. The molecular electrostatic potentials for the substrate inosine, the transition state, and the transition state inhibitor are compared at the van der Waals surface of the molecules. The tautomer of the inhibitor bound to the enzyme bears a striking electrostatic resemblance to the transition state determined by kinetic isotope effect analysis. The spectral and resonance Raman properties of free and enzyme-bound inhibitor have permitted tautomeric assignment of these species and establish that the enzyme substantially changes the electronic distribution of the bound inhibitor toward that of the enzyme-stabilized transition state.