Counter-current chromatography with off-line detection by ultra high performance liquid chromatography/high resolution mass spectrometry in the study of the phenolic profile of Lippia origanoides.

Lippia origanoides (Verbenaceae) is an important Brazilian medicinal plant, also used for culinary purposes. Most chemical studies with this plant have been focused on its volatile composition. In this work, we combined High-Speed Counter-current Chromatography (HSCCC) and High Performance Liquid Chromatography coupled to Ultra Violet detection and High Resolution Mass Spectrometry (HPLC-UV-HRMSn) methodologies to access the non-volatile chemical composition of L. origanoides. The crude ethanol extract of L. origanoides (LOEF) was first analyzed by HPLC-UV-HRMSn and allowed the identification of 7 major compounds. Among them, eriodictyol, naringenin and pinocembrin, were determined and are phytochemical markers of this plant. However, owing to the complexity of this plant matrix, LOEF was fractionated by HSCCC (hexane-ethanol-water, 4:3:1) as a tool for preparative pre-purification, affording a flavonoid-rich fraction. A column screening with the chromatographic stationary phases ZIC-HILIC, monolithic and particulate RP18 was performed. The best column separation was achieved with a Purospher STAR RP18e, which was used for HPLC-DAD-HRMSn studies. By this approach 12 compounds were further identified in addition to the major ones identified in the raw extract. Two of them, 6,8-di-C-hexosyl-luteolin and 6,8-di-C-glucosyl-apigenin, are being reported for the first time in the family Verbenaceae. This work shows the integration of HSCCC as a preparative tool for the fractionation and purification of natural products from a complex plant extract with other analytical techniques, with the purpose of showing each technique's potential.

Comparative evaluation of aspheric toric intraocular lens implantation and limbal relaxing incisions in eyes with cataracts and ≤3 dioptres of astigmatism.

PURPOSE: To compare the visual outcomes of aspheric toric intraocular lens (IOL) implantation and limbal relaxing incisions (LRI) for management of coexisting age-related cataracts and astigmatism. METHODS: In this prospective study, sixty eyes of 60 patients with visually significant cataract and coexisting corneal astigmatism ≤3 dioptres (D) were randomised to undergo phacoemulsification with either aspheric toric IOL or aspheric monofocal IOL with LRI. The main outcome measures were postoperative 3-month uncorrected visual acuity (UCVA), contrast sensitivity, rotational stability of the toric IOL and spectacle independence. RESULTS: The postoperative UCVA, contrast sensitivity and refractive astigmatism were significantly better than the baseline measurements for both groups (p≤0.001). There was no significant difference detected for these parameters between LRI and toric IOL groups postoperatively (p≥0.119). At both postoperative month 1 and 3, the percentages of eyes in need of spectacles were lower in toric group than LRI group (p≤0.030). IOL misalignment was noted in three eyes in the toric IOL group (mean misalignment 7.67±4.04°). On vector analysis, magnitude of error (ME) was negative in the LRI group indicating undercorrection, whereas the ME was close to zero for toric group. CONCLUSIONS: Both toric IOL implantation and LRI were effective in correcting corneal astigmatism ≤3 D during phacoemulsification, while LRI tended to undercorrect astigmatism.

The Association of Delayed Corneal Surface and Visual Recovery After Corneal Transplantation With Longer Donor Storage Time-A Prospective Analysis.

PURPOSE: This study aimed to assess the influence of donor corneal storage time on endothelial cell count (ECC), corneal epithelial recovery, and visual rehabilitation after corneal transplantation in the first postoperative year. DESIGN: A collaborative prospective study involving a local eye bank and a tertiary ophthalmic unit was conducted. METHODS: Donor cornea buttons were stored in Optisol-GS (Chiron Ophthalmics Inc, Irvine, Calif) storage media for a maximum of 14 days before transplantation. Before corneal distribution, the eye bank collected information on death-to-harvesting time, death-to-surgery time, donor central corneal thickness, and donor ECC at various time points. Subjects who underwent penetrating keratoplasty and endothelial keratoplasty were recruited and monitored for 1 year. Postoperative epithelial healing, visual acuity, ECC, and hospital stay were evaluated. RESULTS: Thirty-one eyes of 31 patients completed the study. There was a significant positive correlation between donor storage time and epithelial healing (Spearman ρ = 0.39, P = 0.031). Faster epithelial healing was significantly correlated with posttransplantation visual improvements at months 1, 3, and 6 and shorter hospital stay (Spearman ρ = 0.74, P < 0.001). Mean ECC loss was 23.8% at 12 months posttransplantation. There was no significant correlation between storage time and ECC loss preoperatively and posttransplantation. CONCLUSIONS: The duration of graft storage in Optisol-GS storage media up to 14 days had no significant effects on long-term visual acuity and ECC postoperatively. Shorter storage time had significant correlation with earlier epithelial healing and faster visual rehabilitation.

Comparison of the Surgical Outcomes of Various Methods of Endothelial Keratoplasty.

PURPOSE: The objective of this study was to evaluate the outcomes of various techniques of endothelial keratoplasty (EK) including deep lamellar endothelial keratoplasty (DLEK), Descemet stripping endothelial keratoplasty (DSEK), and Descemet stripping automated endothelial keratoplasty (DSAEK). DESIGN: This was a retrospective comparative case series. METHODS: The medical records of 48 consecutive patients who have undergone EK in a tertiary eye center between January 2005 and June 2011 were reviewed. Information related to demographics, visual acuity, corneal endothelial cell count, and postoperative complications was recorded. RESULTS: The series included 11 eyes with DLEK, 11 eyes with DSEK, and 26 eyes with DSAEK. There was no significant difference in visual outcomes, endothelial cell loss, and postoperative complications between the 3 groups 1 year after surgery. The mean logMAR visual acuity at 12 months was 0.54 (SD, 0.26) for DLEK, 0.55 (SD, 0.47) for DSEK, and 0.63 (SD, 0.48) for DSAEK, respectively. The 6-month endothelial cell density loss was 48.4%, 39.2%, and 47.5% for the DLEK, DSEK, and DSAEK groups, respectively. Early postoperative graft dislocation occurred in 1 (9%) of the DLEK cases, 2 (18%) of the DSEK cases, and 1 (4%) of the DSAEK cases. All of these cases were successfully repositioned. CONCLUSIONS: Despite the various evolution and surgical modifications and development in EK in the past few years, the visual outcomes and postoperative complications between DLEK, DSEK, and DSAEK were comparable.

Comparison between central corneal thickness measurements by ultrasound pachymetry and optical coherence tomography.

PURPOSE: Measurement of central corneal thickness (CCT) plays an important role in both diagnostic and therapeutic assessment of ocular diseases. Although ultrasound pachymetry (U-PACH) is regarded as the golden standard for measurement of CCT, optical coherence tomography (OCT) may offer advantages as it can locate the central cornea with precision with no corneal touch. Nevertheless, the agreement of OCT with U-PACH has not yet been gauged by Bland-Altman analysis. This study compares CCT measurement by OCT with that by U-PACH. METHODS: Healthy subjects without ocular abnormality (except refractive errors less than or equal to -6.0 D), contact lens wear or ocular surgery were recruited. CCT was measured in one eye of normal subjects using OCT and U-PACH. Results were compared using correlation and Bland-Altman plots. RESULTS: Fifty subjects were recruited. Mean +/- SD CCT measured by OCT was 565 +/- 33 microm. This was highly correlated (Pearson's coefficient = 0.934) with the mean thickness measured by U-PACH (543 +/- 33 microm). The coefficients of variation were good and comparable at 7.9% for U-PACH and 3.5% for OCT. Compared with U-PACH, OCT consistently overestimated the CCT by a mean of 23 microm as shown on Bland-Altman plot. CONCLUSION: CCT measured by OCT and U-PACH is highly correlated. With appropriate adjustment factor, OCT agrees well with U-PACH and is a reliable alternative for CCT measurement.

Identification of a novel class of inhibitor of human and Escherichia coli thymidine phosphorylase by in silico screening.

Structure-based computational screening of the National Cancer Institute database of anticancer compounds identified novel non-nucleobase-derived inhibitors of human thymidine phosphorylase as candidates for lead optimization. The hierarchical in silico screening strategy predicted potentially strong low molecular weight ligands exhibiting a range of molecular scaffolds. Of the thirteen ligands assayed for activity, all displayed inhibitory activity against Escherichia coli thymidine phosphorylase. One compound, hydrazine carboxamide 2-[(1-methyl-2,5-dioxo-4-pentyl-4-imidazolidinyl)methylene], was found to inhibit E. coli thymidine phosphorylase with an IC(50) value of 20 microM and an IC(50) value of 77 microM against human thymidine phosphorylase. As this hydantoin derivative lacks the undesirable ionic sites of existing tight-binding nucleobase-derived inhibitors, such as 5-chloro-6-[(2-iminopyrrolidin-1-yl)methyl]uracil hydrochloride, it provides an opportunity for the design of potent thymidine phosphorylase inhibitors with improved pharmacokinetic properties.

Peptoid inhibition of trypanothione reductase as a potential antitrypanosomal and antileishmanial drug lead.

One route to the design of lead compounds for rational drug design approaches to developing drugs against trypanosomiasis, Chagas' disease and leishmaniasis is to develop novel inhibitors of the parasite-specific enzyme trypanothione reductase. A lead inhibitor based on a peptoid structure was designed in the present study based on the known strong competitive inhibition of trypanothione reductase by N-benzoyl-Leu-Arg-Arg-beta-naphthylamide and N-benzyloxycarbonyl-Ala-Arg-Arg-4-methoxy- beta-naphthylamide. In the target peptoid the arginyl residues were replaced by alkylimidazolium units and the benzyloxycarbonyl group by the benzylaminocarbonyl function. The peptoid was synthesised using t-butoxycarbonyl protection chemistry and couplings were activated by 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate. The resulting peptoid was shown to be a competitive inhibitor of recombinant trypanothione reductase from Trypanosoma cruzi with a K(i) value of 179 microM and with only weak inhibition of human erythrocyte glutathione reductase (the inhibition of glutathione reductase was at least 291-fold weaker than of trypanothione reductase).

Specific peptide inhibitors of trypanothione reductase with backbone structures unrelated to that of substrate: potential rational drug design lead frameworks.

By introducing cationic charge sites novel peptide lead inhibitor structures for trypanothione reductase have been designed using molecular modelling methods. The inhibitors showed reversible, linear competitive inhibition and the strongest peptide inhibitor to date was found to be N-benzyloxycarbonyl-Ala-Arg-Arg-4-methoxy-beta-naphthylamide with a Ki value of 2.4 microM and a selectivity for parasitic enzyme (trypanothione reductase) over the host enzyme (human glutathione reductase) of over 3 orders of magnitude.

Increased stability and lifetime of the complex formed between DNA and meta-phenyl-substituted Hoechst dyes as studied by fluorescence titrations and stopped-flow kinetics.

The large increase in fluorescence upon binding of five para- and meta-phenyl substituted hydroxy and methoxy derivatives of the Hoechst dye with poly[d(A-T)], d(CGCGAATTCGCG)2, and its corresponding T4-looped 28-mer hairpin was used to monitor the binding by equilibrium titrations and by stopped-flow kinetics. The affinity increases in the same order for the three DNAs: p-OH

Binding of a desmetallo-porphyrin conjugate of Hoechst 33258 to DNA. III. Strong bonding to single-strand oligonucleotides.

The binding of the conjugate of Hoechst 33258 with 5,10,15,20-tetrakis (1-methyl-4-pyridyl)-21H,23H-porphyrin (PORHOE) to single-strand DNA has been detected by UV-vis spectrophotometry and 1H-NMR. The red-shift of porphyrin Soret band with strong hypochromicity indicates that the porphyrin moiety dominates in the interaction of the PORHOE with ssDNA. The affinity constants of PORHOE for d(GCATACAATTCG) or d(CGAATTGTATGC) were determined to be >10(5) M(-1), with strong cooperativity.

Binding of a porphyrin conjugate of Hoechst 33258 to DNA. I. UV-visible and melting studies detect multiple binding modes to a 12-mer nonself-complementary duplex.

Relative to ligand-free duplex DNA, the melting temperature of the 1:1 complex of the duplex d(CGAATTGTATGC):d(GCATACAATTCG) with the conjugate of Hoechst 33258 with a des-metalloporphyrin, increased from 42 to 60.5 degrees C indicating strong ligand binding. UV-vis spectrophotometric titration detected more than one class of binding site (apparent dissociation constants approximately 0.2 microM for simple noncooperative binding and 1 microM for the simultaneous cooperative mode with Hill coefficient approximately 2).

Binding of a porphyrin conjugate of Hoechst 33258 to DNA. II. NMR spectroscopic studies detect multiple binding modes to a 12-mer nonself-complementary duplex DNA.

We have probed by 1H NMR spectroscopy the molecular basis of the interaction between Hoechst 33258 conjugated to a des-metalloporphyrin and a non self-complementary duplex DNA sequence, designed on the known chemical nuclease selectivity of this system. The imino NMR spectra are consistent with two distinct families of structure, that is, PORHOE binding either way along the duplex. 2D spectral, T2, and linewidth data suggest multiple species within the two conformational families.

Synthetic inhibitors of the processing of pretransfer RNA by the ribonuclease P ribozyme: enzyme inhibitors which act by binding to substrate.

2,2'-p-Phenylene bis[6-(4-methyl-1-piperazinyl)]benzimidazole, 2,2'-bis(3,5-dihydroxyphenyl)-6,6'-bis benzimidazole, and 2,2'-bis(4-hydroxyphenyl)-6,6'-bis benzimidazole are shown by UV-visible and fluorescence spectrophotometry to be strong ligands for tRNA, giving simple, hyperbolic binding isotherms with apparent dissociation constants in the micromolar range. Hydroxyl radical footprinting indicates that they may bind in the D and T loops. On the basis of this tRNA recognition as a rationale, they were tested as inhibitors of the processing of precursor tRNAs by the RNA subunit of Escherichia coli RNase P (M1 RNA). Preliminary studies show that inhibition of the processing of Drosophila tRNA precursor molecules by phosphodiester bond cleavage, releasing the extraneous 5'-portion of RNA and the mature tRNA molecule, was dependent on both the structure of the inhibitor and the structure of the particular tRNA precursor substrate for tRNA(Ala), tRNA(Val), and tRNA(His). In more detailed followup using the tRNA(His) precursor as the substrate, experiments to determine the concentration dependence of the reaction showed that inhibition took time to reach its maximum extent. I(50) values (concentrations for 50% inhibition) were between 5.3 and 20.8 microM, making these compounds among the strongest known inhibitors of this ribozyme, and the first inhibitors of it not based on natural products. These compounds effect their inhibition by binding to the substrate of the enzyme reaction, making them examples of an unusual class of enzyme inhibitors. They provide novel, small-molecule, inhibitor frameworks for this endoribonuclease ribozyme.

Use of an additional hydrophobic binding site, the Z site, in the rational drug design of a new class of stronger trypanothione reductase inhibitor, quaternary alkylammonium phenothiazines.

Improved rationally designed lead drug structures against African trypanosomiasis, Chagas disease, and leishmaniasis were obtained against trypanothione reductase from Trypanosoma cruzi. Substituted-benzyl [3-(2-chloro-4a, 10a-dihydrophenothiazin-10-yl)propyl]dimethylammonium salts, synthesized by Menschutkin quaternization of the tertiary alkylamine omega-nitrogen atom of chlorpromazine, were linear, competitive inhibitors of recombinant trypanothione reductase from T. cruzi, with either trypanothione disulfide or N-benzyloxycarbonyl-L-cysteinylglycyl 3-dimethylaminopropylamide disulfide as substrate. The permanent positive charge on the distal nitrogen atom of the tricyclic side chain contribution to binding was estimated as >/=5.6 kcal.mol(-1) by comparison with the analogue with the cationic nitrogen atom of the quaternary replaced by an ether oxygen atom. A further major contribution to improving K(i) values and inhibition strength was the hydrophobic natures and structures of the N-benzyl substituents. The strongest inhibitor, the [3-(2-chloro-4a,10a-dihydrophenothiazin-10-yl)propyl](3, 4-dichlorobenzyl)dimethylammonium derivative (K(i) 0.12 microM), was approximately 2 orders of magnitude more inhibitory than the parent chlorpromazine. Several of these quaternary phenothiazines completely inhibited T. brucei parasite growth in vitro at <1 microM. Antiparasite activity was not solely determined by inhibition strength against trypanothione reductase, there being a strong contribution from hydrophobicity (for example, benzhydryl-quaternized chlorpromazime had ED(50) < 1 microM). Although active against Leishmania donovani, none of the analogues showed major improvement in this activity relative to chlorpromazine or other nonquaternized phenothiazines. The p-tert-butylbenzyl-quaternized analogue very strongly inhibited (ED(50) < 1 microM) growth of the amastigote stage of T. cruzi.

Refined high-field NMR solution structure of a binary-addressed pyrene/perfluoro-azide complementary DNA oligonucleotide system shows extensive distortion in the central nick region.

The structural analysis of the photoactivated binary system of complementary-addressing nucleic acid sequences (1:2:3) by high-resolution NMR spectroscopy and restrained molecular dynamics is reported. The binary system comprised a 12 base-pair target DNA sequence, pdGTATCAGTTTCT (1), and two hexanucleotides, (dAGAAACp-L-Az (2) and Pyr-pdTGATAC (3)), complementary to neighbouring sites in the target DNA. Oligonucleotide (2) is conjugated with a p-azidotetrafludrobenzyl group (Az) via a linker group (L), and the other oligonucleotide (3) is equipped with the photosensitizing pyrenyl-1-methylamino group (Pyr). We now extend the structural analysis of 1:2:3, which was previously based on qualitative 2D 1H-NMR data and thermodynamic analysis of complex formation from UV-visible thermal denaturation experiments. In the current work structural refinement was performed by separate molecular dynamics runs for six different starting structures based on 318 proton-proton distance-range constraints, evaluated from the 1H-NOESY spectrum (tau(mix) = 200 ms, 600 MHz) using complete relaxation matrix analysis (NMR/TRIAD/MARDIGRAS). Additional Watson-Crick hydrogen bond restraints were included in the calculations based on the detected signals from the exchangeable protons, using REFOPT(NY) methods. The final averaged structure obtained from the six refined co-ordinate sets showed a considerable degree of axis bend (62.5 degrees) with the bending point in the middle of the duplex in the region of the backbone nick between the two short oligonucleotides. The complex behaves dynamically as the equivalent of two short B-DNA-like duplexes displaying a hinge-like flexing at their junction. In all final structures the Pyr function location was very restricted, the aromatic group lying in the duplex minor groove near residues 4T, 5C and 2T. In contrast, the location of the perfluoroazido group was different in all the final structures, indicating the high flexibility of this group in the duplex. The only feature common to all six final azido group orientations was the outside location on the side of the major groove. The distance between the Pyr and Az groups varied from 11 A to 24 A for the six final structures (17 A, final average structure). The dynamics of duplex denaturation for 1:2:3 was probed by monitoring the temperature-induced NMR line broadening of the imino protons in a 1D variable temperature NMR experiment. The melting of 1:2:3 starts both from the ends and from the middle part of the duplex at the backbone break between the two short oligonucleotides reflecting the destabilisation of the pyrene-arylazido nick region in the duplex.

Structure of photoreactive binary system of oligonucleotide conjugates assembled on the target nucleotide sequence.

Recently we have developed an approach to superspecific photomodification of nucleic acids by binary systems of oligonucleotides conjugated to precursor groups capable of assembling into photoactivatable structure upon simultaneous binding of the conjugates to the target. We have investigated the solution structure of a model binary system 1:2:3, where 1 is the target 12-mer 5'-pdGTATCAGTTTCT, 2 is the photoreactive conjugate 5'-dAGAAACp-NH(CH2)2NH-Az and 3 is the sensitizing conjugate 5'-Pyr-pdTGATAC (Az is p-azidotetrafluorobenzoyl group and Pyr is the pyrenyl-1-methylamino group). The photoreaction within this complex results in crosslinking of reagent 2 with N7-position of the G7 residue of the target thus indicating that the photoreactive Az residue is located in the major groove near the G7 residue. The center-to-center distances between the Pyr and Az moieties in complex 1:2:3 independently determined by the Pyr-group fluorescence quenching and the Az-group sensitized photodecomposition were 11.2 and 12.6 A, respectively.

Structural requirements of double and single stranded DNA substrates and inhibitors, including a photoaffinity label, of Fpg protein from Escherichia coli.

Fpg protein (formamidopyrimidine or 8-oxoguanine DNA glycosylase) from E. coli catalyzes excision of several damaged purine bases, including 8-oxoguanine and 2,6-diamino-4-hydroxy-5-N-methylformamidopyrimidine from DNA. In this study the interaction of E. coli Fpg with various specific and nonspecific oligodeoxynucleotides was analyzed. Fpg was shown to remove 8-oxoguanine efficiently, not only from double-stranded, but also from single-stranded oligodeoxynucleotides. The Michaelis constants (KM) of a range of single-stranded oligodeoxynucleotides (0.55-1.3 microM) were shown to be 12-170 times higher that those for corresponding double-stranded oligodeoxynucleotides (KM = 6-60 nM). Depending on the position of the 8-oxoguanine within the oligodeoxynucleotides, relative initial rates of conversion of single-stranded substrates were found to be lower than, comparable to, or higher than those for double-stranded oligodeoxynucleotides. The enzyme can interact effectively not only with specific, but also with nonspecific single-stranded and double-stranded oligodeoxynucleotides, which are competitive inhibitors of the enzyme towards substrate. Fpg became irreversibly labeled after UV-irradiation in the presence of photoreactive analogs of single-stranded and double-stranded oligodeoxynucleotides. Specific and nonspecific single-stranded and double-stranded oligodeoxynucleotides essentially completely prevented the covalent binding of Fpg by the photoreactive analog. All these data argue for similar interactions occurring in the DNA binding cleft of the enzyme with both specific and nonspecific oligodeoxynucleotides. The relative affinities of Fpg for specific and nonspecific oligodeoxynucleotides differ by no more than 2 orders of magnitude. Addition of the second complementary chain increases the affinity of the first single-stranded chain by a factor of approximately 10. It is concluded that Michaelis complex formation of Fpg with DNA containing 8-oxoG cannot alone provide the major part of the enzyme specificity, which is found to lie in the kcat term for catalysis; the reaction rate being increased by 6-7 orders of magnitude by the transition from nonspecific to specific oligodeoxynucleotides.

Synthesis and enzymology of modified N-benzyloxycarbonyl-L-cysteinylglycyl-3,3-dimethylaminopropylamide++ + disulphides as alternative substrates for trypanothione reductase from Trypanosoma cruzi: Part 3.

Kinetic data for alternative substrates of recombinant trypanothione reductase from Trypanosoma cruzi were measured for a series of N-substituted-L-cysteinylglycyl-3-dimethylaminopropylamides, in which the cysteine N-substituent was either a variant of the benzyloxycarbonyl group or was L-phenylalanine or L-tryptophan. Replacing the benzylic ether oxygen atom by CH2 or NH had relatively minor effects on kcat, but raised the value of K(m) 4.5- and 10-fold, respectively. Similarly, relative to the carbobenzoxy group, an N-L-phenylalanyl or N-L-tryptophanyl replacement on the cysteine hardly altered kcat, but increased K(m) values by 16.6 and 7.4 fold, respectively. These observations were consistent with the K(m) values referring primarily to binding for this series of nonspecific substrates.

Rational inhibitor design, synthesis and NMR spectroscopic study by transferred nuclear overhauser spectroscopy of novel inhibitors of cinnamyl alcohol dehydrogenase, a critical enzyme in lignification.

Cinnamyl alcohol dehydrogenase is one of the enzymes controlling the first two committed steps of lignification. Using a 3-dimensional similarity model of this enzyme, a series of novel phosphonates (1-5) was designed as potential inhibitors. Phosphonates 1-5 were synthesized in good yield by reaction of the corresponding cinnamaldehydes with tetraethylmethylene diphosphonate. Monophosphonic acids 6 and 7 were obtained by basic hydrolysis of the corresponding phosphonates while phosphonamidate 8 was synthesized by reacting benzylamine with the iminium salt intermediate of the monophosphonic acid. Using recombinant cinnamyl alcohol dehydrogenase (CAD, EC 1.1.1.195) the inhibitory activity of these compounds was evaluated and compared with that of the carbonyl analogues. Inhibition kinetic studies showed compounds 2 and 3 to be mixed type linear inhibitors while compound 4 was uncompetitive. 1H NMR studies of inhibitor 2, for which Ki and Ki' were 20 and 86 microM, respectively, in the presence of CAD based on selective line-broadening showed an increased interaction of the 3-OMe group of the aromatic ring of the inhibitor with the active site of the CAD. A transferred nuclear overhauser effect spectroscopy (TRNOESY) experiment for inhibitor 2 with CAD was used to determine the conformation of this compound bound to CAD. These results were found to be consistent with the 3-dimensional structural model of the enzyme.