De novo nocturia: A red flag for coronary heart disease patients.

OBJECTIVES: Nocturia is frequent among older patients and has been linked to cardiovascular diseases. The aim of this study was to assess the time relationship between the onset of nocturia and coronary heart disease (CHD). Specifically, this study investigated whether nocturia can be identified as a red flag de novo symptom in patients with CHD. METHODS: This cross-sectional study consisted of patients with CHD-related cardiac complaints who were prospectively recruited from November 2019 till March 2020 at the cardiac catheterization laboratory of the Ghent University Hospital. An analysis was performed to determine the time relationship between nocturia and CHD and to describe the nocturia characteristics. RESULTS: Forty-five patients with nocturia and established CHD were included. Of these patients, 74% (31/42) developed nocturia before their cardiac symptoms occurred, with a median time gap of 57 months (IQR 19-101). Furthermore, 64% (29/45) of them had clinically significant nocturia (≥2 nocturnal voids) and there was a significant correlation between age at which nocturia and cardiac symptoms occurred (r=0.89, p<0.001). CONCLUSION: This is the first study that analysed the time relationship between onset of nocturia and onset of cardiac complaints in patients with CHD. In most of the patients, nocturia had started before they were diagnosed with CHD, meaning that nocturia might precede the development of cardiac symptoms, such as angina and shortness of breath. Keeping this in mind, de novo nocturia may or even should be considered as a red flag for CHD. LEVEL OF EVIDENCE: 4: (cross sectional study with prospectively recruitement) Source: https://www.ciap.health.nsw.gov.au/training/ebp-learning-modules/module1/grading-levels-of-evidence.html.

Community-acquired pneumonia (CAP) hospitalizations and deaths: is there a role for quality improvement through inter-hospital comparisons?

OBJECTIVE: To assess between-hospital variations in standardized in-hospital mortality ratios of community-acquired pneumonia (CAP), and identify possible leads for quality improvement. DESIGN: We used an administrative database to estimate standardized in-hospital mortality ratios for 111 Belgian hospitals, by carrying out a set of hierarchical logistic regression models, intended to disentangle therapeutic attitudes and biases. To facilitate the detection of false-negative/positive results, we added an inconclusive zone to the funnel plots, derived from the results of the study. Data quality was validated by comparison with (i) alternative data from the largest Belgian Sickness Fund, (ii) published German hospital data and (iii) the results of an on-site audit. SETTING: All Belgian hospital discharge records from 2004 to 2007. STUDY PARTICIPANTS: A total of 111 776 adult patients were admitted for CAP. MAIN OUTCOME MEASURE: Risk-adjusted standardized in-hospital mortality ratios. RESULTS: Out of the 111 hospitals, we identified five and six outlying hospitals, with standardized mortality ratios of CAP consistently on the extremes of the distribution, as providing possibly better or worse care, respectively, and 18 other hospitals as having possible quality weaknesses/strengths. At the individuals' level of the analysis, adjusted odds ratios showed the paramount importance of old age, comorbidity and mechanical ventilation. The data compared well with the different validation sources. CONCLUSIONS: Despite the limitations inherent to administrative data, it seemed possible to establish inter-hospital differences in standardized in-hospital mortality ratios of CAP and to identify leads for quality improvement. Monitoring is needed to assess progress in quality.

Neuroprotective properties of anti-apoptotic BCL-2 proteins in 5xFAD mouse model of Alzheimer's disease.

Alzheimer's disease (AD) is the most common cause of dementia. An early feature of the AD pathology is the dysregulation of intracellular Ca2+ signaling in neurons. In particular, increased Ca2+ release from endoplasmic reticulum-located Ca2+ channels, including inositol-1,4,5-trisphosphate type 1 receptors (IP3R1) and ryanodine receptors type 2 (RyR2), have been extensively reported. Known for its anti-apoptotic properties, Bcl-2 also has the ability to bind to and inhibit the Ca2+-flux properties of IP3Rs and RyRs. In this study, the hypothesis that the expression of Bcl-2 proteins can normalize dysregulated Ca2+ signaling in a mouse model of AD (5xFAD) and thereby prevent or slow the progression of AD was examined. Therefore, stereotactic injections of adeno-associated viral vectors expressing Bcl-2 proteins were performed in the CA1 region of the 5xFAD mouse hippocampus. In order to assess the importance of the association with IP3R1, the Bcl-2K17D mutant was also included in these experiments. This K17D mutation has been previously shown to decrease the association of Bcl-2 with IP3R1, thereby impairing its ability to inhibit IP3R1 while not affecting Bcl-2's ability to inhibit RyRs. Here, we demonstrate that Bcl-2 protein expression leads to synaptoprotective and amyloid-protective effects in the 5xFAD animal model. Several of these neuroprotective features are also observed by Bcl-2K17D protein expression, suggesting that these effects are not associated with Bcl-2-mediated inhibition of IP3R1. Potential mechanisms for this Bcl-2 synaptoprotective action may be related to its ability to inhibit RyR2 activity as Bcl-2 and Bcl-2K17D are equally potent in inhibiting RyR2-mediated Ca2+ fluxes. This work indicates that Bcl-2-based strategies hold neuroprotective potential in AD models, though the underlying mechanisms requires further investigation.

The health and economic burden of chickenpox and herpes zoster in Belgium.

Varicella-zoster virus causes chickenpox (CP) and after reactivation herpes zoster (HZ). Vaccines are available against both diseases warranting an assessment of the pre-vaccination burden of disease. We collected data from relevant Belgian databases and performed five surveys of CP and HZ patients. The rates at which a general practitioner is visited at least once for CP and HZ are 346 and 378/100 000 person-years, respectively. The average CP and HZ hospitalization rates are 5·3 and 14·2/100 000 person-years respectively. The direct medical cost for HZ is about twice as large as the direct medical cost for CP. The quality-adjusted life years lost for ambulatory CP patients consulting a physician is more than double that of those not consulting a physician (0·010 vs. 0·004). In conclusion, both diseases cause a substantial burden in Belgium.

Antitumoral and endocrine effects of (+)-vorozole in rats bearing dimethylbenzanthracene-induced mammary tumors.

The antitumoral activity of vorozole, a potent and specific nonsteroidal aromatase inhibitor, against 7,12-dimethylbenz(a)anthracene-induced estrogen-dependent mammary adenocarcinoma was evaluated in 257 Sprague-Dawley rats. Twice daily p.o. administration of 1 and 5 mg/kg of the racemate R 76713 for 42 days induced almost complete regression of tumors, inhibited the appearance of new tumors, and reduced multiplicity of the remaining tumors. Antitumoral effects observed after ovariectomy or treatment with 5 mg/kg twice a day were not significantly different. R 76713, the racemate, (+)-vorozole (both at 2.5 mg/kg twice a day), and ovariectomy all similarly reduced tumor growth at 42 days by 90% or more, lowered the number of existing tumors, and prevented the appearance of new tumors. The less active levo-enantiomer (-)-vorozole at the same dose did not alter tumor growth. Vorozole reduced serum estradiol to the levels measured in ovariectomized animals. Serum progesterone levels were lowered, but to a much lesser extent than after ovariectomy, while serum luteinizing hormone and follicle-stimulating hormone concentrations increased, but also much less than after ovariectomy. On the other hand, the androgen levels, which remained undetectable or decreased after ovariectomy, markedly rose after vorozole treatment. These endocrine changes, observed in intact female rats, were not detected in ovariectomized animals demonstrating the ovarian origin of the endocrine changes induced by vorozole.

A spectroscopic study of the chromatic properties of GafChromicEBT3 films.

PURPOSE: This work provides an interpretation of the chromatic properties of GafChromicEBT3 films based on the chemical nature of the polydiacetylene (PDA) molecules formed upon interaction with ionizing radiation. The EBT3 films become optically less transparent with increasing radiation dose as a result of the radiation-induced polymerization of diacetylene monomers. In contrast to empirical quantification of the chromatic properties, less attention has been given to the underlying molecular mechanism that induces the strong decrease in transparency. METHODS: Unlaminated GafChromicEBT3 films were irradiated with a 6 MV photon beam to dose levels up to 20 Gy. The optical absorption properties of the films were investigated using visible (vis) spectroscopy. The presence of PDA molecules in the active layer of the EBT3 films was investigated using Raman spectroscopy, which probes the vibrational modes of the molecules in the layer. The vibrational modes assigned to PDA's were used in a theoretical vis-absorption model to fit our experimental vis-absorption spectra. From the fit parameters, one can assess the relative contribution of different PDA conformations and the length distribution of PDA's in the film. RESULTS: Vis-spectroscopy shows that the optical density increases with dose in the full region of the visible spectrum. The Raman spectrum is dominated by two vibrational modes, most notably by the ν(C≡C) and the ν(C=C) stretching modes of the PDA backbone. By fitting the vis-absorption model to experimental spectra, it is found that the active layer contains two distinct PDA conformations with different absorption properties and reaction kinetics. Furthermore, the mean PDA conjugation length is found to be 2-3 orders of magnitude smaller than the crystals PDA's are embedded in. CONCLUSIONS: Vis- and Raman spectroscopy provided more insight into the molecular nature of the radiochromic properties of EBT3 films through the identification of the excited states of PDA and the presence of two PDA conformations. The improved knowledge on the molecular composition of EBT3's active layer provides a framework for future fundamental modeling of the dose-response.

Purification and characterisation of the phosphoglycerate kinase isoenzymes of Trypanosoma brucei expressed in Escherichia coli.

The Trypanosoma brucei phosphoglycerate kinase (PGK) glycosomal and cytosolic isoenzymes have been overexpressed in Escherichia coli and purified to near-homogeneity. Both enzymes were similar to the corresponding natural proteins with respect to their physicochemical and kinetic properties. In addition, a mutant of the glycosomal PGK lacking the 20 amino acid long C-terminal extension was overexpressed and purified. Various properties of this truncated glycosomal PGK were examined and it was found that in some aspects the protein behaved quite differently when compared with its natural counterpart. This was notably the case for the apparent Km for 3-phosphoglyceric acid, its sensitivity to inhibitors and its response to salts and guanidine HCl. However, its Vmax was found to be similar to that of the natural glycosomal PGK. These results suggest that the changes in the C-terminus caused a conformational change effecting the 3-phosphoglyceric acid binding site located at the N-terminal domain of the protein.

Inhibition of the glycolytic enzymes in the trypanosome: an approach in the development of new leads in the therapy of parasitic diseases.

Glycolysis in the trypanosome represents an important target for the development of new therapeutic agents due to the fact that this metabolism is essential for the parasite, glucose being its sole source of energy. In addition, different features of this metabolism and those associated with glycolytic enzymes offer opportunities for the development of efficient and selective compounds. Examples are given in this work of inhibitors directed to the enzymes aldolase and glyceraldehyde-phosphate-dehydrogenase and also of molecules acting specifically on the clusters of basic amino-acids present at the surfaces of the glycolytic enzymes in the parasite.

Structure of the complex between trypanosomal triosephosphate isomerase and N-hydroxy-4-phosphono-butanamide: binding at the active site despite an "open" flexible loop conformation.

The structure of triosephosphate isomerase from Trypanosoma brucei complexed with the competitive inhibitor N-hydroxy-4-phosphono-butanamide was determined by X-ray crystallography to a resolution of 2.84 A. Full occupancy binding of the inhibitor is observed only at one of the active sites of the homodimeric enzyme where the flexible loop is locked in a completely open conformation by crystal contacts. There is evidence that the inhibitor also binds to the second active site of the enzyme, but with low occupancy. The hydroxamyl group of the inhibitor forms hydrogen bonds to the side chains of Asn 11, Lys 13, and His 95, whereas each of its three methylene units is involved in nonpolar interactions with the side chain of the flexible loop residue Ile 172. Interactions between the hydroxamyl and the catalytic base Glu 167 are absent. The binding of this phosphonate inhibitor exhibits three unusual features: (1) the flexible loop is open, in contrast with the binding mode observed in eight other complexes between triosephosphate isomerase and various phosphate and phosphonate compounds; (2) compared with these complexes the present structure reveals a 1.5-A shift of the anion-binding site; (3) this is the first phosphonate inhibitor that is not forced by the enzyme into an eclipsed conformation about the P-CH2 bond. The results are discussed with respect to an ongoing drug design project aimed at the selective inhibition of glycolytic enzymes of T. brucei.

An interface point-mutation variant of triosephosphate isomerase is compactly folded and monomeric at low protein concentrations.

Wild-type trypanosomal triosephosphate isomerase (wtTIM) is a very tight dimer. The interface residue His-47 of wtTIM has been mutated into an asparagine. Ultracentrifugation data show that this variant (H47N) only dimerises at protein concentrations above 3 mg/ml. H47N has been characterised at a protein concentration where it is predominantly a monomer. Circular dichroism measurements in the near-UV and far-UV show that this monomer is a compactly folded protein with secondary structure similar as in wtTIM. The thermal stability of the monomeric H47N is decreased compared to wtTIM: temperature gradient gel electrophoresis (TGGE) measurements give Tm-values of 41 degrees C for wtTIM, whereas the Tm-value for the monomeric form of H47N is approximately 7 degrees C lower.

Levamisole plus 5-fluorouracil inhibits the growth of human colorectal xenografts in nude mice.

Fragments of human colorectal adenocarcinomas were inserted under the renal capsule of nude mice. The growth of these tumour grafts was significantly inhibited by the combination of 5-fluorouracil (5-FU) and levamisole. An alternating regimen of levamisole 2.5 mg/kg and 5-FU 20 mg/kg decreased the size of tumour implants by 33-59% and/or increased the number of macroscopically disappeared fragments in the combined group compared with ineffective monotherapy with saline, levamisole or 5-FU. This model could be valuable for investigating the mechanism of action of levamisole and to evaluate the effects of this adjuvant therapy in other oncological settings.

Chemical modification of fructose bisphosphate aldolase from Trypanosoma brucei compared to aldolase from rabbit muscle and Staphylococcus aureus.

Chemical modifications of Class I aldolases from Trypanosoma brucei, rabbit muscle and Staphylococcus aureus with carboxypeptidase A, glyceraldehyde 3-phosphate and cysteine-specific reagents revealed the following differences between the three homologous enzymes. Aldolase from S. aureus was not affected by any of these reagents. Carboxypeptidase-A treatment of rabbit-muscle and T. brucei aldolase inhibited the activity of both enzymes towards fructose-1,6-bisphosphate (Fru(1,6)P2), while the activity towards fructose-1-phosphate (Fru-1-P) was affected only in the case of the trypanosomal enzyme. Moreover carboxypeptidase-A treatment reduced the turnover numbers of these two aldolases for both Fru(1,6)P2 and Fru-1-P to a similar level. Glyceraldehyde 3-phosphate, in the absence of dihydroxyacetone phosphate, also inactivated aldolases from rabbit muscle and T. brucei with second order rate constants of 1054 and 254 min-1 M-1, respectively. Using 5,5'-dithiobis-(2-nitrobenzoic acid) with rabbit-muscle aldolase, a total of 4 thiol groups could be titrated per subunit, resulting in a total inactivation. The presence of substrate completely protected the enzyme from inactivation. Methyl methanethiosulfonate also reacted with four cysteine residues, but this led to very little inactivation. This indicates that the inactivation by modification with DTNB is due to conformational changes in the enzyme. In T. brucei aldolase only one thiol group could be titrated with methyl methanesulfonate and there was no loss of activity. With 5,5'-dithiobis-(2-nitrobenzoic acid) five cysteines were titrated with an immediate and complete loss of activity.(ABSTRACT TRUNCATED AT 250 WORDS)

Some kinetic properties of pyruvate kinase from Trypanosoma brucei.

We have studied the kinetics of the allosteric interactions of pyruvate kinase from Trypanosoma brucei. The kinetics for phosphoenolpyruvate depended strongly on the nature of the bivalent metal ions. Pyruvate kinase activated by Mg2+ had the highest catalytic activity, but also the highest S0.5 for phosphoenolpyruvate, while the opposite was true for pyruvate kinase activated by Mn2+. The reaction rates of Mg(2+)-pyruvate kinase and Mn(2+)-pyruvate kinase were clearly allosteric with respect to phosphoenolpyruvate, while the kinetics with Co(2+)-pyruvate kinase were hyperbolic. However, Co(2+)-pyruvate kinase was still sensitive to heterotropic activation. Trypanosomal pyruvate kinase is unique in that the best activator was fructose 2,6-bisphosphate. Ribulose 1,5-bisphosphate and 5-phosphorylribose 1-pyrophosphate were also strong heterotropic activators, which were much more effective than fructose 1,6-bisphosphate and glucose 1,6-bisphosphate. In the presence of the heterotropic activators, the sigmoidal kinetics with respect to phosphoenolpyruvate and the bivalent metal ions were modified as were the concentrations of phosphoenolpyruvate and the bivalent metal ions needed to attain the maximal activity. Maximal activities were not significantly changed with Mg2+ and Mn2+ as the activating metal ions. Moreover, with Co2+ and fructose 2,6-bisphosphate or ribulose 1,5-bisphosphate or 5-phosphorylribose 1-pyrophosphate, the maximal activity was significantly reduced. Ribulose 1,5-bisphosphate and 5-phosphorylribose 1-pyrophosphate resembled fructose 2,6-bisphosphate rather than fructose 1,6-bisphosphate and glucose 1,6-bisphosphate in their action in that the K0.5 values for the former 3 compounds increased when Mg2+ was replaced by Co2+, while the K0.5 for fructose 1,6-bisphosphate and glucose 1,6-bisphosphate increased.(ABSTRACT TRUNCATED AT 250 WORDS)

Kinetic properties of fructose bisphosphate aldolase from Trypanosoma brucei compared to aldolase from rabbit muscle and Staphylococcus aureus.

The kinetic properties of aldolase from Trypanosoma brucei were studied in comparison with aldolase from rabbit muscle and Staphylococcus aureus. The 3 enzymes displayed a similar broad pH optimum for the cleavage of fructose 1,6-bisphosphate (Fru(1,6)P2) and a similar narrow pH optimum for the cleavage of fructose 1-phosphate (Fru-1-P). However, small alterations in the maximal cleavage rate at more extreme pH values yielded disparities between the pH curves. The reaction catalyzed by the aldolases from T. brucei and S. aureus proceeded via an ordered sequence, as described for the rabbit-muscle enzyme. We determined for the 3 enzymes the kinetic parameters for both the cleavage and the formation of Fru(1,6)P2 and for the cleavage of Fru-1-P. The trypanosomal enzyme differed in its higher ratio of the maximal rate of Fru(1,6)P2-cleavage vs. the maximal rate of Fru(1,6)P2-formation, its higher affinity towards dihydroxyacetone phosphate, and its higher turnover number for the cleavage of Fru-1-P. At ionic strengths above 0.1 M the kinetic parameters of the trypanosomal enzyme followed the limited form of the Debye-Hückel equation. At ionic strengths below 0.1 M the enzyme revealed a characteristic deviation: the apparent Km for Fru(1,6)P2 increased with decreasing salt concentration. The trypanosomal aldolase was competitively inhibited by adenine nucleotides and phosphates. This inhibition occurred in the same concentration range as observed for the rabbit-muscle enzyme, while the bacterial enzyme was less affected.

Characterization of pyruvate kinase of Trypanosoma brucei and its role in the regulation of carbohydrate metabolism.

Pyruvate kinase from Trypanosoma brucei is a labile enzyme, losing its activity within several hours. In mixtures containing 50 mM triethanolamine buffer, pH 7.2, 25% glycerol and 0.5 mM inorganic phosphate the enzyme remained active and could be purified to homogeneity with a specific activity of 417 units mg-1 and a yield of 65%. The enzyme has an activation energy of 31.9 kJ mol-1. Magnesium and potassium ions are essential for activity. Cobalt or manganese ions replace Mg2+ but this leads to a decrease in maximal velocity. Potassium ions can be substituted by ammonium ions, while sodium ions behave as a competitive inhibitor with respect to both K+ and NH4+. All metal ions studied displayed sigmoidal kinetics. The enzyme is activated, with decreasing efficiency by fructose 2-phosphorothioate 6-phosphate, fructose 2,6-bisphosphate, fructose 1,6-bisphosphate and glucose 1,6-bisphosphate. They all display hyperbolic kinetics. Glycerate 2,3-bisphosphate, glyceraldehyde 3-phosphate, CoASAc, oxalate, AMP, ADP, and ATP inhibit the enzyme. At substrate saturation PK was activated by Pi up to a concentration of 0.8 mM. At higher Pi concentrations the enzyme is inhibited. The enzyme is unaffected by most amino acids, only phenylalanine stimulates and tyrosine inhibits.

Pyruvate kinase of Leishmania mexicana mexicana. Cloning and analysis of the gene, overexpression in Escherichia coli and characterization of the enzyme.

Leishmania mexicana mexicana contains two tandemly arranged genes for pyruvate kinase (PYK). The 5' located gene codes for a polypeptide with a molecular mass of 54,370. The calculated net charge and isoelectric point of the polypeptide are -6 and 6.5, respectively. Its amino-acid sequence is 73.7% identical to that of the Trypanosoma brucei PYK and 46.4-49.8% to the enzyme of mammalian cells. The second gene appears not to be functional, because its 5' and 3' extremities have undergone recombinations. L. m. mexicana PYK has been overexpressed in Escherichia coli, using a T7 expression system. Approximately 30% of the protein was detected in the soluble cell fraction. It has been highly purified by chromatography over DEAE-Sephacel and Affigel Blue. From a 1-1 culture 6 mg enzyme was obtained with a specific activity of 224 units mg-1. The protein has a subunit molecular mass of 59,000, as determined by SDS/PAGE, and an isoelectric point of 5.9. Some kinetic properties of the enzyme have been measured and compared with those reported for the T. brucei enzyme. The kinetics of both enzymes are very similar, the most important aspect being their activation by fructose 2,6-bisphosphate. Nevertheless, some differences were observed; the T. brucei enzyme is activated by the effector in a cooperative manner, whereas the activation of the L. m. mexicana enzyme is not cooperative.

Synthesis and activity of inhibitors highly specific for the glycolytic enzymes from Trypanosoma brucei.

Most glycosomal enzymes of Trypanosoma brucei carry a relatively high number of positive charges. In at least 3 of the enzymes some of the charges unique to these enzymes are concentrated in 2 distinct areas on the enzymes' surface, about 4 nm apart [4] and these positively charged structural elements have been suggested to be the site of interaction with the trypanocidal drug Suramin. We have synthesized a series of symmetrical long chain molecules with negative charges or strong dipoles at each end. Several of these compounds inhibited the glycosomal enzymes more strongly than Suramin. They also exhibited a specificity for the trypanosome enzymes, when compared with homologous enzymes from other organisms. By varying the chain length of the active compounds, a 4-nm distance between the molecules' extremes proved optimal for inhibition. Tetra-substituted compounds were better than di-substituted. Modifications introduced at the two ends indicated that a planar orientation, with an amide bond linking a phenyl ring to the chain, is preferred. Inhibition kinetics for some of the enzymes indicated the existence of multi-site interactions with the inhibitors.

Cloning and characterization of the NAD-linked glycerol-3-phosphate dehydrogenases of Trypanosoma brucei brucei and Leishmania mexicana mexicana and expression of the trypanosome enzyme in Escherichia coli.

A polyclonal antiserum raised against the purified glycosomal glycerol-3-phosphate dehydrogenase of Trypanosoma brucei brucei has been used to identify the corresponding cDNA clone in a T.b. brucei expression library. This cDNA was subsequently used to obtain genomic clones containing glycerol-3-phosphate dehydrogenase genes. Two tandemly arranged genes were detected in these clones. Characterization of one of the genes showed that it codes for a polypeptide of 353 amino acids, with a molecular mass of 37,651 Da and a calculated net charge of +8. Using the T.b. brucei gene as a probe, a corresponding glycerol-3-phosphate dehydrogenase gene was also identified in a genomic library of Leishmania mexicana mexicana. The L.m. mexicana gene codes for a polypeptide of 365 amino acids, with a molecular mass of 39,140 Da and a calculated net charge of +8. The amino-acid sequences of both polypeptides are 63% identical and carry a type-1 peroxisomal targeting signal (PTS1) SKM and -SKL at their respective C-termini. Moreover, the L.m. mexicana polypeptide also carries a short N-terminal extension reminiscent of a mitochondrial transit sequence. Subcellular localisation analysis showed that in L.m. mexicana the glycerol-3-phosphate dehydrogenase activity co-fractionated both with mitochondria and with glycosomes. This is not the case in T. brucei, where the enzyme is predominantly glycosomal. The two trypanosomatid sequences resemble their prokaryotic homologues (32-36%) more than their eukaryotic counterparts (25-31%) and carry typical prokaryotic signatures. The possible reason for this prokaryotic nature of a trypanosomatid glycerol-3-phosphate dehydrogenase is discussed.

Selective inhibition of trypanosomal glyceraldehyde-3-phosphate dehydrogenase by protein structure-based design: toward new drugs for the treatment of sleeping sickness.

Within the framework of a project aimed at rational design of drugs against diseases caused by trypanosomes and related hemoflagellate parasites, selective inhibitors of trypanosomal glycolysis were designed, synthesized, and tested. The design was based upon the crystallographically determined structures of the NAD:glyceraldehyde-3-phosphate dehydrogenase complexes of humans and Trypanosoma brucei, the causative agent of sleeping sickness. After one design cycle, using the adenosine part of the NAD cofactor as a lead, the following encouraging results were obtained: (1) a 2-methyl substitution, targeted at a small pocket near Val 36, improves inhibition of the parasite enzyme 12.5-fold; (2) an 8-(thien-2-yl) substitution, aimed at Leu 112 of the parasite enzyme, where the equivalent residue in the mammalian enzyme is Val 100, results in a 167-fold better inhibition of the trypanosomal enzyme, while the inhibition of the human enzyme is improved only 13-fold; (3) exploitation of a "selectivity cleft" created by a unique backbone conformation in the trypanosomal enzyme near the adenosine ribose yields a considerable improvement in selectivity: 2'-deoxy-2'-(3-methoxybenzamido)adenosine inhibits the human enzyme only marginally but enhances inhibition of the parasite enzyme 45-fold when compared with adenosine. The designed inhibitors are not only better inhibitors of T. brucei GAPDH but also of the enzyme from Leishmania mexicana.

Synthesis and structure-activity relationships of analogs of 2'-deoxy-2'-(3-methoxybenzamido)adenosine, a selective inhibitor of trypanosomal glycosomal glyceraldehyde-3-phosphate dehydrogenase.

In continuation of a project aimed at the structure-based design of drugs against sleeping sickness, analogs of 2'-deoxy-2'-(3-methoxybenzamido)adenosine (1) were synthesized and tested to establish structure-activity relationships for inhibiting glycosomal glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Compound 1 was recently designed using the NAD:GAPDH complexes of the human enzyme and that of Trypanosoma brucei, the causative agent of sleeping sickness. In an effort to exploit an extra hydrophobic domain due to Val 207 of the parasite enzyme, several new 2'-amido-2'-deoxyadenosines were synthesized. Some of them displayed an interesting improvement in inhibitory activity compared to 1. Carbocyclic or acyclic analogs showed marked loss of activity, illustrating the importance of the typical (C-2'-endo) puckering of the ribose moiety. We also describe the synthesis of a pair of compounds that combine the beneficial effects of a 2- and 8-substituted adenine moiety on potency with the beneficial effect of a 2'-amido moiety on selectivity. Unfortunately, in both cases, IC50 values demonstrate the incompatibility of these combined modifications. Finally, introduction of a hydrophobic 5'-amido group on 5'-deoxyadenosine enhances the inhibition of the protozoan enzyme significantly, although the gain in selectivity is mediocre.