robenidine and bis((3-(aminoiminomethyl)phenyl)methylene)carbonimidic-dihydrazide-trihydrochloride

Trypanosomatids depend on spermidine for growth and survival. Consequently, enzymes involved in spermidine synthesis and utilization, i.e. arginase, ornithine decarboxylase (ODC), S-adenosylmethionine decarboxylase (AdoMetDC), spermidine synthase, trypanothione synthetase (TryS), and trypanothione reductase (TryR), are promising targets for drug development. The ODC inhibitor alpha-difluoromethylornithine (DFMO) is about to become a first-line drug against human late-stage gambiense sleeping sickness. Another ODC inhibitor, 3-aminooxy-1-aminopropane (APA), is considerably more effective than DFMO against Leishmania promastigotes and amastigotes multiplying in macrophages. AdoMetDC inhibitors can cure animals infected with isolates from patients with rhodesiense sleeping sickness and leishmaniasis, but have not been tested on humans. The antiparasitic effects of inhibitors of polyamine and trypanothione formation, reviewed here, emphasize the relevance of these enzymes as drug targets. By taking advantage of the differences in enzyme structure between parasite and host, it should be possible to design new drugs that can selectively kill the parasites.

Topics: Adenosine; Adenosylmethionine Decarboxylase; Animals; Biogenic Polyamines; Chagas Disease; Eflornithine; Enzyme Inhibitors; Glutathione; Humans; Leishmania; Leishmaniasis; Ornithine Decarboxylase Inhibitors; Robenidine; Spermidine; Spermidine Synthase; Trypanocidal Agents; Trypanosoma brucei gambiense; Trypanosoma brucei rhodesiense; Trypanosoma cruzi; Trypanosomiasis, African

The energetics for binding of a diphenyl diamidine antitrypanosomal agent CGP 40215A to DNA have been studied by spectroscopy, isothermal titration calorimetry, and surface plasmon resonance biosensor methods. Both amidines are positively charged under experimental conditions, but the linking group for the two phenyl amidines has a pK(a) of 6.3 that is susceptible to a protonation process. Spectroscopic studies indicate an increase of 2.7 pK(a) units in the linking group when the compound binds to an A/T minor-groove site. Calorimetric titrations in different buffers and pH conditions support the proton-linkage process and are in a good agreement with spectroscopic titrations. The two methods established a proton-uptake profile as a function of pH. The exothermic enthalpy of complex formation varies with different pH conditions. The observed binding enthalpy increases as a function of temperature indicating a negative heat capacity change that is typical for DNA minor-groove binders. Solvent accessible surface area calculations suggest that surface burial accounts for about one-half of the observed intrinsic negative heat capacity change. Biosensor and calorimetric experiments indicate that the binding affinities vary with pH values and salt concentrations due to protonation and electrostatic interactions. The surface plasmon resonance binding studies indicate that the charge density per phosphate in DNA hairpins is smaller than that in polymers. Energetic contributions from different factors were also estimated for the ligand/DNA complex.

Topics: Amidines; Buffers; DNA; Hydrogen-Ion Concentration; Models, Molecular; Nucleic Acid Conformation; Protons; Robenidine; Surface Plasmon Resonance; Temperature

S-Adenosylmethionine decarboxylase (AdoMetDC) is a pyruvoyl-dependent enzyme that catalyzes an essential step in polyamine biosynthesis. The polyamines are required for cell growth, and the biosynthetic enzymes are targets for antiproliferative drugs. The function of AdoMetDC is regulated by the polyamine-precursor putrescine in a species-specific manner. AdoMetDC from the protozoal parasite Trypanosoma cruzi requires putrescine for maximal enzyme activity, but not for processing to generate the pyruvoyl cofactor. The putrescine-binding site is distant from the active site, suggesting a mechanism of allosteric regulation. To probe the structural basis by which putrescine stimulates T. cruzi AdoMetDC we generated mutations in both the putrescine-binding site and the enzyme active site. The catalytic efficiency of the mutant enzymes, and the binding of the diamidine inhibitors, CGP 48664A and CGP 40215, were analyzed. Putrescine stimulates the k(cat)/K(m) for wild-type T. cruzi AdoMetDC by 27-fold, and it stimulates the binding of both inhibitors (IC(50)s decrease 10-20-fold with putrescine). Unexpectedly CGP 48664A activated the T. cruzi enzyme at low concentrations (0.1-10 microM), while at higher concentrations (>100 microM), or in the presence of putrescine, inhibition was observed. Analysis of the mutant data suggests that this inhibitor binds both the putrescine-binding site and the active site, providing evidence that the putrescine-binding site of the T. cruzi enzyme has broad ligand specificity. Mutagenesis of the active site identified residues that are important for putrescine stimulation of activity (F7 and T245), while none of the active site mutations altered the apparent putrescine-binding constant. Mutations of residues in the putrescine-binding site that resulted in reduced (S111R) and enhanced (F285H) catalytic efficiency were both identified. These data provide evidence for coupling between residues in the putrescine-binding site and the active site, consistent with a mechanism of allosteric regulation.

Topics: Adenosylmethionine Decarboxylase; Allosteric Regulation; Amidines; Amino Acid Sequence; Animals; Humans; Indans; Molecular Sequence Data; Mutagenesis, Site-Directed; Putrescine; Robenidine; Sequence Alignment; Trypanosoma cruzi

The intraerythrocytic development of Plasmodium falciparum correlates with increasing levels of the polyamines putrescine, spermidine, and spermine in the infected red blood cells; and compartmental analyses revealed that the majority is associated with the parasite. Since depletion of cellular polyamines is a promising strategy for inhibition of parasite proliferation, new inhibitors of polyamine biosynthesis were tested for their antimalarial activities. The ornithine decarboxylase (ODC) inhibitor 3-aminooxy-1-aminopropane (APA) and its derivatives CGP 52622A and CGP 54169A as well as the S-adenosylmethionine decarboxlyase (AdoMetDC) inhibitors CGP 40215A and CGP 48664A potently affected the bifunctional P. falciparum ODC-AdoMetDC, with K(i) values in the low nanomolar and low micromolar ranges, respectively. Furthermore, the agents were examined for their in vitro plasmodicidal activities in 48-h incubation assays. APA, CGP 52622A, CGP 54169A, and CGP 40215A were the most effective, with 50% inhibitory concentrations below 3 microM. While the effects of the ODC inhibitors were completely abolished by the addition of putrescine, growth inhibition by the AdoMetDC inhibitor CGP 40215A could not be antagonized by putrescine or spermidine. Moreover, CGP 40215A did not affect the cellular polyamine levels, indicating a mechanism of action against P. falciparum independent of polyamine synthesis. In contrast, the ODC inhibitors led to decreased cellular putrescine and spermidine levels in P. falciparum, supporting the fact that they exert their antimalarial activities by inhibition of the bifunctional ODC-AdoMetDC.

Topics: Adenosylmethionine Decarboxylase; Amidines; Animals; Cells, Cultured; Erythrocytes; Humans; Indans; Ornithine Decarboxylase Inhibitors; Plasmodium falciparum; Polyamines; Propylamines; Robenidine

Many dicationic amidine compounds bind in the DNA minor groove and have excellent biological activity against a range of infectious diseases. Para-substituted aromatic diamidines such as furamidine, which is currently being tested against trypanosomiasis in humans, and berenil, which is used in animals, are typical examples of this class. Recently, a meta-substituted diamidine, CGP 40215A, has been found to have excellent antitrypanosomal activity. The compound has a linear, conjugated linking group that can be protonated under physiological conditions when the compound interacts with DNA. Structural and molecular dynamics analysis of the DNA complex indicated an unusual AT-specific complex that involved water-mediated H-bonds between one amidine of the compound and DNA bases at the floor of the minor groove. To investigate this unique system in more detail DNase I footprinting, surface plasmon resonance biosensor techniques, linear dichroism, circular dichroism, ultraviolet-visible spectroscopy, and additional molecular dynamics simulations have been conducted. Spectrophotometric titrations of CGP 40215A binding to poly(dAT)(2) have characteristics of DNA-binding-induced spectral changes as well as effects due to binding-induced protonation of the compound linker. Both footprinting and surface plasmon resonance results show that this compound has a high affinity for AT-rich sequences of DNA but very weak binding to GC sequences. The dissociation kinetics of the CGP 40215A-DNA complex are much slower than with similar diamidines such as berenil. The linear dichroism results support a minor-groove complex for the compound in AT DNA sequences. Molecular dynamics studies complement the structural analysis and provide a clear picture of the importance of water in mediating the dynamic interactions between the ligand and the DNA bases in the minor groove.

Topics: Base Sequence; Binding Sites; Computer Simulation; Kinetics; Macromolecular Substances; Models, Chemical; Models, Molecular; Molecular Conformation; Molecular Sequence Data; Nucleic Acid Conformation; Poly dA-dT; Polydeoxyribonucleotides; Robenidine; Structure-Activity Relationship; Water

A combination of biophysical techniques has been used to characterize the interaction of an antitrypanosomal agent, CGP 40215A, with DNA. The results from a broad array of methods (DNase I footprinting, surface plasmon resonance, X-ray crystallography, and molecular dynamics) indicate that this compound binds to the minor groove of AT DNA sequences. Despite its unusual linear shape that is not complementary to that of the DNA groove, a high binding affinity was observed in comparison with other similar but more curved diamidine compounds. The amidine groups at both ends of the ligand and the -NH groups on the linker are involved in extensive and dynamic H-bonds to the DNA bases. Complementary and consistent results were obtained from both the X-ray and molecular dynamics studies; both of these methods reveal direct and water-mediated H-bonds between the ligand and the DNA.

Topics: DNA; Models, Molecular; Molecular Conformation; Nucleic Acid Conformation; Pentamidine; Robenidine

CGP 40 215 is an inhibitor of S-adenosylmethionine decarboxylase, a key enzyme in trypanosomal polyamine biosynthesis. It is highly active against Trypanosoma brucei rhodesiense and T. b. gambiense in vitro and in the corresponding rodent models, and therefore was a promising candidate for further development as a new drug against human African trypanosomiasis. We conducted initial pharmacokinetic and efficacy studies in African green monkeys: based on two dose-finding studies, an infection-treatment and a pharmacokinetic study in eight monkeys infected with T. b. rhodesiense in the 1st stage of infection. PK analysis revealed curative drug levels in the serum but complete absence of the drug in the cerebrospinal fluid. No adverse effects of the drug were observed, although in rats CGP 40 215 had caused hypotension. The following PK parameters were calculated using a two-compartment model: t1/2=1.8 h, VSS/f=0.4 l/kg, CL/f=3.0 ml/min x kg and AUC=21 900 ng x h/ml. Six of the eight monkeys were cured, one animal relapsed on day 222 and one animal died of unknown reasons, but was aparasitaemic. The study confirmed the curative potential of CGP 40 215 for 1st stage T. b. rhodesiense infection. Unfortunately, it was also found that the compound did not pass the blood-brain barrier, a pre-requisite for cure of 2nd stage (CNS) infection. As the majority of sleeping sickness patients seeking treatment are in the 2nd stage of the disease, further development of the compound was stopped.

Topics: Adenosylmethionine Decarboxylase; Animals; Blood Pressure; Blood-Brain Barrier; Chlorocebus aethiops; Disease Models, Animal; Dose-Response Relationship, Drug; Drug Evaluation, Preclinical; Drug Monitoring; Female; Robenidine; Time Factors; Trypanocidal Agents; Trypanosoma brucei rhodesiense; Trypanosomiasis, African

Treatment with the drugs berenil, dibromopropamidine, pentamidine and CGP-4O-215 were found to alter levels of intracellular polyamines of Acanthamoeba polyphagia trophozoites in organisms. While the polyamine, spermidine, consistently remained higher in the control organisms incubated at 8, 16 and 32 h respectively, the level fluctuated significantly from below 5% to 40% in drug-treated organisms. A novel polyamine (polyamine F), yet to be identified, representing 80% of the total polyamine extracts and seen in control organisms after 48 h of incubation (stationary phase), was seen much earlier in the drug treated organisms. Pentamidine, dibromopropamidine and CGP-4O-215 induced the appearance of the novel polyamine in the trophozoites after only 8 h of incubation with the drugs while induction by berenil occurred after 32 h. It is suggested that diamidine drugs either induce encystment or alter the pathway of polyamine metabolism in Acanthamoeba. If inducing encystment is the main mode of reaction, then drugs that induce early encystment will be less effective in treatment than those that delay it.

Topics: Acanthamoeba; Animals; Antiprotozoal Agents; Benzamidines; Diminazene; Humans; Pentamidine; Polyamines; Pyridines; Robenidine

A series of novel aromatic derivatives based on the structure of methylglyoxal bis(guanylhydrazone) (MGBG) was examined for trypanocidal activities in human and veterinary trypanosomes of African origin. One agent, CGP 40215A, a bicyclic analog of MGBG which also resembles the diamidines diminazene (Berenil) and pentamidine, was curative of infections by 19 isolates of Trypanosoma brucei subspecies as well as a Trypanosoma congolense isolate. Several of these isolates were resistant to standard trypanocides. Curative doses were < or = 25 mg/kg of body weight/day for 3 days in these acute laboratory model infections. In addition, CGP 40215A also cured a model central nervous system infection in combination with the ornithine decarboxylase inhibitor DL-alpha-difluoromethylornithine (DFMO; Ornidyl, eflornithine). Curative combinations were 14 days of oral 2% DFMO (approximately 5 g/kg/day) plus 5, 10, or 25 mg/kg/day for 3 or 7 days given by intraperitoneal injection or with a miniosmotic pump. Combinations were most effective if CGP 40215A was given in the second half or at the end of the DFMO regimen. MGBG has modest activity as an inhibitor of trypanosome S-adenosylmethionine decarboxylase (50% inhibitory concentration [IC50]. 130 microM), while CGP 40215A was a more active inhibitor (IC50, 20 microM). Preincubation of trypanosomes with CGP 40215A for 1 h caused a reduction in spermidine content (36%) and an increase in putrescine content (20%), indicating that one possible mechanism of its action may be inhibition of polyamine biosynthesis.

Topics: Adenosylmethionine Decarboxylase; Animals; Female; Mice; Mice, Inbred ICR; Mitoguazone; Random Allocation; Robenidine; Structure-Activity Relationship; Trypanocidal Agents; Trypanosoma brucei brucei; Trypanosoma brucei rhodesiense; Trypanosoma congolense; Trypanosomiasis, African

CGP 40215A, specific S-adenosylmethionine decarboxylase (AdoMetDC) inhibitor was found to inhibit the growth of Leishmania donovani promastigotes (strain UR6) in a dose-dependent manner with an IC50 of 18 microM. The growth inhibition was reversed with 100 microM of spermidine and spermine. The growth inhibition in vitro by this inhibitor was accompanied by a significant decrease in AdoMetDC activity and spermidine levels. CGP 40215A was more potent than other AdoMetDC inhibitors, Berenil or methyl glyoxal (bis) guanyl hydrazone. The combination of CGP 40215A with other polyamine biosynthetic inhibitors like DL-alpha-difluoromethylornithine or the bis (benzyl) polyamine analogue (MDL 27695) shows an accentuated inhibitory effect on leishmanial growth.

Topics: Adenosylmethionine Decarboxylase; Animals; Antiprotozoal Agents; Enzyme Inhibitors; Leishmania donovani; Robenidine