ABSTRACT
There is an urgent need for the development of new drugs to treat Chagas' disease, which is caused by the protozoan parasite Trypanosoma cruzi. The enzyme dihydrofolate reductase (DHFR) has been a very successful drug target in a number of diseases and we decided to investigate it as a potential drug target for Chagas' disease. A homology model of the enzyme was used to search the Cambridge Structural Database using the program DOCK 3.5. Compounds were then tested against the enzyme and the whole parasite. Compounds were also screened against the related parasite, Trypanosoma brucei.
Subject(s)
Enzyme Inhibitors/chemistry , Enzyme Inhibitors/pharmacology , Tetrahydrofolate Dehydrogenase/metabolism , Trypanosoma cruzi/drug effects , Trypanosoma cruzi/enzymology , Animals , Cell Line , Chagas Disease/drug therapy , Chagas Disease/parasitology , Databases as Topic , Disease Models, Animal , Drug Design , Drug Evaluation, Preclinical , Enzyme Inhibitors/therapeutic use , Folic Acid Antagonists/chemistry , Folic Acid Antagonists/pharmacology , Folic Acid Antagonists/therapeutic use , Inhibitory Concentration 50 , Mice , Muscles/cytology , Rats , Trypanosoma brucei rhodesiense/drug effectsABSTRACT
The C-terminal domain of the Escherichia coli Ada protein (Ada-C) aids in the maintenance of genomic integrity by efficiently repairing pre-mutagenic O:(6)-alkylguanine lesions in DNA. Structural and thermodynamic studies were carried out to obtain a model of the DNA-binding process. Nuclear magnetic resonance (NMR) studies map the DNA-binding site to helix 5, and a loop region (residues 151-160) which form the recognition helix and the 'wing' of a helix-turn-wing motif, respectively. The NMR data also suggest the absence of a large conformational change in the protein upon binding to DNA. Hence, an O:(6)-methylguanine (O:(6)meG) lesion would be inaccessible to active site nucleophile Cys146 if the modified base remained stacked within the DNA duplex. The experimentally determined DNA-binding face of Ada-C was used in combination with homology modelling, based on the catabolite activator protein, and the accepted base-flipping mechanism, to construct a model of how Ada-C binds to DNA in a productive manner. To complement the structural studies, thermodynamic data were obtained which demonstrate that binding to unmethylated DNA was entropically driven, whilst the demethylation reaction provoked an exothermic heat change. Methylation of Cys146 leads to a loss of structural integrity of the DNA-binding subdomain.
Subject(s)
DNA/metabolism , Escherichia coli/enzymology , O(6)-Methylguanine-DNA Methyltransferase/chemistry , O(6)-Methylguanine-DNA Methyltransferase/metabolism , Amino Acid Motifs , Amino Acid Sequence , Binding Sites , Calorimetry , DNA/chemistry , DNA/genetics , DNA Methylation , DNA Repair , DNA, Single-Stranded/chemistry , DNA, Single-Stranded/genetics , DNA, Single-Stranded/metabolism , DNA-Binding Proteins/chemistry , DNA-Binding Proteins/metabolism , Entropy , Models, Molecular , Molecular Sequence Data , Mutation/genetics , Nuclear Magnetic Resonance, Biomolecular , Nucleic Acid Conformation , Protein Binding , Protein Structure, Secondary , TitrimetryABSTRACT
A series of new substituted-aryl phosphoramidate derivatives of the anti-HIV drug d4T were synthesized as membrane-soluble nucleotide prodrugs, to extend and quantify the SAR observed for an earlier series of related derivatives. All of the compounds were found to be significantly more potent against HIV in cell culture than the nucleoside analogue d4T, and most were also found to be significantly more potent than the parent phosphoramidate. A Hansch type QSAR analysis was applied to the combined series of 21 compounds. The results of this analysis revealed anti-HIV activity to be principally dependent on lipophilicity in a quadratic manner, with terms representing substituent steric bulk and electronic effects having a minimal significance.
Subject(s)
Amides/chemical synthesis , Anti-HIV Agents/chemical synthesis , Phosphoric Acids/chemical synthesis , Prodrugs/chemical synthesis , Stavudine/analogs & derivatives , Amides/chemistry , Amides/pharmacology , Anti-HIV Agents/chemistry , Anti-HIV Agents/pharmacology , Cell Line , Dideoxynucleotides , HIV-1/drug effects , HIV-2/drug effects , Phosphoric Acids/chemistry , Phosphoric Acids/pharmacology , Prodrugs/chemistry , Prodrugs/pharmacology , Stavudine/chemistry , Stavudine/metabolism , Structure-Activity RelationshipABSTRACT
This paper describes the design and synthesis of potential inhibitors of Trypanosoma cruzi dihydrofolate reductase using a structure-based approach. A model of the structure of the T. cruzi enzyme was compared with the structure of the human enzyme. The differences were used to design modifications of methotrexate to produce compounds which should be selective for the parasite enzyme. The derivatives of methotrexate were synthesised and tested against the enzyme and intact parasites.
Subject(s)
Drug Design , Enzyme Inhibitors/chemistry , Folic Acid Antagonists/chemistry , Tetrahydrofolate Dehydrogenase/drug effects , Trypanosoma cruzi/enzymology , Animals , Binding Sites , Cell Line , Enzyme Inhibitors/chemical synthesis , Folic Acid Antagonists/chemical synthesis , Humans , Mice , Molecular Structure , Recombinant Proteins/antagonists & inhibitorsABSTRACT
Dihydrofolate reductase has successfully been used as a drug target in the area of anti-cancer, anti-bacterial and anti-malarial chemotherapy. Little has been done to evaluate it as a drug target for treatment of the trypanosomiases and leishmaniasis. A crystal structure of Leishmania major dihydrofolate reductase has been published. In this paper, we describe the modelling of Trypanosoma cruzi and Trypanosoma brucei dihydrofolate reductases based on this crystal structure. These structures and models have been used in the comparison of protozoan, bacterial and human enzymes in order to highlight the different features that can be used in the design of selective anti-protozoan agents. Comparison has been made between residues present in the active site, the accessibility of these residues, charge distribution in the active site, and the shape and size of the active sites. Whilst there is a high degree of similarity between protozoan, human and bacterial dihydrofolate reductase active sites, there are differences that provide potential for selective drug design. In particular, we have identified a set of residues which may be important for selective drug design and identified a larger binding pocket in the protozoan than the human and bacterial enzymes.
Subject(s)
Folic Acid Antagonists/pharmacology , Leishmania major/enzymology , Tetrahydrofolate Dehydrogenase/drug effects , Trypanosoma brucei brucei/enzymology , Trypanosoma cruzi/enzymology , Amino Acid Sequence , Animals , Binding Sites , Folic Acid Antagonists/chemistry , Humans , Molecular Sequence Data , Sequence Homology, Amino Acid , Solvents , Tetrahydrofolate Dehydrogenase/chemistry , Tetrahydrofolate Dehydrogenase/metabolismABSTRACT
The cholera toxin from Vibrio cholerae (CT) and the 80% homologous heat-labile toxin of Escherichia coli (LT) are two well-known cases of sugar-binding proteins. The GM1:toxin complexes were chosen as test cases for the elaboration of a computational approach to the modeling of protein-saccharide interactions. The reliability of the method was evaluated on the LT:lactose complex. A model of this complex was built by performing a MC/EM conformational search of the sugar moiety within the binding pocket of LT, using the AMBER* force field and the GB/SA solvation model. The results are a reasonable reproduction of the reported X-ray structure of the complex. The same protocol was then applied to the LT:GM1 complex. The calculations were performed on a substructure that includes the protein shell within 5 A from GM1, three water molecules solvating Glu-51 carboxylate, and two water molecules at crystallographic sites 2 and 3. A satisfactory agreement was found with the recently published X-ray structure of the CT:GM1 complex. All the relevant interactions between the sugar and the residues involved in binding are well reproduced by the calculations. These results suggest that the substructure here identified can be taken as a realistic representation of the toxin binding surface and that the method presented in this paper can be used as a predictive tool in designing artificial LT (CT) binders and thus potential anticholera drugs.
