ABSTRACT
The synthesis of [1-[(5-hydroxy-4-(phenylmethyl)-3-oxazolidinyl)carbonyl]-2-ethylpropy lcarbamic acid phenylmethyl ester (2; MDL 104,903), a potent inhibitor of calpain, is described. Synthesis of related compounds, which offer insights into the mechanism of action for 2, are also described, as is an O-acetyl prodrug derivative of 2.
Subject(s)
Calpain/antagonists & inhibitors , Carbamates/pharmacology , Oxazoles/pharmacology , Protease Inhibitors/pharmacology , Carbamates/chemistry , Magnetic Resonance Spectroscopy , Oxazoles/chemistry , Protease Inhibitors/chemistryABSTRACT
In the present study we characterize key activities of an agent designed to simultaneously inhibit angiotensin I-converting enzyme (ACE) and neutral endopeptidase (NEP). MDL 100,240 is a thioester prodrug of MDL 100,173, which is a potent competitive inhibitor of both ACE and NEP in vitro. MDL 100,240 was shown in an ex vivo study to inhibit both of these enzymes in rat kidney. When administered to anesthetized rats, MDL 100,240 enhanced the effect of infused ANP on blood pressure, diuresis and natriuresis and of infused bradykinin on blood pressure. Moreover, MDL 100,173 and MDL 100,240 inhibited the pressor response to angiotensin I. These results indicate that MDL 100,173 and its prodrug, MDL 100,240, produced effects, in vivo, consistent with inhibition of both ACE and NEP.
Subject(s)
Angiotensin-Converting Enzyme Inhibitors/pharmacology , Benzazepines/pharmacology , Neprilysin/antagonists & inhibitors , Peptidyl-Dipeptidase A/metabolism , Pyridines/pharmacology , Animals , Atrial Natriuretic Factor/pharmacology , Bradykinin/pharmacology , Male , Molecular Structure , Neprilysin/metabolism , Prodrugs/pharmacology , Rats , Rats, Sprague-DawleyABSTRACT
Autosomal recessive spinal muscular atrophy (SMA) has been mapped to a 6-cM interval on chromosome 5q12-13.3, flanked proximally by locus D5S6 and distally by locus D5S112. In this study we describe the isolation of two new microsatellite markers (EF1/2a and EF13/14) near locus D5S125, which lies 2 cM distal to D5S6. We show by linkage analysis and the study of the recombinants in 55 SMA pedigrees that the disease lies in the 4-cM interval between EF1/2a and D5S112. Fluorescence in situ analysis of cosmids from D5S6, EF1/2 and D5S112 confirms the genetic order and relative distance of markers. The microsatellites EF1/2a and EF13/14 are the first highly polymorphic PCR-based proximal markers in SMA to be described, and will be of value in prenatal prediction of the disorder.
Subject(s)
Chromosomes, Human, Pair 5 , DNA, Satellite/genetics , Prenatal Diagnosis/methods , Spinal Muscular Atrophies of Childhood/diagnosis , Spinal Muscular Atrophies of Childhood/genetics , Base Sequence , Chromosome Mapping , Female , Fetal Diseases/diagnosis , Fetal Diseases/genetics , Genetic Linkage , Genetic Markers , Humans , In Situ Hybridization, Fluorescence , Male , Molecular Sequence Data , Pedigree , Polymerase Chain Reaction , Polymorphism, Genetic , Predictive Value of Tests , Pregnancy , Recombination, GeneticSubject(s)
Angiotensin-Converting Enzyme Inhibitors/chemistry , Blood Pressure/drug effects , Neprilysin/chemistry , Oligopeptides/chemical synthesis , Sympathomimetics/chemical synthesis , Animals , Neprilysin/antagonists & inhibitors , Oligopeptides/pharmacology , Protein Conformation , Rats , Rats, Inbred SHR , Structure-Activity Relationship , Sympathomimetics/analogs & derivatives , Sympathomimetics/pharmacologyABSTRACT
Chromosome fragility in two families not exhibiting amplification of the CGG trinucleotide associated with the fragile X site has been examined. Fluorescence in situ hybridisation with cosmid DNA from loci immediately flanking FRAXA and other distal loci have confirmed that cytogenetic fragility in these subjects is the result of expression of a new folate sensitive fragile X site, FRAXE.
Subject(s)
Chromosome Fragility , Fragile X Syndrome/genetics , X Chromosome , Chromosome Fragile Sites , Chromosome Mapping , DNA Mutational Analysis , DNA Probes , Folic Acid/pharmacology , Genetic Linkage , Humans , In Situ Hybridization, Fluorescence , In Vitro Techniques , Male , Phenotype , Repetitive Sequences, Nucleic Acid , X Chromosome/drug effectsABSTRACT
Although autosomal recessive spinal muscular atrophy (SMA) has been mapped to chromosome 5q12-q13, there is for this region no genetic map based on highly informative markers. In this study we present the mapping of two previously reported microsatellite markers in 40 CEPH and 31 SMA pedigrees. We also describe the isolation of a new microsatellite marker at the D5S112 locus. The most likely order of markers (with recombination fractions given in parentheses) is 5cen-D5S6-(.02)-D5S125-(.04)-(JK53CA1/2,D5S11 2)-(.04)-D5S39-qter. The relative order of D5S6, D5S112, and D5S39 was confirmed by in situ hybridization. Multipoint linkage analysis in 31 SMA families indicates that the SMA locus lies in the 6-cM interval between D5S6 and JK53CA1/2, D5S112.
Subject(s)
Chromosome Mapping/methods , Chromosomes, Human, Pair 5 , Spinal Muscular Atrophies of Childhood/genetics , Base Sequence , Chromosomes, Fungal , Cloning, Molecular , DNA/analysis , DNA, Satellite/analysis , Female , Genetic Linkage/genetics , Genetic Markers , Humans , Infant , Lod Score , Male , Molecular Sequence Data , Nucleic Acid Hybridization , Pedigree , Polymerase Chain Reaction , X ChromosomeABSTRACT
A novel route to activated phenolic sulfoxide analogs of sparsomycin has been developed. These analogs display an enhanced "preincubation effect" as inhibitors of peptide-bond formation. This time-dependent component of inhibition, which is postulated to result from an enzyme-mediated Pummerer rearrangement, is the dominant route to inhibition in these activated analogs.
Subject(s)
Acyltransferases/antagonists & inhibitors , Antibiotics, Antineoplastic/chemical synthesis , Peptidyl Transferases/antagonists & inhibitors , Sparsomycin/analogs & derivatives , Sparsomycin/chemical synthesis , Computer Graphics , Escherichia coli , In Vitro Techniques , Models, Molecular , Peptide Chain Elongation, Translational/drug effects , Puromycin/metabolism , Ribosomes/drug effects , Sparsomycin/pharmacology , Stereoisomerism , Structure-Activity RelationshipABSTRACT
Inhibition of rabbit lung angiotensin I-converting enzyme was studied with two inhibitors that combined tricyclic mimics of a substrate C-terminal dipeptide recognition unit with a 4-phenylbutanoic acid fragment. The overall inhibition constant for [4S-[4 alpha, 7 alpha(R*),12b beta]]-7-[S-(1-carboxy-3-phenylpropyl) amino]-1,2,3,4,6,7,8,12b-octahydro-6-oxopyrido[2,1-a] [2] benzazepine-4-carboxylic acid (MDL 27,088) was approximately 4 pM, whereas that for [4R-[4 alpha, 7 alpha(S*), 12b beta]]-7-[S-(1-carboxy-3-phenylpropyl)amino]-3,4,6,7,8, 12b-hexahydro-6-oxo-1H-[1,4]thiazino[3,4-a] [2]benzazepine-4-carboxylic acid (MDL 27,788) was estimated to be 46 pM. The formation of an initial complex of target enzyme and MDL 27,088 and its slower isomerization to a second complex were characterized kinetically. Both compounds appear to be among the most potent inhibitors known for this enzyme.
Subject(s)
Angiotensin-Converting Enzyme Inhibitors/pharmacology , Benzazepines/pharmacology , Lung/enzymology , Pyridines/pharmacology , Thiazines/pharmacology , Kinetics , Molecular Structure , Peptidyl-Dipeptidase A/metabolism , Protein BindingABSTRACT
Peptidyl transferase activity of Trypanosoma brucei brucei polyribosomes was competitively inhibited by analogs of sparsomycin (Ki = 1-100 microM). The analogs were also potent inhibitors of [3H]-leucine and [3H]mannose incorporation into the proteins of intact trypanosomes with little or no effect on overall respiratory rate, suggesting a specific site of action for these analogs on protein synthesis. The peptidyl transferase inhibitors were effective at low concentrations at limiting the proliferation of trypanosomes both in vitro and in vivo. The potency of the compounds as inhibitors of cell proliferation was positively correlated with their efficacy as inhibitors of peptidyl transferase activity. One compound, MDL 20828 (1 mg/kg), increased the survival time of T. b. brucei-infected mice 4-fold in the absence of any overt drug toxicity to the hosts.
Subject(s)
Acyltransferases/antagonists & inhibitors , Antibiotics, Antineoplastic/pharmacology , Peptidyl Transferases/antagonists & inhibitors , Protein Biosynthesis , Sparsomycin/pharmacology , Trypanosoma brucei brucei/enzymology , Animals , Glycoproteins/biosynthesis , Leucine/metabolism , Mice , Polyribosomes/enzymology , Sparsomycin/analogs & derivatives , Trypanosoma brucei brucei/drug effects , Trypanosoma brucei brucei/growth & development , Trypanosomiasis, African/drug therapyABSTRACT
Sparsomycin, ScRs configuration, was the most potent of the four possible stereoisomers as a competitive inhibitor of peptide bond formation. In addition, the configuration of the two chiral centers dictated whether the compound exhibited time- and temperature-dependent inhibition of peptidyl transferase when incubated with polysomes prior to enzyme assay. The data corroborate the thesis that a peptidyl transferase-mediated acylation of the pivotal sulfoxide moiety and subsequent Pummerer rearrangement play a significant role in the inhibitory properties of sparsomycin.
Subject(s)
Acyltransferases/antagonists & inhibitors , Antibiotics, Antineoplastic/pharmacology , Escherichia coli/enzymology , Peptidyl Transferases/antagonists & inhibitors , Sparsomycin/pharmacology , Kinetics , Molecular Conformation , Stereoisomerism , ThermodynamicsABSTRACT
Nontoxic analogs of sparsomycin were competitive inhibitors of puromycin in the peptidyl transferase assay with Escherichia coli polysomes. The sensitivity of HeLa cells in vitro to the analogs was used as a preliminary index of cellular toxicity. In vitro killing of HeLa cells by this class of compounds correlated well with in vivo 50% lethal doses. The data indicate that modification of the hydrophobic sulfoxide substituent on sparsomycin decreases the toxicity of the molecule for mammalian cells by several hundredfold. Such modifications have less of an effect on the inhibitory activity of the compounds for peptidyl transferase. The differential effects of an analog active against bacterial but not mammalian cells was due to a decreased uptake of the compound by HeLa cells.
Subject(s)
Antibiotics, Antineoplastic/pharmacology , Sparsomycin/pharmacology , Animals , Escherichia coli/drug effects , Escherichia coli/enzymology , HeLa Cells , Humans , Lethal Dose 50 , Mice , Peptidyl Transferases/antagonists & inhibitors , Sparsomycin/analogs & derivatives , Sparsomycin/toxicity , Structure-Activity Relationship , Sulfoxides/pharmacologyABSTRACT
An analog of the peptidyl transferase inhibitor sparsomycin was a competitive inhibitor (Ki = 1.8 microM) of peptidyl-puromycin synthesis on E. coli polysomes. Preincubation of polysomes with the compound enhanced the degree of inhibition of peptide bond formation. A model for the involvement of a histidine residue in peptidyl transferase activity is presented as a result of our observations which include direct association of [3H] labelled analog with 70S ribosomes. The correct oxidation state of sulfur in the compound was necessary for the "preincubation effect" and entry of the compound into bacterial cells.
