ABSTRACT
Deletions within human chromosome 4p16.3 cause Wolf-Hirschhorn syndrome (WHS), which is characterized by severe mental and developmental defects. It is thought that haploinsufficiency of more than one gene contributes to the complex phenotype. We have cloned and characterized a novel gene (LETM1) that is deleted in nearly all WHS patients. LETM1 encodes a putative member of the EF-hand family of Ca(2+)-binding proteins. The protein contains two EF-hands, a transmembrane domain, a leucine zipper, and several coiled-coil domains. On the basis of its possible Ca(2+)-binding property and involvement in Ca(2+) signaling and/or homeostasis, we propose that haploinsufficiency of LETM1 may contribute to the neuromuscular features of WHS patients.
Subject(s)
EF Hand Motifs/genetics , Gene Deletion , Amino Acid Sequence , Animals , Base Sequence , Chromosome Mapping , Cloning, Molecular , DNA, Complementary/genetics , Developmental Disabilities/genetics , Exons , Gene Expression , Humans , In Situ Hybridization, Fluorescence , Intellectual Disability/genetics , Introns , Mice , Molecular Sequence Data , Neuromuscular Diseases/genetics , Phenotype , Sequence Homology, Amino Acid , Species Specificity , SyndromeABSTRACT
The design, synthesis, and crystallographic analysis of protein-inhibitor complexes is described for a novel series of nonpeptidic HIV protease (HIV Pr)inhibitors. Beginning with a cocrystal structure of a Phe-Pro peptidomimetic bound to the HIV Pr, design was initiated that resulted in the substituted 2-butanol compound 8 as the lead compound (Ki = 24.5 microM, racemic mixture). Modifications on the initial compound were then made on the basis of its cocrystal structure with HIV Pr and inhibition data, resulting in compounds with enhanced potency against the enzyme (compound 18, Ki = 0.48 microM). These inhibitors were found to bind to the enzyme essentially as predicted on the basis of the original design hypothesis. Stereospecific synthesis of individual enantiomers confirmed the prediction of a binding preference for the S alcohol stereochemistry. Modest antiviral activity was demonstrated for several of the more potent HIV Pr inhibitors in a HIV-1 infected CEM-SS cell line.
Subject(s)
Amides/chemistry , Antiviral Agents/chemistry , Butanols/pharmacology , HIV Protease Inhibitors/chemistry , HIV-1/drug effects , Amides/pharmacology , Antiviral Agents/pharmacology , Butanols/chemistry , Cell Line , Crystallography, X-Ray , Drug Design , HIV Protease Inhibitors/pharmacology , HIV-1/enzymology , Humans , Models, Molecular , Structure-Activity RelationshipABSTRACT
We have cloned the cDNA for the human homolog of the rat AP17 gene, a small chain of the clathrin-associated protein complex AP-2. The cDNA is highly conserved between rat and human. Human AP17, gene symbol CLAPS2 (clathrin-associated/assembly/adaptor protein, small 3, 17 kDa), was assigned to chromosome region 19q13.2-->q13.3.
Subject(s)
Adaptor Protein Complex 2 , Adaptor Protein Complex sigma Subunits , Chromosome Mapping , Chromosomes, Human, Pair 19/genetics , Clathrin , Nerve Tissue Proteins/genetics , Phosphoproteins/genetics , Adaptor Proteins, Vesicular Transport , Amino Acid Sequence , Animals , Base Sequence , Cloning, Molecular , DNA, Complementary/genetics , Gene Library , Humans , In Situ Hybridization, Fluorescence , Kidney/metabolism , Molecular Sequence Data , Nerve Tissue Proteins/chemistry , Phosphoproteins/chemistry , Rats , Sequence Homology, Nucleic AcidABSTRACT
A class of potent nonpeptidic inhibitors of human immunodeficiency virus protease has been designed by using the three-dimensional structure of the enzyme as a guide. By employing iterative protein cocrystal structure analysis, design, and synthesis the binding affinity of the lead compound was incrementally improved by over four orders of magnitude. An inversion in inhibitor binding mode was observed crystallographically, providing information critical for subsequent design and highlighting the utility of structural feedback in inhibitor optimization. These inhibitors are selective for the viral protease enzyme, possess good antiviral activity, and are orally available in three species.
Subject(s)
Drug Design , HIV Protease Inhibitors/chemistry , HIV Protease Inhibitors/pharmacology , HIV/drug effects , Administration, Oral , Amino Acid Sequence , Animals , Benzamides/chemistry , Biological Availability , Dogs , Drug Evaluation , HIV/enzymology , HIV Protease Inhibitors/administration & dosage , HIV Protease Inhibitors/pharmacokinetics , Haplorhini , Mice , Models, Molecular , Molecular Sequence Data , Rats , Species Specificity , Structure-Activity RelationshipABSTRACT
Antifolate inhibitors of thymidylate synthase (TS) have primarily been based on the structure of folic acid. This paper describes the identification and development of novel 6,7-imidazotetrahydroquinoline TS inhibitors by iterative ligand design, synthesis, and crystallographic analysis of protein-inhibitor complexes. Beginning with a high-resolution crystal structure of E. coli TS (TS, EC 2.1.1.45), an imidazotetrahydroquinoline inhibitor was designed de novo to occupy the folate binding pocket. Structural modifications of the initial compound 1h (Ki approximately 5 microM human/E. coli TS) were then made on the basis of feedback from additional cocrystal structures and activity data. An amino group in the 2-position of the imidazole was found to increase the potency of the series by 1-2 orders of magnitude. Other substitutions on the imidazole ring (1-CH3, 2-CH3, 2-NHCH3, 2-SCH3) generally led to weaker inhibition. Additional improvements in activity were obtained by modification of the substituents on the tetrahydroquinoline nitrogen, bringing the Ki of three of the compounds below 15 nM against the human TS enzyme. The compounds were tested for cytotoxicity and were shown to inhibit the growth of three tumor cell lines in vitro.