ABSTRACT
Imidazolemethyl diaryl ethers are potent inhibitors of farnesyl-protein transferase. The SNAr displacement reaction used to prepare these diaryl ethers was amenable to rapid parallel synthesis of FPTase inhibitors. The use of a broad range of commercially available phenols quickly identified compounds which proved active in cells.
Subject(s)
Alkyl and Aryl Transferases/antagonists & inhibitors , Phenyl Ethers/pharmacology , Alkyl and Aryl Transferases/metabolism , Animals , Binding, Competitive , Cell Line , Enzyme Inhibitors/chemical synthesis , Enzyme Inhibitors/pharmacology , Imidazoles/chemistry , Inhibitory Concentration 50 , Peptide Library , Phenyl Ethers/chemical synthesis , Rats , Structure-Activity RelationshipABSTRACT
The design and syntheses of non-thiol inhibitors of farnesyl-protein transferase are described. Substitutions on an imidazolylmethyl-AMBA-methionine template gave a highly potent and cell-active inhibitor.
Subject(s)
Alkyl and Aryl Transferases/antagonists & inhibitors , Benzamides/chemistry , Benzamides/pharmacology , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/pharmacology , Alkyl and Aryl Transferases/chemistry , Alkyl and Aryl Transferases/metabolism , Animals , Benzamides/metabolism , Binding Sites , Cell Division/drug effects , Drug Design , Drug Evaluation, Preclinical , Imidazoles/chemistry , Inhibitory Concentration 50 , Models, Molecular , Molecular Structure , Protein Conformation , Rats , Structure-Activity Relationship , Sulfhydryl Compounds/chemistry , Sulfhydryl Compounds/pharmacologySubject(s)
Alkyl and Aryl Transferases , Antineoplastic Agents/chemical synthesis , Enzyme Inhibitors/chemical synthesis , Piperazines/chemical synthesis , Piperazines/pharmacology , Transferases/antagonists & inhibitors , Animals , Antineoplastic Agents/pharmacology , Cell Death/drug effects , Cell Division/drug effects , Cell Line, Transformed , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/pharmacology , Farnesyltranstransferase , Mice , Mice, Nude , Molecular Conformation , Molecular Structure , Neoplasm Transplantation , Polyisoprenyl Phosphates/metabolism , Protein Prenylation , Protein Processing, Post-Translational/drug effects , Rats , Sarcoma, Experimental/drug therapy , Sarcoma, Experimental/pathology , Sesquiterpenes , ras Proteins/metabolismABSTRACT
A series of highly potent, structurally novel, non-nucleoside RT inhibitors has been described. Low nanomolar concentrations of 5-chloro-3-(phenylsulfonyl)-indole-2-carboxamide (1) inhibit the HIV-1 RT enzyme in vitro and HTLVIIIb viral spread in MT-4 human T-lymphoid cells. Good oral bioavailability was observed in rhesus monkeys upon oral dosing of 1 as a suspension in methocel. When compared to other non-nucleoside inhibitors (e.g. 15-18), 1 possesses improved inhibitory potency with respect to the wild-type RT, as well as the K103N and Y181C mutant enzymes. Additional studies within this class of inhibitors are in progress.
Subject(s)
Antiviral Agents/pharmacology , HIV-1/enzymology , Indoles/pharmacology , Reverse Transcriptase Inhibitors , Sulfoxides/pharmacology , Animals , Antiviral Agents/chemistry , Base Sequence , Biological Availability , HIV/drug effects , HIV Reverse Transcriptase , Indoles/chemistry , Indoles/pharmacokinetics , Macaca mulatta , Molecular Sequence Data , Molecular Structure , Sulfoxides/chemistry , Sulfoxides/pharmacokineticsABSTRACT
The binding of fibrinogen to its platelet receptor, the glycoprotein IIb-IIIa complex, is mediated, in part, by an Arg-Gly-Asp (RGD) sequence within the fibrinogen A alpha chain. PAC1 is an IgM-kappa murine monoclonal antibody that binds to the platelet fibrinogen receptor, and its binding is inhibited by both fibrinogen and RGD-containing peptides. To identify the regions of PAC1 that interact with the fibrinogen receptor, we determined the mRNA sequences of PAC1 immunoglobulin heavy and light chain variable regions. Five out of the six complementarity-determining regions (CDRs) of PAC1 had entirely germline sequences with no regions of similarity to fibrinogen. However, CDR3 of the PAC1 heavy chain (H-CDR3) was very large and unique due to the insertion of a novel D region segment. H-CDR3 contained a sequence, Arg-Tyr-Asp (RYD), that, if present in the proper conformation, might behave like the RGD sequence in fibrinogen. A 21-residue synthetic peptide encompassing the H-CDR3 region inhibited fibrinogen-dependent platelet aggregation as well as the binding of PAC1 (Ki = 10 microM) and fibrinogen (Ki = 5 microM) to activated platelets. The RYD region of H-CDR3 appeared to be central to its function, because substitution of the tyrosine with glycine increased the inhibitory potency of the peptide by 10-fold, while replacing the tyrosine with D-alanine or inverting the RYD sequence sharply reduced the inhibitory potency. Thus, the linear sequence, RYD, within H-CDR3 of PAC1 appears to mimic the RGD receptor recognition sequence in fibrinogen. This type of immunologic approach could be useful in studying the structural basis of other receptor-ligand interactions.
Subject(s)
Antibodies, Monoclonal , Fibrinogen/genetics , Platelet Membrane Glycoproteins/metabolism , Amino Acid Sequence , Antigen-Antibody Complex , Base Sequence , Blood Platelets/metabolism , Fibrinogen/immunology , Fibrinogen/metabolism , Humans , Kinetics , Macromolecular Substances , Molecular Sequence Data , Platelet Membrane Glycoproteins/immunologyABSTRACT
The mature proteins of retroviruses originate as a result of proteolytic cleavages of polyprotein precursors. Retroviruses encode proteases responsible for several of these processing events, making them potential antiviral drug targets. A 99-amino acid HIV-1 protease, produced by chemical synthesis or by expression in bacteria, is shown here to hydrolyze peptides corresponding to all of the known cleavage sites in the HIV-1 gag and pol polyproteins. It does not hydrolyze peptides corresponding to an env cleavage site or a distantly related retroviral gag cleavage site.
Subject(s)
HIV-1/enzymology , Peptide Hydrolases/metabolism , Retroviridae Proteins/metabolism , Amino Acid Sequence , Antigens, Viral , Gene Products, gag , HIV-1/genetics , Hydrolysis , Kinetics , Peptide Hydrolases/chemical synthesis , Peptide Hydrolases/genetics , Substrate SpecificityABSTRACT
Retroviral proteins, including those from the human immunodeficiency virus (HIV), are synthesized as polyprotein precursors that require proteolytic cleavage to yield the mature viral proteins. A 99-residue polypeptide, encoded by the 5' end of the pol gene, has been proposed as the processing protease of HIV. The chemical synthesis of the 99-residue peptide was carried out by the solid-phase method, and the isolated product was found to exhibit specific proteolytic activity upon folding under reducing conditions. Upon size-exclusion chromatography, enzymatic activity was eluted at a point consistent with a dimeric molecular size. Specificity was demonstrated by the cleavage of the natural substrate HIV gag p55 into gag p24 and gag p17, as well as cleavage of small peptide substrates representing processing sites of HIV fusion proteins. The proteolytic action of the synthetic product could be inhibited by pepstatin, an aspartic protease inhibitor.
Subject(s)
Endopeptidases/chemical synthesis , Amino Acid Sequence , Endopeptidases/analysis , HIV Protease , Molecular Sequence DataABSTRACT
Native rat atrial natriuretic peptide (NANP) was shown to bind with high affinity and to increase intracellular levels of cGMP in cultured rat Leydig tumor cells. A linear analog of NANP which lacks the disulfide-linked bridge structure also bound with high affinity but did not increase levels of intracellular cGMP or antagonize the increase of this cyclic nucleotide by NANP. These data are consistent with the existence of two functional subpopulations of ANP receptors on cultured rat Leydig tumor cells; one which is capable of activating guanylate cyclase and one which is not linked to this enzyme.
Subject(s)
Leydig Cell Tumor/metabolism , Receptors, Cell Surface/metabolism , Amino Acid Sequence , Animals , Atrial Natriuretic Factor/analogs & derivatives , Atrial Natriuretic Factor/metabolism , Atrial Natriuretic Factor/pharmacology , Cyclic GMP/biosynthesis , Disulfides , Enzyme Activation , Guanylate Cyclase/metabolism , Male , Molecular Sequence Data , Rats , Receptors, Atrial Natriuretic Factor , Tumor Cells, CulturedABSTRACT
The actions of atrial natriuretic factor (ANF) on the vascular wall are diverse and show a profound regional heterogeneity. ANF is a potent relaxant of aortic smooth muscle, a response which is associated with activation of particulate guanylate cyclase and elevation in tissue levels of cyclic GMP. However, many large and small muscular arteries and most veins are unresponsive to the peptide. The regional vascular heterogeneity may be due to an altered distribution of high affinity receptors and (or) alterations in the coupling of receptor activation to elevations in cyclic 3',5'-guanosine monophosphate (cGMP). Species differences exist in the structural requirements for receptor activation as well as the effects of infused ANF on peripheral resistance. Although the relaxation to ANF in vitro does not require an intact endothelium, endothelial cells contain multiple receptor subtypes for ANF. Differences amongst tissues and (or) species in the receptor profile for ANF may, in part, explain some of heterogeneity in responsiveness to ANF.
