ABSTRACT
Severe acute respiratory syndrome (SARS) is an emerging infectious disease associated with a high rate of mortality. The SARS-associated coronavirus (SARS-CoV) has been identified as the etiological agent of the disease. Although public health procedures have been effective in combating the spread of SARS, concern remains about the possibility of a recurrence. Various approaches are being pursued for the development of efficacious therapeutics. One promising approach is to develop small molecule inhibitors of the essential major polyprotein processing protease 3Clpro. Here we report a complete description of the tetrapeptide substrate specificity of 3Clpro using fully degenerate peptide libraries consisting of all 160,000 possible naturally occurring tetrapeptides. The substrate specificity data show the expected P1-Gln P2-Leu specificity and elucidate a novel preference for P1-His containing substrates equal to the expected preference for P1-Gln. These data were then used to develop optimal substrates for a high-throughput screen of a 2000 compound small-molecule inhibitor library consisting of known cysteine protease inhibitor scaffolds. We also report the 1.8 A X-ray crystal structure of 3Clpro bound to an irreversible inhibitor. This inhibitor, an alpha,beta-epoxyketone, inhibits 3Clpro with a k3/Ki of 0.002 microM(-1) s(-1) in a mode consistent with the substrate specificity data. Finally, we report the successful rational improvement of this scaffold with second generation inhibitors. These data provide the foundation for a rational small-molecule inhibitor design effort based upon the inhibitor scaffold identified, the crystal structure of the complex, and a more complete understanding of P1-P4 substrate specificity.
Subject(s)
Antiviral Agents/isolation & purification , Antiviral Agents/pharmacology , Cysteine Proteinase Inhibitors/pharmacology , Dipeptides/pharmacology , Epoxy Compounds/pharmacology , Oligopeptides/pharmacology , Severe acute respiratory syndrome-related coronavirus/enzymology , Viral Proteins/antagonists & inhibitors , Amino Acid Substitution , Animals , Antiviral Agents/chemistry , Antiviral Agents/metabolism , Catalytic Domain/drug effects , Chlorocebus aethiops , Coronavirus 3C Proteases , Crystallography, X-Ray , Cysteine Endopeptidases/chemistry , Cysteine Proteinase Inhibitors/chemistry , Cysteine Proteinase Inhibitors/classification , Dipeptides/chemistry , Dipeptides/isolation & purification , Dipeptides/metabolism , Epoxy Compounds/chemistry , Epoxy Compounds/isolation & purification , Epoxy Compounds/metabolism , Models, Molecular , Oligopeptides/chemistry , Oligopeptides/isolation & purification , Oligopeptides/metabolism , Peptide Library , Protein Structure, Tertiary , Severe acute respiratory syndrome-related coronavirus/drug effects , Structure-Activity Relationship , Substrate Specificity , Vero Cells , Viral Proteins/chemistry , Virus Replication/drug effectsABSTRACT
Cysteine protease activity of African trypanosome parasites is a target for new chemotherapy using synthetic protease inhibitors. To support this effort and further characterize the enzyme, we expressed and purified rhodesain, the target protease of Trypanosoma brucei rhodesiense (MVAT4 strain), in reagent quantities from Pichia pastoris. Rhodesain was secreted as an active, mature protease. Site-directed mutagenesis of a cryptic glycosylation motif not previously identified allowed production of rhodesain suitable for crystallization. An invariable ER(A/V)FNAA motif in the pro-peptide sequence of rhodesain was identified as being unique to the genus Trypanosoma. Antibodies to rhodesain localized the protease in the lysosome and identified a 40-kDa protein in long slender forms of T. b. rhodesiense and all life-cycle stages of T. b. brucei. With the latter parasite, protease expression was five times greater in short stumpy trypanosomes than in the other stages. Radiolabeled active site-directed inhibitors identified brucipain as the major cysteine protease in T. b. brucei. Peptidomimetic vinyl sulfone and epoxide inhibitors designed to interact with the S2, S1 and S' subsites of the active site cleft revealed differences between rhodesain and the related trypanosome protease cruzain. Using fluorogenic dipeptidyl substrates, rhodesain and cruzain had acid pH optima, but unlike some mammalian cathepsins retained significant activity and stability up to pH 8.0, consistent with a possible extracellular function. S2 subsite mapping of rhodesain and cruzain with fluorogenic peptidyl substrates demonstrates that the presence of alanine rather than glutamate at S2 prevents rhodesain from cleaving substrates in which P2 is arginine.
Subject(s)
Cysteine Endopeptidases/genetics , Cysteine Endopeptidases/metabolism , Lysosomes/enzymology , Trypanosoma brucei rhodesiense/enzymology , Animals , Binding Sites/genetics , Cysteine Endopeptidases/chemistry , Cysteine Endopeptidases/isolation & purification , Epoxy Compounds/pharmacology , Molecular Sequence Data , Mutagenesis, Site-Directed , Protease Inhibitors/pharmacology , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Sequence Analysis, DNA , Sulfones/pharmacology , Trypanosoma brucei rhodesiense/genetics , Trypanosoma brucei rhodesiense/growth & developmentABSTRACT
A new family of potent N-alkoxyvinylsulfonamide inhibitors of cruzain have been developed. Inhibitor 13 has a second order inactivation rate constant of 6,480,000s(-1)M(-1) versus cruzain, and is also highly effective against Trypanosoma cruzi trypomastigotes in a tissue culture assay.
Subject(s)
Cysteine Proteinase Inhibitors/chemical synthesis , Protozoan Proteins/antagonists & inhibitors , Sulfonamides/chemical synthesis , Sulfonamides/pharmacology , Trypanocidal Agents/chemical synthesis , Trypanosoma cruzi/drug effects , Animals , Cysteine Endopeptidases/metabolism , Cysteine Proteinase Inhibitors/chemistry , Cysteine Proteinase Inhibitors/pharmacology , Parasitic Sensitivity Tests , Protozoan Proteins/metabolism , Sulfonamides/chemistry , Trypanocidal Agents/chemistry , Trypanocidal Agents/pharmacologyABSTRACT
Propargylic 1,2-anti-diol derivatives 2 and 10 are prepared in high yield and excellent diastereoselectivity by addition of alpha-alkoxypropargylstannanes 4a and 4b to aldehydes in the presence of BuSnCl(3). We also introduce the use of KF on Celite as a convenient and mild reagent for removal of the organotin waste products of these reactions. Reaction: see text.
Subject(s)
Alkynes/chemical synthesis , Propanols/chemical synthesis , Aldehydes/chemistry , Biological Factors/chemical synthesis , Stereoisomerism , Tin Compounds/chemistryABSTRACT
An efficient, eight-step synthesis of L-rhodinosyl acetate derivative 3 is described. The synthesis originates from methyl (S)-lactate and involves a highly stereoselective, chelate-controlled addition of allyltributylstannane to the lactaldehyde derivative 7. The beta-anomeric configuration of 3 was established with high selectivity by acetylation of the pyranose precursor with Ac(2)O and Et(3)N in CH(2)Cl(2). Preliminary studies of glycosidation reactions of 3 and L-rhodinosyl acetate 10 containing a 3-O-TES ether revealed that these compounds are highly reactive glycosidating agents and that trialkylsilyl triflates are effective glycosylation promoters. The best conditions for reactions with 15 as the acceptor involved use of diethyl ether as the reaction solvent and 0.2 equiv of TES-OTf at -78 degrees C. However, the TES ether protecting group of 10 proved to be too labile under these reaction conditions, and mixtures of 16a, 17, and 18a are obtained in reactions of 10 and 15. Disaccharide 17 arises via in situ cleavage of the TES ether of disaccharide 16a, while trisaccharide 18a results from a glycosidation of in situ generated 17 (or of 16a itself) with a second equivalent of 10. These problems were largely suppressed by using 3 with a 3-O-TBS ether protecting group as the glycosyl donor and 0.2 equiv of TES-OTf as the reaction promoter. Attempts to selectively glycosylate the C(3)-OH of diol acceptors 20 or 28 gave a 70:30 mixture of 21 and 22 in the reaction of 20 and a 43:27:30 mixture of regioisomeric trisaccharides 29 and 30 and tetrasaccharide 31 from the glycosidation reaction of 28. However, excellent results were obtained in the glycosidation of differentially protected disaccharide 34 using 1.5 equiv of 3 and 0.05 equiv of TBS-OTf in CH(2)Cl(2) at -78 degrees C. The latter step is an important transformation in the recently reported synthesis of the landomycin A hexasaccharide unit.
Subject(s)
Aminoglycosides , Anti-Bacterial Agents/chemical synthesis , Antibiotics, Antineoplastic/chemical synthesis , Glycosides/chemistry , Hexoses/chemistry , Monosaccharides/chemical synthesis , Anti-Bacterial Agents/chemistry , Antibiotics, Antineoplastic/chemistry , Monosaccharides/chemistryABSTRACT
The carboxypeptidase and endopeptidase activities of cathepsins X and B, as well as their inhibition by E-64 derivatives, have been investigated in detail and compared. The results clearly demonstrate that cathepsins X and B do not share similar activity profiles against substrates and inhibitors. Using quenched fluorogenic substrates, we show that cathepsin X preferentially cleaves substrates through a monopeptidyl carboxypeptidase pathway, while cathepsin B displays a preference for the dipeptidyl pathway. The preference for one or the other pathway is about the same for both enzymes, i. e. approximately 2 orders of magnitude. Cleavage of a C-terminal dipeptide of a substrate by cathepsin X can be observed under conditions that preclude efficient monopeptidyl carboxypeptidase activity. In addition, an inhibitor designed to exploit the unique structural features responsible for the carboxypeptidase activity of cathepsin X has been synthesized and tested against cathepsins X, B and L. Although of moderate potency, this E-64 derivative is the first reported example of a cathepsin X-specific inhibitor. By comparison, CA074 was found to inactivate cathepsin B at least 34000-fold more efficiently than cathepsin X.
Subject(s)
Cathepsin B/antagonists & inhibitors , Cathepsins/antagonists & inhibitors , Cathepsin B/chemistry , Cathepsin B/metabolism , Cathepsin K , Cathepsins/chemistry , Cathepsins/metabolism , Cysteine Endopeptidases/chemistry , Cysteine Endopeptidases/metabolism , Cysteine Proteinase Inhibitors/chemistry , Cysteine Proteinase Inhibitors/pharmacology , Drug Design , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/pharmacology , Exopeptidases/metabolism , Fluorescent Dyes , Humans , Leucine/analogs & derivatives , Leucine/chemistry , Leucine/pharmacology , Models, Chemical , Substrate SpecificityABSTRACT
[figure: see text] An efficient and highly concise synthesis of 6, corresponding to the C(1)-C(11) fragment of formamicin (1), has been accomplished by a route utilizing a diastereoselective lactate aldol reaction to set the C(6) tertiary ether and the TES-OTf mediated transketalization of the C(6) tertiary methoxymethyl ether and the C(25) PMB ether to set the seven-membered methylene acetal unit (see 37-->38).
Subject(s)
Anti-Bacterial Agents/chemical synthesis , Antifungal Agents/chemical synthesis , Macrolides , Actinomycetales/chemistryABSTRACT
[see reaction]. A synthesis of tetracycle 2 corresponding to the C(11)-C(26) fragment of pectenotoxin II is described. The synthesis features two highly stereoselective [3 + 2]-annulation reactions of chiral allylsilanes, generated via allylboration of aldehydes with the chiral gamma-silylallylborane 4 or the gamma-silylallylboronate 19, for construction of the highly substituted C and E rings.
Subject(s)
Furans/chemical synthesis , Pyrans/chemical synthesis , Furans/chemistry , Macrolides , Pyrans/chemistry , StereoisomerismABSTRACT
Several new cysteine proteases of the papain family have been discovered in the past few years. To help in the assignment of physiological roles and in the design of specific inhibitors, a clear picture of the specificities of these enzymes is needed. One of these novel enzymes, cathepsin X, displays a unique specificity, cleaving single amino acid residues at the C-terminus of substrates very efficiently. In this study, the carboxypeptidase activities and substrate specificity of cathepsins X and B have been investigated in detail and compared. Using quenched fluorogenic substrates and HPLC measurements, it was shown that cathepsin X preferentially cleaves substrates through a monopeptidyl carboxypeptidase pathway, while cathepsin B displays a preference for the dipeptidyl pathway. The preference for one or the other pathway is about the same for both enzymes, i.e., approximately 2 orders of magnitude, a result supported by molecular modeling of enzyme-substrate complexes. Cleavage of a C-terminal dipeptide of a substrate by cathepsin X can become more important under conditions that preclude efficient monopeptidyl carboxypeptidase activity, e.g., nonoptimal interactions in subsites S(2)-S(1). These results confirm that cathepsin X is designed to function as a monopeptidyl carboxypeptidase. Contrary to a recent report [Klemencic, I., et al. (2000) Eur. J. Biochem. 267, 5404-5412], it is shown that cathepsins X and B do not share similar activity profiles, and that reagents are available to clearly distinguish the two enzymes. In particular, CA074 was found to inactivate cathepsin B at least 34000-fold more efficiently than cathepsin X. The insights obtained from this and previous studies have been used to produce an inhibitor designed to exploit the unique structural features responsible for the carboxypeptidase activity of cathepsin X. Although of moderate potency, this E-64 derivative is the first reported example of a cathepsin X-specific inhibitor.
Subject(s)
Cathepsin B/metabolism , Cathepsins/metabolism , Cysteine Endopeptidases/metabolism , Endopeptidases/metabolism , Cathepsin B/antagonists & inhibitors , Cathepsin B/chemistry , Cathepsin K , Cathepsins/antagonists & inhibitors , Cathepsins/chemistry , Chromogenic Compounds/metabolism , Cysteine Endopeptidases/chemistry , Cysteine Proteinase Inhibitors/chemical synthesis , Cysteine Proteinase Inhibitors/metabolism , Endopeptidases/chemistry , Enzyme Inhibitors/chemical synthesis , Enzyme Inhibitors/metabolism , Exopeptidases/chemistry , Exopeptidases/metabolism , Humans , Hydrolysis , Kinetics , Models, Molecular , Oligopeptides/metabolism , Spectrometry, Fluorescence , Substrate Specificity , ThermodynamicsABSTRACT
Lewis acid catalyzed intramolecular Diels-Alder reactions of trienes (E,E,Z)-1a-d, (E,E,Z)-4a-d, and (E,Z,Z)-7a,b are described. Trienes containing enal or enone dienophiles cyclize in excellent yield under mild conditions using substoichiometric amounts of MeAlCl(2), in most cases with high levels of diastereoselectivity. The thermal IMDA reactions of 1a, 4a, and 7a require forcing conditions and proceed in low yield with reversed stereoselectivity in the cases of 1a and 4a.
Subject(s)
Acids, Acyclic/chemistry , Indicators and Reagents , Acids, Acyclic/chemical synthesis , Catalysis , Kinetics , Models, Molecular , Molecular Conformation , Molecular Structure , StereoisomerismABSTRACT
A synthesis of the C(29)-C(45) bis-pyran subunit 2 of spongistatin 1 (1a) is described. The synthesis proceeds in 19 steps from the chiral aldehyde ent-7, and features highly diastereoselective alpha-alkoxyallylation reactions using the gamma-alkoxy substituted allylstannanes 17 and 19, as well as a thermodynamically controlled intramolecular Michael addition to close the F-ring pyran. The E ring was assembled via the Mukaiyama aldol reaction of F-ring methyl ketone 3 and the 2,3-syn aldehyde 4.
Subject(s)
Ethers, Cyclic/chemistry , Lactones/chemistry , Macrolides , Pyrans/chemical synthesis , Pyrans/chemistryABSTRACT
[reaction: see text]Thallium(I) ethoxide promotes Suzuki cross couplings for a range of vinyl- and arylboronic acids with vinyl and aryl halide partners in good to excellent yields. This reagent offers distinct advantages over thallium(I) hydroxide in terms of commercial availability, stability, and ease of use.
Subject(s)
Organometallic Compounds/chemistry , Boronic Acids/chemical synthesis , Indicators and Reagents , Solvents , Vinyl Compounds/chemical synthesisABSTRACT
BACKGROUND: Cysteine proteases of the papain superfamily are present in nearly all groups of eukaryotes and play vital roles in a wide range of biological processes and diseases, including antigen and hormone processing, bacterial infection, arthritis, osteoporosis, Alzheimer's disease and cancer-cell invasion. Because they are critical to the life-cycle progression of many pathogenic protozoa, they represent potential targets for selective inhibitors. Chagas' disease, the leading cause of death due to heart disease in Latin American countries, is transmitted by Trypanosoma cruzi. Cruzain is the major cysteine protease of T cruzi and has been the target of extensive structure-based drug design. RESULTS: High-resolution crystal structures of cruzain bound to a series of potent phenyl-containing vinyl-sulfone, sulfonate and sulfonamide inhibitors have been determined. The structures show a consistent mode of interaction for this family of inhibitors based on a covalent Michael addition formed at the enzyme's active-site cysteine, hydrophobic interactions in the S2 substrate-binding pocket and a strong constellation of hydrogen bonding in the S1' region. CONCLUSIONS: The series of vinyl-sulfone-based inhibitors examined in complex with cruzain was designed to probe recognition and binding potential of an aromatic-rich region of the enzyme. Analysis of the interactions formed shows that aromatic interactions play a less significant role, whereas the strength and importance of hydrogen bonding in the conformation adopted by the inhibitor upon binding to the enzyme was highlighted. A derivative of one inhibitor examined is currently under development as a therapeutic agent against Chagas' disease.
Subject(s)
Chagas Disease/enzymology , Cysteine Endopeptidases/chemistry , Protein Conformation , Protozoan Proteins/chemistry , Trypanosoma/chemistry , Animals , Binding Sites , Cysteine Endopeptidases/metabolism , Humans , Molecular Sequence Data , Protozoan Proteins/metabolism , Structure-Activity Relationship , Substrate Specificity , Trypanosoma/enzymologyABSTRACT
[reaction: see text] A highly convergent three-component coupling strategy for the stereocontrolled synthesis of 2,3,5-trisubstituted tetrahydrofurans is described. After allylboration of the first aldehyde with 1, the chiral, nonracemic allylsilanes 2 are coupled with a second aldehyde or ketone with Lewis acid catalysis to give tetrahydrofurans 3 or 4 with excellent selectivity. The 2,5-stereochemistry is controlled by operating under nonchelate (e.g., 3) or chelate (e.g., 4) conditions.
Subject(s)
Allyl Compounds/chemistry , Boronic Acids/chemistry , Furans/chemical synthesis , Organosilicon Compounds/chemistryABSTRACT
[formula: see text] The final stages of a total synthesis of mycalamide A are described. A key step is the aldol reaction (mismatched) of imide 4 and aldehyde 5 which provided a ca. 5:4 mixture of aldols 10a and 10b, with incorrect C(7) stereochemistry. Elaboration of the 10a-10b mixture to mycalamide A required epimerization of C(7) at the stage of beta-keto imide 11. Alternatively, Swern oxidation of the 10a-10b mixture under conditions that minimize C(7) epimerization led to 7-epi-mycalamide A selectively.
Subject(s)
Pyrans/chemical synthesis , Antineoplastic Agents/chemical synthesis , Antineoplastic Agents/chemistry , Indicators and Reagents , Marine Toxins/chemical synthesis , Marine Toxins/chemistry , Models, Molecular , Molecular Conformation , Pyrans/chemistryABSTRACT
[formula: see text] A highly diastereoselective synthesis of 3, corresponding to the C(5)-C(21) segment of tedanolide, has been accomplished by a route utilizing the aldol reaction of aldehyde 4 and the beta,gamma-unsaturated methyl ketone 5.
Subject(s)
Anti-Bacterial Agents/chemical synthesis , Antibiotics, Antineoplastic/chemical synthesis , Porifera/chemistry , Aldehydes/chemistry , Animals , Lactones/chemical synthesis , Macrolides , Molecular Conformation , Peptide Fragments/chemical synthesis , Peptide Fragments/chemistry , StereoisomerismABSTRACT
[formula: see text] 2-Deoxy-2-iodo- and 2-deoxy-2-bromoglucopyranosyl trichloroacetimidates 8-10 and 22 are extremely useful precursors of 2-deoxy-beta-glycosides. These reactive glycosyl donors undergo highly stereoselective glycosidation reactions at -78 degrees C with a range of glycosyl acceptors using TBS-OTf as the activating agent. beta-Glycosides are obtained with > or = 19:1 selectivity in six of the seven examples reported herein.
Subject(s)
Chloroacetates , Glycosides/chemical synthesis , Acetamides , Carbohydrate Sequence , Glycosides/chemistry , Molecular Sequence Data , Trichloroacetic Acid/chemistryABSTRACT
[formula: see text] The reactions of glycals with ceric(IV) ammonium nitrate and sodium iodide in the presence of acetic acid provides 2-deoxy-2-iodo-alpha-mannopyranosyl acetates with good stereoselectivity. In the majority of the cases examined, the selectivity was considerably better than that from reactions using N-iodosuccinimide and HOAc.
Subject(s)
Acetates/chemical synthesis , Cerium/chemistry , Glucose/chemistry , Mannose/chemistry , Sodium Iodide/chemistry , Acetates/chemistryABSTRACT
[formula: see text] TMS-OTf- or TBS-OTf-promoted glycosidation reactions of 2-deoxy-2-iodo-alpha-mannopyranosyl acetates 8-10 and the 2-deoxy-2-iodo-alpha-talopyranosyl acetate 11 provide the corresponding 2-deoxy-2-iodo-alpha-pyranosides, precursors to 2-deoxy-alpha-glycosides, as the only observed reaction products.
Subject(s)
Acetates/chemistry , Glycosides/chemical synthesis , Mannose/chemistry , Carbohydrate Sequence , Molecular Sequence Data , StereoisomerismABSTRACT
The inhibition of cysteine proteases is being studied as a strategy to combat parasitic diseases such as Chagas' disease, leishmaniasis, and malaria. Cruzain is the major cysteine protease of Trypanosoma cruzi, the etiologic agent of Chagas' disease. A crystal structure of cruzain, covalently inactivated by fluoromethyl ketone inhibitor 1 (Cbz-Phe-Ala-FMK), was used as a template to design potential inhibitors. Conformationally constrained gamma-lactams containing electrophilic aldehyde (12, 17, 18, 25, 26, and 29) or vinyl sulfone (43, 44, and 46) units were synthesized. Constrained lactam 26 had IC50 values of ca. 20 nM against the Leishmania major protease and ca. 50 nM versus falcipain, an important cysteine protease isolated from Plasmodium falciparum. However, all of the conformationally constrained inhibitors were weak inhibitors of cruzain, compared to unconstrained peptide aldehyde (e.g. 5 ) and vinyl sulfone inhibitors (e.g. 48, which proved to be an excellent inhibitor of cruzain with an apparent second order inhibition rate constant (k(inact)/Ki) of 634,000s(-1)M(-1). A significant reduction in activity was also observed with acyclic inhibitors 30 and 51 containing alpha-methyl phenylalanine residues at the P2 position. These data indicate that the pyrrolidinone ring, especially the quarternary center at P2, interferes with the normal substrate binding mode with cruzain, but not with falcipain or the leishmania protease.