Narrow substrate specificity and sensitivity toward ligand-binding site mutations of human T-cell Leukemia virus type 1 protease

Human T-cell leukemia virus type 1 (HTLV-1) is associated with a number of human diseases; therefore, its protease is a potential target for chemotherapy. To compare the specificity of HTLV-1 protease with that of human immunodeficiency virus type 1 (HIV-1) protease, oligopeptides representing naturally occurring cleavage sites in various retroviruses were tested. The number of hydrolyzed peptides as well as the specificity constants suggested a substantially broader specificity of the HIV protease. Amino acid residues of HTLV-1 protease substrate-binding sites were replaced by equivalent ones of HIV-1 protease. Most of the single and multiple mutants had altered specificity and a dramatically reduced folding and catalytic capability, suggesting that mutations are not well tolerated in HTLV-1 protease. The catalytically most efficient mutant was that with the flap residues of HIV-1 protease. The inhibition profile of the mutants was also determined for five inhibitors used in clinical practice and inhibitor analogs of HTLV-1 cleavage sites. Except for indinavir, the HIV-1 protease inhibitors did not inhibit wild type and most of the mutant HTLV-1 proteases. The wild type HTLV-1 protease was inhibited by the reduced peptide bond-containing substrate analogs, whereas the mutants showed various degrees of weakened binding capability. Most interesting, the enzyme with HIV-1-like residues in the flap region was the most sensitive to the HIV-1 protease inhibitors and least sensitive to the HTLV-1 protease inhibitors, indicating that the flap plays an important role in defining the specificity differences of retroviral proteases.     

Crystal structure of lysine sulfonamide inhibitor reveals the displacement of the conserved flap water molecule in human immunodeficiency virus type 1 protease

Human immunodeficiency virus type 1 (HIV-1) protease has been continuously evolving and developing resistance to all of the protease inhibitors. This requires the development of new inhibitors that bind to the protease in a novel fashion. Most of the inhibitors that are on the market are peptidomimetics, where a conserved water molecule mediates hydrogen bonding interactions between the inhibitors and the flaps of the protease. Recently a new class of inhibitors, lysine sulfonamides, was developed to combat the resistant variants of HIV protease. Here we report the crystal structure of a lysine sulfonamide. This inhibitor binds to the active site of HIV-1 protease in a novel manner, displacing the conserved water and making extensive hydrogen bonds with every region of the active site.     

Extensive post-translational processing of potato tuber storage proteins and vacuolar targeting

Potato tuber storage proteins were obtained from vacuoles isolated from field-grown starch potato tubers cv. Kuras. Vacuole sap proteins fractionated by gel filtration were studied by mass spectrometric analyses of trypsin and chymotrypsin digestions. The tuber vacuole appears to be a typical protein storage vacuole absent of proteolytic and glycolytic enzymes. The major soluble storage proteins included 28 Kunitz protease inhibitors, nine protease inhibitors 1, eight protease inhibitors 2, two carboxypeptidase inhibitors, eight patatins and five lipoxygenases (lox), which all showed cultivar-specific sequence variations. These proteins, except for lox, have typical endoplasmic reticulum (ER) signal peptides and putative vacuolar sorting determinants of either the sequence or structure specific type or the C-terminal type, or both. Unexpectedly, sap protein variants imported via the ER showed multiple molecular forms because of extensive and unspecific proteolytic cleavage of exposed N- and C-terminal propeptides and surface loops, in spite of the abundance of protease inhibitors. Some propeptides are potential novel vacuolar targeting peptides. In the insoluble vacuole fraction two variants of phytepsin (aspartate protease) were identified. These are most probably the processing enzymes of potato tuber vacuolar proteins. Database Proteome data have been submitted to the PRIDE database under accession number 17707.     

Locking the two ends of tetrapeptidic HTLV-I protease inhibitors inside the enzyme

Adult T-cell leukemia and tropical spastic paraparesis/HTLV-I-associated myelopathy are only some of the more common end results of an infection with a human T-cell leukemia virus type 1 (HTLV-I). Expanding from our previous reports, we synthesized all different permutations of tetrapeptidic HTLV-I protease inhibitors using at least eight P(3)-cap and five P(1)(')-cap moieties. The inhibitors exhibited over 97% inhibition against HIV-1 protease and a wide range of inhibitory activity against HTLV-I protease.     

Synthesis and activity of tetrapeptidic HTLV-I protease inhibitors possessing different P3-cap moieties

The causative agent behind adult T-cell leukemia and tropical spastic paraparesis/HTLV-I-associated myelopathy is the human T-cell leukemia virus type 1 (HTLV-I). Tetrapeptidic HTLV-I protease inhibitors were designed on a previously reported potent inhibitor KNI-10516, with modifications at the P(3)-cap moieties. All the inhibitors showed high HIV-1 protease inhibitory activity (over 98% inhibition at 50nM) and most exhibited highly potent inhibition against HTLV-I protease (IC(50) values were less than 100nM).     

High molecular mass type i.v. collagen-specific metalloprotease from human carcinoma tissue

A protease degrading type IV collagen was purified more than 8000-fold from human stomach carcinoma tissue. This protease degraded type IV collagen, while type I, II, III and V collagen, laminin, fibronectin, casein, albumin and hemoglobin were not affected. This enzyme had a pH optimum of pH 7.0-8.0 and was inhibited completely by EDTA and o-phenanthroline, but not by seryl, thiol and carboxyl protease inhibitors. Furthermore, the molecular mass of this enzyme was estimated to be 1 MDa by Sepharose 6B column and HPLC-gel filtration. The molecular mass and substrate specificity of this metalloprotease from human carcinoma tissue indicate it to be a new protease.     

Phenotypic hypersusceptibility to multiple protease inhibitors and low replicative capacity in patients who are chronically infected with human immunodeficiency virus type 1

Increased susceptibility to the protease inhibitors saquinavir and amprenavir has been observed in human immunodeficiency virus type 1 (HIV-1) with specific mutations in protease (V82T and N88S). Increased susceptibility to ritonavir has also been described in some viruses from antiretroviral agent-naive patients with primary HIV-1 infection in association with combinations of amino acid changes at polymorphic sites in the protease. Many of the viruses displaying increased susceptibility to protease inhibitors also had low replication capacity. In this retrospective study, we analyze the drug susceptibility phenotype and the replication capacity of virus isolates obtained at the peaks of viremia during five consecutive structured treatment interruptions in 12 chronically HIV-1-infected patients. Ten out of 12 patients had at least one sample with protease inhibitor hypersusceptibility (change 相似文献   

Design and synthesis of several small-size HTLV-I protease inhibitors with different hydrophilicity profiles

The human T cell leukemia/lymphotropic virus type 1 (HTLV-I) is clinically associated with adult T cell leukemia/lymphoma, HTLV-I associated myelopathy/tropical spastic paraparesis, and a number of other chronic inflammatory diseases. To stop the replication of the virus, we developed highly potent tetrapeptidic HTLV-I protease inhibitors. In a recent X-ray crystallography study, several of our inhibitors could not form co-crystal complexes with the protease due to their high hydrophobicity. In the current study, we designed, synthesized and evaluated the HTLV-I protease inhibition potency of compounds with hydrophilic end-capping moieties with the aim of improving pharmaceutic and pharmacokinetic properties.     

Seco-prolinenitrile inhibitors of dipeptidyl peptidase IV define minimal pharmacophore requirements at P1

A series of seco-prolinenitrile-containing dipeptides were synthesized and assayed as inhibitors of the N-terminal sequence-specific serine protease dipeptidyl peptidase IV, a promising new target for treatment of type 2 diabetes. The inhibitors described herein assess the minimum structural requirements at P1 for this enzyme, resulting in the identification of inhibitors with low nM potency.     

The binding energetics of first- and second-generation HIV-1 protease inhibitors: implications for drug design

KNI-764 is a powerful HIV-1 protease inhibitor with a reported low susceptibility to the effects of protease mutations commonly associated with drug resistance. In this paper the binding thermodynamics of KNI-764 to the wild-type and drug-resistant mutant V82F/I84V are presented and the results compared to those obtained with existing clinical inhibitors. KNI-764 binds to the wild-type HIV-1 protease with very high affinity (3.1 x 10(10) M(-1) or 32 pM) in a process strongly favored by both enthalpic and entropic contributions to the Gibbs energy of binding (Delta G = -RTlnK(a)). When compared to existing clinical inhibitors, the binding affinity of KNI-764 is about 100 fold higher than that of indinavir, saquinavir, and nelfinavir, but comparable to that of ritonavir. Unlike the existing clinical inhibitors, which bind to the protease with unfavorable or only slightly favorable enthalpy changes, the binding of KNI-764 is strongly exothermic (-7.6 kcal/mol). The resistant mutation V82F/I84V lowers the binding affinity of KNI-764 26-fold, which can be accounted almost entirely by a less favorable binding enthalpy to the mutant. Since KNI-764 binds to the wild type with extremely high affinity, even after a 26-fold decrease, it still binds to the resistant mutant with an affinity comparable to that of other inhibitors against the wild type. These results indicate that the effectiveness of this inhibitor against the resistant mutant is related to two factors: extremely high affinity against the wild type achieved by combining favorable enthalpic and entropic interactions, and a mild effect of the protease mutation due to the presence of flexible structural elements at critical locations in the inhibitor molecule. The conclusions derived from the HIV-1 protease provide important thermodynamic guidelines that can be implemented in general drug design strategies.     

Human Immunodeficiency Virus Type 1 Protease Genotypes and In Vitro Protease Inhibitor Susceptibilities of Isolates from Individuals Who Were Switched to Other Protease Inhibitors after Long-Term Saquinavir Treatment

An understanding of the mechanisms of virologic cross-resistance between human immunodeficiency virus type 1 protease inhibitors is important for the establishment of effective treatment strategies for patients who no longer respond to their initial protease inhibitor. Protease gene sequencing results from patients treated with saquinavir showed significant increases in the frequency of the G48V protease mutation in patients receiving higher doses of the drug. In addition, all six patients who developed the G48V mutation during saquinavir therapy developed the V82A mutation either on continued saquinavir or after a switch to nelfinavir or indinavir. In vitro susceptibility assays showed that all 13 isolates with reduced susceptibilities to two or more protease inhibitors had either the G48V or L90M mutation, along with an average of six other protease mutations. Reduced susceptibility to nelfinavir was found in 14 isolates, but only 1 possessed the D30N mutation. These results suggest that mutations selected in vivo by initial saquinavir therapy may provide more cross-resistance to the other protease inhibitors than has been previously reported.     

Identification and monitoring of protease activity in recombinant Saccharomyces cerevisiae

An assay for the detection of yeast (Saccharomyces cerevisiae) protease activity, using partially purified yeast-derived recombinant hepatitis B surface antigen (rHBsAg) as substrate, was developed to monitor proteolysis of rHBsAg that may occur through fermentation and isolation. The method consists of incubating small amounts of yeast lysate (protease source) with the substrate at 35 degrees C for about 16 h. Substrate proteolysis is assessed by subjecting the incubation mixtures to SDS-PAGE followed by silver-staining. The type of protease responsible for particular cleavages can be identified by treating the yeast lysates with specific protease inhibitors prior to incubation with substrate. The treatment of lysates with PMSF indicated that while many lysates possessed only serine protease activity (Protease B), some possessed proteolytic activity that could not be quenched with high levels of PMSF or other serine protease inhibitors. The use of the aspartyl protease inhibitor Pepstatin A in conjunction with PMSF virtually eliminated all proteolytic activity in these lysates, indicating that an aspartyl protease (Protease A) is expressed under some fermentation conditions. The relative amount of each protease in a lysate can be determined semiquantitatively by scanning the SDS gels densitometrically and plotting the ratio of degradates to intact antigen in the presence and absence of protease inhibitors. This method was used successfully to monitor the time-dependent expression of these proteases throughout production-scale fermentations. The impact of fermentation and purification changes on those proteases specifically responsible for the rHBsAg degradation can be easily evaluated.     

Novel arylsulfonamides possessing sub-picomolar HIV protease activities and potent anti-HIV activity against wild-type and drug-resistant viral strains

A novel series of P1' chain-extended arylsufonamides was synthesized and evaluated for wild-type HIV protease inhibitory activity and in vitro antiviral activity against wild type virus and two protease inhibitor-resistant mutant viruses. All of the compounds showed dramatic increases in enzyme activity as compared to the currently marketed HIV protease inhibitors amprenavir, indinavir, and nelfinavir. In addition, significant improvements in antiviral potencies against wild type and the two mutant viruses were also realized.     

The structure of Bowman-Birk type protease inhibitor A-II from peanut (Arachis hypogaea) at 3.3 A resolution

The structure of Bowman-Birk type protease inhibitor (A-II from peanut) is described at 3.3 A resolution. The molecules form a tetramer with 222 local symmetry in our crystals. Each monomer has an elongated shape with approximate dimensions of 45 X 15 X 15 A and consists of two distinct domains. The three-dimensional structures of the two domains are similar and are related by the intramolecular approximate twofold rotation axis. The two independent protease binding sites protrude from the molecular body on opposite sides. A scheme for the molecular evolution of the double-headed Bowman-Birk type protease inhibitors is proposed, based on the three-dimensional structure.     

Role of hydroxyl group and R/S configuration of isostere in binding properties of HIV-1 protease inhibitors.

The crystal structure of the complex between human immunodeficiency virus type 1 (HIV-1) protease and a peptidomimetic inhibitor of ethyleneamine type has been refined to R factor of 0.178 with diffraction limit 2.5 A. The peptidomimetic inhibitor Boc-Phe-Psi[CH2CH2NH]-Phe-Glu-Phe-NH2 (denoted here as OE) contains the ethyleneamine replacement of the scissile peptide bond. The inhibitor lacks the hydroxyl group which is believed to mimic tetrahedral transition state of proteolytic reaction and thus is suspected to be necessary for good properties of peptidomimetic HIV-1 protease inhibitors. Despite the missing hydroxyl group the inhibition constant of OE is 1.53 nm and it remains in the nanomolar range also towards several available mutants of HIV-1 protease. The inhibitor was found in the active site of protease in an extended conformation with a unique hydrogen bond pattern different from hydroxyethylene and hydroxyethylamine inhibitors. The isostere nitrogen forms a hydrogen bond to one catalytic aspartate only. The other aspartate forms two weak hydrogen bridges to the ethylene group of the isostere. A comparison with other inhibitors of this series containing isostere hydroxyl group in R or S configuration shows different ways of accommodation of inhibitor in the active site. Special attention is devoted to intermolecular contacts between neighbouring dimers responsible for mutual protein adhesion and for a special conformation of Met46 and Phe53 side chains not expected for free protein in water solution.     

Protease inhibitors in Bombyx mori silk might participate in protecting the pupating larva from microbial infection

Pupae inside cocoons rarely suffer from disease. It is apparent that some factors in the cocoon exert antimicrobial effects whereby the pupae inside can be protected from microbial infection. In the present study, we investigated the expression of cocoon protease inhibitors using immunoblotting and activity staining. Enzymatic hydrolysis of cocoon proteins in vitro was performed to characterize their roles in protecting the cocoon from microbial proteases. We found that some protease inhibitors, particularly trypsin inhibitor‐like (TIL)‐type protease inhibitors, can be secreted into the cocoon layer during the spinning process, thereby providing effective protection to the cocoon and pupa by inhibiting the extracellular proteases that can be secreted by pathogens.     

Interactions of ketoamide inhibitors on HCV NS3/4A protease target: molecular docking studies

HCV infection in more than 200 million individuals worldwide is a principal health problem. Prior to the development of HCV protease inhibitor combination therapy, HCV infected patients were treated with pegylated interferon-α and ribavirin. The adverse side effects associated with this type of treatment may lead to the discontinuation of treatment in certain number of patients. Currently, the inhibitors of NS3/4A Protease were found promising candidates for the treatment of HCV infection. There are several inhibitors of HCV NS3/4A protease that are passing through clinical improvement showing good potency against HCV infections in a number of patients. To further recognize binding interactions and activity trend, the molecular docking studies were performed on a number of HCV NS3/4A protease ketoamide inhibitors via MOE docking protocol. The docking analysis resulted in the detection of important ligand interactions with respect to binding site of target proteinand produced good correlation coefficient (r² = 0.690) between docking score and biological activities. These molecular docking results should, in our view, contribute for further optimization of ketoamide derivatives as NS3/4A protease inhibitors.     

Expression and enzymatic characterization of recombinant human kallikrein 14

Human kallikrein 14 (KLK14) is a member of the human kallikrein gene family of serine proteases, and its protein, hK14, has recently been suggested to serve as a new ovarian and breast cancer marker. To gain insights into hK14's physiological functions, the active recombinant enzyme was obtained in an enzymatically pure state for biochemical and enzymatic characterizations. We studied its substrate specificity and behavior to various protease inhibitors, and identified candidate physiological substrates. hK14 had trypsin-like activity with a strong preference for Arg over Lys in the P1 position, and its activity was inhibited by typical serine protease inhibitors. The protease degraded casein, fibronectin, gelatin, collagen type I, collagen type IV, fibrinogen, and high-molecular-weight kininogen. Furthermore, it rapidly hydrolyzed insulin-like growth factor binding protein-3 (IGFBP-3). These findings suggest that hK14 may be implicated in tumor progression in ovarian carcinoma.     

Synthesis and biological evaluation of novel small non-peptidic HIV-1 PIs: the benzothiophene ring as an effective moiety

Synthesis and biological evaluation of a new series of potential HIV-1 protease inhibitors incorporating different heterocycles are described. The variation of heteroatom in such molecules has displayed totally different biological activities and a benzothiophene containing inhibitor has shown high potency against wild type HIV-1 protease with IC(50)=60 nM, thanks to the lower desolvation penalty to be payed by such hydrophobic moiety.