Resolution and prevention of feline immunodeficiency virus-induced neurological deficits by treatment with the protease inhibitor TL-3

In vivo tests were performed to assess the influence of the protease inhibitor TL-3 on feline immunodeficiency virus (FIV)-induced central nervous system (CNS) deficits. Twenty cats were divided into four groups of five animals each. Group 1 received no treatment, group 2 received TL-3 only, group 3 received FIV strain PPR (FIV-PPR) only, and group 4 received FIV-PPR and TL-3. Animals were monitored for immunological and virological status, along with measurements of brain stem auditory evoked potential (BAEP) changes. Groups 1 and 2 remained FIV negative, and groups 3 and 4 became virus positive and seroconverted by 3 to 5 weeks postinoculation. No adverse effects were noted with TL-3 only. The average peak viral load for the virus-only group 3 animals was 1.32 x 10(6) RNA copies/ml, compared to 6.9 x 10(4) copies/ml for TL-3-treated group 4 cats. Group 3 (virus-only) cats exhibited marked progressive delays in BAEPs starting at 2 weeks post virus exposure, which is typical of infection with FIV-PPR. In contrast, TL-3-treated cats of group 4 exhibited BAEPs similar to those of control and drug-only cats. At 97 days postinfection, treatments were switched; i.e., group 4 animals were taken off TL-3 and group 3 animals were treated with TL-3. BAEPs in group 3 animals returned to control levels, while BAEPs in group 4 animals remained at control levels. After 70 days on TL-3, group 3 was removed from the drug treatment regimen. Delays in BAEPs immediately increased to levels observed prior to TL-3 treatment. The findings show that early TL-3 treatment can effectively eliminate FIV-induced changes in the CNS. Furthermore, TL-3 can counteract FIV effects on the CNS of infected cats, although continued treatment is required to maintain unimpaired CNS function.     

Non-active site changes elicit broad-based cross-resistance of the HIV-1 protease to inhibitors.

Three high level, cross-resistant variants of the HIV-1 protease have been analyzed for their ability to bind four protease inhibitors approved by the Food and Drug Administration (saquinavir, ritonavir, indinavir, and nelfinavir) as AIDS therapeutics. The loss in binding energy (DeltaDeltaG(b)) going from the wild-type enzyme to mutant enzymes ranges from 2.5 to 4.4 kcal/mol, 40-65% of which is attributed to amino acid substitutions away from the active site of the protease and not in direct contact with the inhibitor. The data suggest that non-active site changes are collectively a major contributor toward engendering resistance against the protease inhibitor and cannot be ignored when considering cross-resistance issues of drugs against the HIV-1 protease.     

Genotypic changes in human immunodeficiency virus type 1 protease associated with reduced susceptibility and virologic response to the protease inhibitor tipranavir

Tipranavir is a novel, nonpeptidic protease inhibitor of human immunodeficiency virus type 1 (HIV-1) with activity against clinical HIV-1 isolates from treatment-experienced patients. HIV-1 genotypic and phenotypic data from phase II and III clinical trials of tipranavir with protease inhibitor-experienced patients were analyzed to determine the association of protease mutations with reduced susceptibility and virologic response to tipranavir. Specific protease mutations were identified based on stepwise multiple-regression analyses of phase II study data sets. Validation included analyses of phase III study data sets to determine if the same mutations would be selected and to assess how these mutations contribute to multiple-regression models of tipranavir-related phenotype and of virologic response. A tipranavir mutation score was developed from these analyses, which consisted of a unique string of 16 protease positions and 21 mutations (10V, 13V, 20M/R/V, 33F, 35G, 36I, 43T, 46L, 47V, 54A/M/V, 58E, 69K, 74P, 82L/T, 83D, and 84V). HIV-1 isolates displaying an increasing number of these tipranavir resistance-associated mutations had a reduced phenotypic susceptibility and virologic response to tipranavir. Regression models for predicting virologic response in phase III trials revealed that each point in the tipranavir score was associated with a 0.16-log10 copies/ml-lower virologic response to tipranavir at week 24 of treatment. A lower number of points in the tipranavir score and a greater number of active drugs in the background regimen were predictive of virologic success. These analyses demonstrate that the tipranavir mutation score is a potentially valuable tool for predicting the virologic response to tipranavir in protease inhibitor-experienced patients.     

Molecular evolution of the wound-induced serine protease inhibitor wip1 in Zea and related genera

Plant defense mechanisms have been the subject of intensive investigation. However, little is known about their long-term evolutionary dynamics. We investigated the molecular diversity of a wound-induced serine protease inhibitor, wip1, in the genus Zea, as well as the divergence of wip1 among four genera, Zea, Tripsacum, Sorghum, and Oryza, in order to gain insight into the long-term evolution of plant defense. The specific objectives of this study were to determine (1) whether wip1 has a history of positive or balancing selection, as has been shown for genes involved in plant defense against pathogens, and (2) if the evolutionary histories of wip1 inhibitory loops, which come into closest contact with proteases, differ from the evolutionary history of other parts of this gene. The Zea polymorphism data are consistent with a neutral evolutionary history. In contrast, relative-rate tests suggest a nonneutral evolutionary history. This inconsistency may indicate that selection acting on wip1 is episodic or that wip1 evolves in response to selection favoring novel alleles. We also detected significant heterogeneity in the evolutionary rates of the two inhibitory loops of wip1-one inhibitory loop is highly conserved, whereas the second has diverged rapidly. Because these two inhibitory loops are predicted to have very similar biochemical functions, the significantly different evolutionary histories suggest that these loops have different ecological functions.     

Tigutcystatin, a cysteine protease inhibitor from Triatoma infestans midgut expressed in response to Trypanosoma cruzi

The insect Triatoma infestans is a vector of Trypanosoma cruzi, the etiological agent of Chagas disease. A cDNA library was constructed from T. infestans anterior midgut, and 244 clones were sequenced. Among the EST sequences, an open reading frame (ORF) with homology to a cystatin type 2 precursor was identified. Then, a 288-bp cDNA fragment encoding mature cystatin (lacking signal peptide) named Tigutcystatin was cloned fused to a N-terminal His tag in pET-14b vector, and the protein expressed in Escherichia coli strain Rosetta gami. Tigutcystatin purified and cleaved by thrombin to remove His tag presented molecular mass of 11 kDa and 10,137 Da by SDS–PAGE and MALDI-TOF mass spectrometry, respectively. Purified Tigutcystatin was shown to be a tight inhibitor towards cruzain, a T. cruzi cathepsin L-like enzyme (K_i = 3.29 nM) and human cathepsin L (K_i = 3.78 nM). Tissue specific expression analysis showed that Tigutcystatin was mostly expressed in anterior midgut, although amplification in small intestine was also detected by semi quantitative RT-PCR. qReal time PCR confirmed that Tigutcystatin mRNA is significantly up-regulated in anterior midgut when T. infestans is infected with T. cruzi. Together, these results indicate that Tigutcystatin may be involved in modulation of T. cruzi in intestinal tract by inhibiting parasite cysteine proteases, which represent the virulence factors of this protozoan.     

The staphostatin family of cysteine protease inhibitors in the genus Staphylococcus as an example of parallel evolution of protease and inhibitor specificity

Staphostatins constitute a family of staphylococcal cysteine protease inhibitors sharing a lipocalin-like fold and a unique mechanism of action. Each of these cytoplasmic proteins is co-expressed from one operon, together with a corresponding target extracellular cysteine protease (staphopain). To cast more light on staphostatin/staphopain interaction and the evolution of the encoding operons, we have cloned and characterized a staphopain (StpA2aur CH-91) and its inhibitor (StpinA2aur CH-91) from a novel staphylococcal thiol protease operon (stpAB2CH-91) identified in S. aureus strain CH-91. Furthermore, we have expressed a staphostatin from Staphylococcus warneri (StpinBwar) and characterized its target protease (StpBwar). Analysis of the reciprocal interactions among novel and previously described members of the staphostatin and staphopain families demonstrates that the co-transcribed protease is the primary target for each staphostatin. Nevertheless, the inhibitor derived from one species of Staphylococcus can inhibit the staphopain from another species, although the Ki values are generally higher and inhibition only occurs if both proteins belong to the same subgroup of either S. aureus staphopain A/staphostatin A (alpha group) or staphopain B/staphostatin B (beta group) orthologs. This indicates that both subgroups arose in a single event of ancestral allelic duplication, followed by parallel evolution of the protease/inhibitor pairs. The tight coevolution is likely the result of the known deleterious effects of uncontrolled staphopain action.     

Secretory leukocyte protease inhibitor (SLPI), a multifunctional protein in the host defense response

Secretory leukocyte protease inhibitor (SLPI), a ∼12 kDa nonglycosylated cationic protein, is emerging as an important regulator of innate and adaptive immunity and as a component of tissue regenerative programs. First described as an inhibitor of serine proteases such as neutrophil elastase, this protein is increasingly recognized as a molecule that benefits the host via its anti-proteolytic, anti-microbial and immunomodulatory activities. Here, we discuss the diverse functions of SLPI. Moreover, we review several novel layers of SLPI-mediated control that protect the host from excessive/dysregulated inflammation typical of infectious, allergic and autoinflammatory diseases and that support healing responses through affecting cell proliferation, differentiation and apoptosis.     

Proteolysis of the calcium-dependent protease inhibitor by myocardial calcium-dependent protease

Bovine heart peak II calcium-dependent protease was capable of hydrolyzing its specific inhibitor protein at high molar ratios of protease to inhibitor. The proteolysis was inhibited by leupeptin and required millimolar calcium. Thus, it appeared to be attributable to the calcium-dependent protease and not to possible contaminating proteases in the purified preparations of inhibitor or calcium-dependent protease. Incubation of the purified inhibitor with the calcium-dependent protease produced a discrete pattern of inhibitor fragments on Western blots developed with an inhibitor-specific monoclonal antibody. Traces of similar or identical lower molecular weight immunoreactive material could be observed in Western blots of bovine heart extracts, and the immunoreactivity present as these lower molecular weight forms could be increased by incubation of the extracts with calcium ion. These results suggest that the inhibitor can be proteolyzed to low molecular weight forms which can be detected in cardiac tissue extracts, and that calcium-dependent protease(s) may be responsible for this phenomenon.     

A loop-mimetic inhibitor of the HCV-NS3 protease derived from a minibody

We have been interested for some time in establishing a strategy for deriving lead compounds from macromolecule ligands such as minibody variants. A minibody is a minimized antibody variable domain whose two loops are amenable to combinatorial mutagenesis. This approach can be especially useful when dealing with 'difficult' targets. One such target is the NS3 protease of hepatitis C virus (HCV), a human pathogen that is believed to infect about 100 million individuals worldwide and for which an effective therapy is not yet available. Based on known inhibitor specificity (residues P6-P1) of NS3 protease, we screened a number of minibodies from our collection and we were able to identify a competitive inhibitor of this enzyme. We thus validated an aspect of recognition by HCV NS3 protease, namely that an acid anchor is necessary for inhibitor activity. In addition, the characterization of the minibody inhibitor led to the synthesis of a constrained hexapeptide mimicking the bioactive loop of the parent macromolecule. The cyclic peptide is a lead compound prone to rapid optimization through solid phase combinatorial chemistry. We therefore confirmed that the potential of turning a protein ligand into a low molecular weight active compound for lead discovery is achievable and can complement more traditional drug discovery approaches.     

A derivative of the plasma protease inhibitor alpha(2)-macroglobulin regulates the response to peripheral nerve injury

Peripheral nerve injury induces endoneural inflammation, controlled by diverse cytokines and extracellular mediators. Although inflammation is coupled to axonal regeneration, fulminant inflammation may increase nerve damage and neuropathic pain. alpha(2)-Macroglobulin (alpha2M) is a plasma protease inhibitor, cytokine carrier, and ligand for cell-signaling receptors, which exists in two well-characterized conformations and in less well-characterized intermediate states. Previously, we generated an alpha2M derivative (alpha(2)-macroglobulin activated for cytokine binding; MAC) similar in structure to alpha(2)M conformational intermediates, which binds tumor necrosis factor-alpha (TNF-alpha) and interleukin-1beta (IL-1beta), and inhibits endotoxin toxicity. In this study, we report that the continuum of cytokines that bind to MAC includes IL-6 and IL-18. MAC inhibited TNF-alpha-induced p38 mitogen-activated protein kinase activation and cell death in cultured Schwann cells. When administered by i.p. injection to mice with sciatic nerve crush injury, MAC decreased inflammation and preserved axons. Macrophage infiltration and TNF-alpha expression also are decreased. MAC inhibited TNF-alpha expression in the chronic constriction injury model of nerve injury. When MAC was prepared using a mutated recombinant alpha2M, which does not bind to the alpha2M receptor, low-density lipoprotein receptor-related protein-1, activity in the chronic constriction injury model was blocked. These studies demonstrate that an alpha2M derivative is capable of regulating the response to peripheral nerve injury by a mechanism that requires low-density lipoprotein receptor-related protein-1.     

Effect of E-64, thiol protease inhibitor, on the secondary anti-SRBC response in vitro

E-64, L-trans-epoxysuccinyl- leucylamido (4-guanidino) butane, a specific inhibitor of thiol proteases originally isolated from the culture of a fungus, was examined in connection with the immune responses to the splenocytes of mice. In cultures of C3H/He mouse splenocytes, E-64 and its analogues showed mitogenic activity, and some of them enhanced the lymphocyte blast transformation induced by a suboptimal concentration of concanavalin A. E-64 caused a significant suppressive effect on the secondary anti-SRBC responses when 7- or 14-day-primed BDF1 mouse splenocytes were cultured with SRBC, while it induced no effect on cultured splenocytes either from mice treated with cyclophosphamide, from mice sensitized with dinitrophenyl-Ficoll. The results with E-64 and its close analogues revealed that their effects on the immune response roughly correlated with their inhibitory activity against thiol protease. These results suggest that a thiol protease might be involved in the process of secondary immune response in mouse splenocytes.     

Ddi1, a eukaryotic protein with the retroviral protease fold

Retroviral aspartyl proteases are homodimeric, whereas eukaryotic aspartyl proteases tend to be large, monomeric enzymes with 2-fold internal symmetry. It has been proposed that contemporary monomeric aspartyl proteases evolved by gene duplication and fusion from a primordial homodimeric enzyme. Recent sequence analyses have suggested that such "fossil" dimeric aspartyl proteases are still encoded in the eukaryotic genome. We present evidence for retention of a dimeric aspartyl protease in eukaryotes. The X-ray crystal structure of a domain of the Saccharomyces cerevisiae protein Ddi1 shows that it is a dimer with a fold similar to that of the retroviral proteases. Furthermore, the double Asp-Thr-Gly-Ala amino acid sequence motif at the active site of HIV protease is found with identical geometry in the Ddi1 structure. However, the putative substrate binding groove is wider in Ddi1 than in the retroviral proteases, suggesting that Ddi1 accommodates bulkier substrates. Ddi1 belongs to a family of proteins known as the ubiquitin receptors, which have in common the ability to bind ubiquitinated substrates and the proteasome. Ubiquitin receptors contain an amino-terminal ubiquitin-like (UBL) domain and a carboxy-terminal ubiquitin-associated (UBA) domain, but Ddi1 is the only representative in which the UBL and UBA domains flank an aspartyl protease-like domain. The remarkable structural similarity between the central domain of Ddi1 and the retroviral proteases, in the global fold and in active-site detail, suggests that Ddi1 functions proteolytically during regulated protein turnover in the cell.     

The role of the S-S bridge in retroviral protease function and virion maturation

Retroviral proteases are translated as a part of Gag-related polyproteins, and are released and activated during particle release. Mason-Pfizer monkey virus (M-PMV) Gag polyproteins assemble into immature capsids within the cytoplasm of the host cells; however, their processing occurs only after transport to the plasma membrane and subsequent release. Thus, the activity of M-PMV protease is expected to be highly regulated during the replication cycle. It has been proposed that reversible oxidation of protease cysteine residues might be responsible for such regulation. We show that cysteine residues in M-PMV protease can form an intramolecular S-S bridge. The disulfide bridge shifts the monomer/dimer equilibrium in favor of the dimer, and increases the proteolytic activity significantly. To investigate the role of this disulfide bridge in virus maturation and replication, we engineered an M-PMV clone in which both protease cysteine residues were replaced by alanine (M-PMV(PRC7A/C106A)). Surprisingly, the cysteine residues were dispensable for Gag polyprotein processing within the virus, indicating that even low levels of protease activity are sufficient for polyprotein processing during maturation. However, the long-term infectivity of M-PMV(PRC7A/C106A) was noticeably compromised. These results show clearly that the proposed redox mechanism does not rely solely on the formation of the stabilizing S-S bridge in the protease. Thus, in addition to the protease disulfide bridge, reversible oxidation of cysteine and/or methionine residues in other domains of the Gag polyprotein or in related cellular proteins must be involved in the regulation of maturation.     

Synthesis and evaluation of phenylisoserine derivatives for the SARS-CoV 3CL protease inhibitor

Synthesis and evaluation of new scaffold phenylisoserine derivatives connected with the essential functional groups against SARS CoV 3CL protease are described. The phenylisoserine backbone was found by simulation on GOLD software and the structure activity relationship study of phenylisoserine derivatives gave SK80 with an IC₅₀ value of 43 μM against SARS CoV 3CL R188I mutant protease.