Protease Resistance Research Articles

Virological efficacy of PI monotherapy for HIV-1 in clinical practice.

BackgroundClinical trials of PI monotherapy indicate that most participants maintain viral suppression and emergent protease resistance is rare. However, outcomes among patients receiving PI monotherapy for clinical reasons, such as toxicity or adherence issues, are less well studied.MethodsAn observational study of patients attending an HIV treatment centre in London, UK, who had received PI monotherapy between 2004 and 2013, was conducted using prospectively collected clinical data and genotypic resistance reports. Survival analysis techniques were used to examine the times to virological failure and treatment discontinuation.ResultsNinety-five patients had PI monotherapy treatment for a median duration of 126 weeks. Virological failure occurred during 64% of episodes and 8% of patients developed emergent protease mutations. We estimate failure occurs in half of episodes within 2 years following initiation. Where PI monotherapy was continued following virological failure, 68% of patients achieved viral re-suppression. Despite a high incidence of virological failure, many patients continued PI monotherapy and 79% of episodes were ongoing at the end of the study. The type of PI used, the presence of baseline protease mutations and the plasma HIV RNA at initiation did not have a significant impact on treatment outcomes.ConclusionsThere was a higher incidence of virological failure and emerging resistance in our UK clinical setting than described in PI monotherapy clinical trials and other European observational studies. Despite this, many patients continued PI monotherapy and regained viral suppression, indicating this strategy remains a viable option in certain individuals following careful clinical evaluation.

A Helix-Stabilizing Linker Improves Subcutaneous Bioavailability of a Helical Peptide Independent of Linker Lipophilicity.

Stabilized peptides address several limitations to peptide-based imaging agents and therapeutics such as poor stability and low affinity due to conformational flexibility. There is also active research in developing these compounds for intracellular drug targeting, and significant efforts have been invested to determine the effects of helix stabilization on intracellular delivery. However, much less is known about the impact on other pharmacokinetic parameters such as plasma clearance and bioavailability. We investigated the effect of different fluorescent helix-stabilizing linkers with varying lipophilicity on subcutaneous (sc) bioavailability using the glucagon-like peptide-1 (GLP-1) receptor ligand exendin as a model system. The stabilized peptides showed significantly higher protease resistance and increased bioavailability independent of linker hydrophilicity, and all subcutaneously delivered conjugates were able to successfully target the islets of Langerhans with high specificity. The lipophilic peptide variants had slower absorption and plasma clearance than their respective hydrophilic conjugates, and the absolute bioavailability was also lower likely due to the longer residence times in the skin. Their ease and efficiency make double-click helix stabilization chemistries a useful tool for increasing the bioavailability of peptide therapeutics, many of which suffer from rapid in vivo protease degradation. Helix stabilization using linkers of varying lipophilicity can further control sc absorption and clearance rates to customize plasma pharmacokinetics.

FcRn binding is not sufficient for achieving systemic therapeutic levels of immunoglobulin G after oral delivery of enteric-coated capsules in cynomolgus macaques.

Although much speculation has surrounded intestinally expressed FcRn as a means for systemic uptake of orally administered immunoglobulin G (IgG), this has not been validated in translational models beyond neonates or in FcRn‐expressing cells in vitro. Recently, IgG1 intestinal infusion acutely in anesthetized cynomolgus resulted in detectable serum monoclonal antibody (mAb) levels. In this study, we show that IgG2 has greater protease resistance to intestinal enzymes in vitro and mice in vivo, due to protease resistance in the hinge region. An IgG2 mAb engineered for FcRn binding, was optimally formulated, lyophilized, and loaded into enteric‐coated capsules for oral dosing in cynomolgus. Small intestinal pH 7.5 was selected for enteric delivery based on gastrointestinal pH profiling of cynomolgus by operator‐assisted IntelliCap System®. Milling of the lyophilized IgG2 M428L FcRn‐binding variant after formulation in 10 mmol/L histidine, pH 5.7, 8.5% sucrose, 0.04% PS80 did not alter the physicochemical properties nor the molecular integrity compared to the batch released in PBS. Size 3 hard gel capsules (23.2 mg IgG2 M428L ~3 mg/kg) were coated with hydroxypropyl methylcellulose acetate succinate for rapid dissolution at pH 7.5 in small intestine and FcRn binding of encapsulated mAb confirmed. Initial capsule dosing by endoscopic delivery into the small intestine achieved 0.2 + 0.1 ng/mL (n = 5) peak at 24 h. Weekly oral capsule dosing for 6 weeks achieved levels of 0.4 + 0.2 ng/mL and, despite increasing the dose and frequency, remained below 1 ng/mL. In conclusion, lyophilized milled mAb retains FcRn binding and molecular integrity for small intestinal delivery. The low systemic exposure has demonstrated the limitations of intestinal FcRn in non‐human primates and the unfeasibility of employing this for therapeutic levels of mAb. Local mAb delivery with limited systemic exposure may be sufficient as a therapeutic for intestinal diseases.

Efficient Absorption of X-Hydroxyproline (Hyp)-Gly after Oral Administration of a Novel Gelatin Hydrolysate Prepared Using Ginger Protease.

Recent studies have reported that oral intake of gelatin hydrolysate has various beneficial effects, such as reduction of joint pain and lowering of blood sugar levels. In this study, we produced a novel gelatin hydrolysate using a cysteine-type ginger protease having unique substrate specificity with preferential peptide cleavage with Pro at the P2 position. Substantial amounts of X-hydroxyproline (Hyp)-Gly-type tripeptides were generated up to 2.5% (w/w) concomitantly with Gly-Pro-Y-type tripeptides (5%; w/w) using ginger powder. The in vivo absorption of the ginger-degraded gelatin hydrolysate was estimated using mice. The plasma levels of collagen-derived oligopeptides, especially X-Hyp-Gly, were significantly high (e.g., 2.3-fold for Glu-Hyp-Gly, p < 0.05) compared with those of the control gelatin hydrolysate, which was prepared using gastrointestinal proteases and did not contain detectable X-Hyp-Gly. This study demonstrated that orally administered X-Hyp-Gly was effectively absorbed into the blood, probably due to the high protease resistance of this type of tripeptide.

Protease-resistant modified human β-hexosaminidase B ameliorates symptoms in GM2 gangliosidosis model.

GM2 gangliosidoses, including Tay-Sachs and Sandhoff diseases, are neurodegenerative lysosomal storage diseases that are caused by deficiency of β-hexosaminidase A, which comprises an αβ heterodimer. There are no effective treatments for these diseases; however, various strategies aimed at restoring β-hexosaminidase A have been explored. Here, we produced a modified human hexosaminidase subunit β (HexB), which we have termed mod2B, composed of homodimeric β subunits that contain amino acid sequences from the α subunit that confer GM2 ganglioside-degrading activity and protease resistance. We also developed fluorescent probes that allow visualization of endocytosis of mod2B via mannose 6-phosphate receptors and delivery of mod2B to lysosomes in GM2 gangliosidosis models. In addition, we applied imaging mass spectrometry to monitor efficacy of this approach in Sandhoff disease model mice. Following i.c.v. administration, mod2B was widely distributed and reduced accumulation of GM2, asialo-GM2, and bis(monoacylglycero)phosphate in brain regions including the hypothalamus, hippocampus, and cerebellum. Moreover, mod2B administration markedly improved motor dysfunction and a prolonged lifespan in Sandhoff disease mice. Together, the results of our study indicate that mod2B has potential for intracerebrospinal fluid enzyme replacement therapy and should be further explored as a gene therapy for GM2 gangliosidoses.

Structural Design of Oligopeptides for Intestinal Transport Model

Glycyl-sarcosine (Gly-Sar) is a well-known model substrate for the intestinal uptake of dipeptides through peptide transporter 1 (PepT1). However, there are no other model peptides larger than tripeptides to evaluate their intestinal transport ability. In this study, we designed new oligopeptides based on the Gly-Sar structure in terms of protease resistance. Gly-Sar-Sar was found to be an appropriate transport model for tripeptides because it does not degrade during the transport across the rat intestinal membrane, while Gly-Gly-Sar was degraded to Gly-Sar during the 60 min transport. Caco-2 cell transport experiments revealed that the designed oligopeptides based on Gly-Sar-Sar showed a significantly (p < 0.05) lower transport ability by factors of 1/10-, 1/25-, and 1/40-fold for Gly-Sar-Sar, Gly-Sar-Sar-Sar, and Gly-Sar-Sar-Sar-Sar, respectively, compared to Gly-Sar (apparent permeability coefficient: 38.6 ± 11.4 cm/s). Cell experiments also showed that the designed tripeptide and Gly-Sar were transported across Caco-2 cell via PepT1, whereas the tetra- and pentapeptides were transported through the paracellular tight-junction pathway.

Effectiveness of Ritonavir-Boosted Protease Inhibitor Monotherapy in Clinical Practice Even with Previous Virological Failures to Protease Inhibitor-Based Regimens.

Background and ObjectiveSignificant controversy still exists about ritonavir-boosted protease inhibitor monotherapy (mtPI/rtv) as a simplification strategy that is used up to now to treat patients that have not experienced previous virological failure (VF) while on protease inhibitor (PI) -based regimens. We have evaluated the effectiveness of two mtPI/rtv regimens in an actual clinical practice setting, including patients that had experienced previous VF with PI-based regimens.MethodsThis retrospective study analyzed 1060 HIV-infected patients with undetectable viremia that were switched to lopinavir/ritonavir or darunavir/ritonavir monotherapy. In cases in which the patient had previously experienced VF while on a PI-based regimen, the lack of major HIV protease resistance mutations to lopinavir or darunavir, respectively, was mandatory. The primary endpoint of this study was the percentage of participants with virological suppression after 96 weeks according to intention-to-treat analysis (non-complete/missing = failure).ResultsA total of 1060 patients were analyzed, including 205 with previous VF while on PI-based regimens, 90 of whom were on complex therapies due to extensive resistance. The rates of treatment effectiveness (intention-to-treat analysis) and virological efficacy (on-treatment analysis) at week 96 were 79.3% (CI95, 76.8−81.8) and 91.5% (CI95, 89.6–93.4), respectively. No relationships were found between VF and earlier VF while on PI-based regimens, the presence of major or minor protease resistance mutations, the previous time on viral suppression, CD4+ T-cell nadir, and HCV-coinfection. Genotypic resistance tests were available in 49 out of the 74 patients with VFs and only four patients presented new major protease resistance mutations.ConclusionSwitching to mtPI/rtv achieves sustained virological control in most patients, even in those with previous VF on PI-based regimens as long as no major resistance mutations are present for the administered drug.

Contribution of Gag and Protease to HIV-1 Phenotypic Drug Resistance in Pediatric Patients Failing Protease Inhibitor-Based Therapy.

Protease inhibitors (PIs) are used as a first-line regimen in HIV-1-infected children. Here we investigated the phenotypic consequences of amino acid changes in Gag and protease on lopinavir (LPV) and ritonavir (RTV) susceptibility among pediatric patients failing PI therapy. The Gag-protease from isolates from 20 HIV-1 subtype C-infected pediatric patients failing an LPV and/or RTV-based regimen was phenotyped using a nonreplicativein vitroassay. Changes in sensitivity to LPV and RTV relative to that of the matched baseline (pretherapy) sample were calculated. Gag and protease amino acid substitutions associated with PI failure were created in a reference clone by site-directed mutagenesis and assessed. Predicted phenotypes were determined using the Stanford drug resistance algorithm. Phenotypic resistance or reduced susceptibility to RTV and/or LPV was observed in isolates from 10 (50%) patients, all of whom had been treated with RTV. In most cases, this was associated with protease resistance mutations, but substitutions at Gag cleavage and noncleavage sites were also detected. Gag amino acid substitutions were also found in isolates from three patients with reduced drug susceptibilities who had wild-type protease. Site-directed mutagenesis confirmed that some amino acid changes in Gag contributed to PI resistance but only in the presence of major protease resistance-associated substitutions. The isolates from all patients who received LPV exclusively were phenotypically susceptible. Baseline isolates from the 20 patients showed a large (47-fold) range in the 50% effective concentration of LPV, which accounted for most of the discordance seen between the experimentally determined and the predicted phenotypes. Overall, the inclusion of thegaggene and the use of matched baseline samples provided a more comprehensive assessment of the effect of PI-induced amino acid changes on PI resistance. The lack of phenotypic resistance to LPV supports the continued use of this drug in pediatric patients.

Synonymous Codons Direct Cotranslational Folding toward Different Protein Conformations

In all genomes, most amino acids are encoded by more than one codon. Synonymous codons can modulate protein production and folding, but the mechanism connecting codon usage to protein homeostasis is not known. Here we show that synonymous codon variants in the gene encoding gamma-B crystallin, a mammalian eye-lens protein, modulate the rates of translation and cotranslational folding of protein domains monitored in real time by Förster resonance energy transfer and fluorescence-intensity changes. Gamma-B crystallins produced from mRNAs with changed codon bias have the same amino acid sequence but attain different conformations, as indicated by altered invivo stability and invitro protease resistance. 2D NMR spectroscopic data suggest that structural differences are associated with different cysteine oxidation states of the purified proteins, providing a link between translation, folding, and the structures of isolated proteins. Thus, synonymous codons provide a secondary code for protein folding in the cell.

NS3 protease polymorphisms and genetic barrier to drug resistance of distinct hepatitis C virus genotypes from worldwide treatment‐naïve subjects

Hepatitis C virus (HCV) NS3 protease inhibitors have been primarily designed against genotype 1, the one with the lowest response to dual therapy. However, less evidence of their efficacy on non-1 genotypes is available, and any such information is mostly concentrated on genotypes 2-4. This study evaluated HCV protease resistance profiles in the major six HCV genotypes and identified genetic barrier (GB) profiles to each available protease inhibitor across HCV strains from different locations worldwide. We obtained 15099 HCV sequences from treatment-naïve subjects retrieved at the Los Alamos HCV Sequence Database. The wild-type codons of different HCV genotypes were used to analyse the smallest number of nucleotide substitution steps required for changing that codon to the closest one associated with drug resistance. The 36L and 175L RAVs were found as genetic signatures of genotypes 2-5, while the 80K RAV was found in all genotype 5 sequences. Genotypes 4 and 6 showed a higher GB to RAV mutations conferring resistance to telaprevir, while genotypes 2-5 presented baseline resistance to that drug, carrying the 36L mutation. Genotype 4 had a higher GB to simeprevir resistance, requiring three substitutions to acquire the 155K mutation. Subtype 1b showed a higher GB than subtype 1a to resistance for most PIs, with RAVs at codons 36 and 155. Geographic disparities were also found in frequencies of certain RAVs in genotypes 2 and 3. Under a scenario of unprecedented evolution of anti-HCV direct-acting agents, the genetic composition of the circulating HCV sequences should be evaluated worldwide to choose the most appropriate/feasible therapeutic schemes with the highest genetic barriers to resistance.

In-depth glycoproteomic characterisation of grape berry vacuolar invertase using a combination of mass spectrometry-based approaches

Vacuolar invertase is a key enzyme of sugar metabolism in grape berries. A full characterisation of this highly N-glycosylated protein is required to help understand its biological and biochemical significance in grapes. We have developed a mass spectrometry (MS)-based glycoproteomic approach wherein deglycosylated peptides are analysed by LC–MS/MS, while intact glycopeptides are characterised using a dedicated MS method to determine the attachment sites and micro-heterogeneity. For grape invertase, in parallel with deglycosylated peptides analysis, different enzymatic digestions were performed and glycopeptide detection was improved by enrichment method, nanoLC–MS and oxonium glycan ions. This MS-based glycoproteomic approach demonstrates that vacuolar invertase is glycosylated at all twelve potential N-glycosylation sites. Glycosylation is heterogeneous, with twelve glycoforms identified at six of the sites. The identification of several types of N-glycans is a major result to correlate with the surface and foaming properties of wine, the solubility, allergenicity, and protease resistance of wine proteins.

Serum Stability and Affinity Optimization of an M2 Macrophage-Targeting Peptide (M2pep).

Tumor associated macrophages (TAMs) are a major stromal component of the tumor microenvironment in several cancers. TAMs are a potential target for adjuvant cancer therapies due to their established roles in promoting proliferation of cancer cells, angiogenesis, and metastasis. We previously discovered an M2 macrophage-targeting peptide (M2pep) which was successfully used to target and deliver a pro-apoptotic KLA peptide to M2-like TAMs in a CT-26 colon carcinoma model. However, the effectiveness of in vivo TAM-targeting using M2pep is limited by its poor serum stability and low binding affinity. In this study, we synthesized M2pep derivatives with the goals of increasing serum stability and binding affinity. Serum stability evaluation of M2pepBiotin confirmed its rapid degradation attributed to exolytic cleavage from the N-terminus and endolytic cleavages at the W10/W11 and S16/K17 sites. N-terminal acetylation of M2pepBiotin protected the peptide against the exolytic degradation while W10w and K(17,18,19)k substitutions were able to effectively protect endolytic degradation at their respective cleavage sites. However, no tested amino acid changes at the W10 position resulted in both protease resistance at that site and retention of binding activity. Therefore, cyclization of M2pep was investigated. Cyclized M2pep better resisted serum degradation without compromising binding activity to M2 macrophages. During the serum stability optimization process, we also discovered that K9R and W10Y substitutions significantly enhanced binding affinity of M2pep. In an in vitro binding study of different M2pep analogs pre-incubated in mouse serum, cyclic M2pep with K9R and W10Y modifications (cyclic M2pep(RY)) retained the highest binding activity to M2 macrophages over time due to its improved serum stability. Finally, we evaluated the in vivo accumulation of sulfo-Cy5-labeled M2pep and cyclic M2pep(RY) in both the CT-26 and 4T1 breast carcinoma models. Cyclic M2pep(RY) outperformed M2pep in both tumor localization and selective accumulation in M2-like TAMs. In conclusion, we report cyclic M2pep(RY) as our lead M2pep analog with improved serum stability and M2 macrophage-binding activity. Its enhanced utility as an in vivo M2-like-TAM-targeting agent was demonstrated in two tumor models, and is expected to be applicable for other tumor models or in models of M2 macrophage-related diseases.

Covalent Label Transfer between Peroxisomal Importomer Components Reveals Export-driven Import Interactions

Peroxisomes are vital metabolic organelles found in almost all eukaryotic organisms, and they rely exclusively on import of their matrix protein content from the cytosol. In vitro import of proteins into isolated peroxisomal fractions has provided a wealth of knowledge on the import process. However, the common method of protease protection garnered no information on the import of an N-terminally truncated PEX5 (PEX5C) receptor construct or peroxisomal malate dehydrogenase 1 (pMDH1) cargo protein into sunflower peroxisomes because of high degrees of protease susceptibility or resistance, respectively. Here we present a means for analysis of in vitro import through a covalent biotin label transfer and employ this method to the import of PEX5C. Label transfer demonstrates that the PEX5C construct is monomeric under the conditions of the import assay. This technique was capable of identifying the PEX5-PEX14 interaction as the first interaction of the import process through competition experiments. Labeling of the peroxisomal protein import machinery by PEX5C demonstrated that this interaction was independent of added cargo protein, and, strikingly, the interaction between PEX5C and the import machinery was shown to be ATP-dependent. These important mechanistic insights highlight the power of label transfer in studying interactions, rather than proteins, of interest and demonstrate that this technique should be applied to future studies of peroxisomal in vitro import.

An Update on HIV-1 Protease Inhibitor Resistance

Protease inhibitors (PIs) are a potent class of antiretroviral agents which have been used to successfully treat many patients with HIV disease. As with other classes of antiretroviral agents, drug resistance has been described with each of the PIs. PI resistance can occur in newly infected individuals due to transmission of drug resistant strains or, more commonly, evolves from the selective pressure of antiretroviral therapy. When present, PI resistance results in an increased likelihood of drug failure. Individual protease gene mutations have been identified which are associated with phenotypic resistance to these agents. However, protease mutations can exert different effects on individual PIs, as some will produce resistance to one agent and may enhance susceptibility to another. In patients failing PIs, particularly in the setting of prior treatment with multiple agents, protease resistance patterns may be complex and result in broad cross-resistance to the class. Strategies to limit the development of resistance to these agents and manage its occurrence are important for the on-going care of individuals with HIV infection.

Highly efficient and easy protease-mediated protein purification.

As both research on and application of proteins are rarely focused on the resistance towards nonspecific proteases, this property remained widely unnoticed, in particular in terms of protein purification and related fields. In the present study, diverse aspects of protease-mediated protein purification (PMPP) were explored on the basis of the complementary proteases trypsin and proteinase K as well as the model proteins green fluorescent protein (GFP) from Aequorea victoria, lipase A from Candida antarctica (CAL-A), a transaminase from Aspergillus fumigatus (AspFum), quorum quenching lactonase AiiA from Bacillus sp., and an alanine dehydrogenase from Thermus thermophilus (AlaDH). While GFP and AiiA were already known to be protease resistant, the thermostable enzymes CAL-A, AspFum, and AlaDH were selected due to the documented correlation between thermostability and protease resistance. As proof of principle for PMPP, recombinant GFP remained unaffected whereas most Escherichia coli (E. coli) host proteins were degraded by trypsin. PMPP was highly advantageous compared to the widely used heat-mediated purification of commercial CAL-A. The resistance of AspFum towards trypsin was improved by rational protein design introducing point mutation R20Q. Trypsin also served as economical and efficient substitute for site-specific endopeptidases for the removal of a His-tag fused to AiiA. Moreover, proteolysis of host enzymes with interfering properties led to a strongly improved sensitivity and accuracy of the NADH assay in E. coli cell lysate for AlaDH activity measurements. Thus, PMPP is an attractive alternative to common protein purification methods and facilitates also enzyme characterization in cell lysate.

Bioengineering Lantibiotics for Therapeutic Success.

Several examples of highly modified antimicrobial peptides have been described. While many such peptides are non-ribosomally synthesized, ribosomally synthesized equivalents are being discovered with increased frequency. Of the latter group, the lantibiotics continue to attract most attention. In the present review, we discuss the implementation of in vivo and in vitro engineering systems to alter, and even enhance, the antimicrobial activity, antibacterial spectrum and physico-chemical properties, including heat stability, solubility, diffusion and protease resistance, of these compounds. Additionally, we discuss the potential applications of these lantibiotics for use as therapeutics.

Exploring the cause of initially reactive bovine brains on rapid tests for BSE

ABSTRACTBovine spongiform encephalopathy (BSE) is an invariably fatal prion disease of cattle. The identification of the zoonotic potential of BSE prompted safety officials to initiate surveillance testing for this disease. In Canada, BSE surveillance is primarily focused on high risk cattle including animals which are dead, down and unable to rise, diseased or distressed. This targeted surveillance results in the submission of brain samples with a wide range of tissue autolysis and associated contaminants. These contaminants have the potential to interfere with important steps of surveillance tests resulting in initially positive test results requiring additional testing to confirm the disease status of the animal.The current tests used for BSE screening in Canada utilize the relative protease resistance of the prion protein gained when it misfolds from PrPC to PrPSc as part of the disease process. Proteinase K completely digests PrPC in normal brains, but leaves most of the PrPSc in BSE positive brains intact which is detected using anti-prion antibodies. These tests are highly reliable but occasionally give rise to initially reactive/false positive results. Test results for these reactive samples were close to the positive/negative cut-off on a sub set of test platforms. This is in contrast to all of the previous Canadian positive samples whose numeric values on these same test platforms were 10 to 100 fold greater than the test positive/negative cut-off. Here we explore the potential reason why a sample is repeatedly positive on a sub-set of rapid surveillance tests, but negative on other test platforms.In order to better understand and identify what might cause these initial reactions, we have conducted a variety of rapid and confirmatory assays as well as bacterial isolation and identification on BSE positive, negative and initially reactive samples. We observed high levels of viable bacterial contamination in initially reactive samples suggesting that the reactivity may be related to bacterial factors. Several bacteria isolated from the initially reactive samples have characteristics of biofilm forming bacteria and this extracellular matrix might play a role in preventing complete digestion of PrPC in these samples.

Improving Properties of Recombinant SsoPox by Site-Specific Pegylation.

SsoPox, a ~35 kDa enzyme from Sulfolobus solfataricus, can hydrolyze and inactivate a variety of organophosphate (OP)-compounds. The enzyme is a potential candidate for the development of prophylactic and therapeutic agent against OP-poisoning in humans. However, the therapeutic use of recombinant SsoPox suffers from certain limitations associated with the use of recombinant protein pharmaceuticals. Some of these limitations could be overcome by conjugating SsoPox enzyme with polyethylene glycol (PEG). In this study, we report generation and in vitro characterization of N-terminal mono-PEGylated rSsoPox(2p) (a variant of rSsoPox(wt) having enhanced OP-hydrolyzing activity). The enzyme was PEGylated with mPEG-propionaldehyde and the PEGylated protein was isolated using ion-exchange chromatography. Compared with the unmodified enzyme, mono-PEGylation of rSsoPox results in improvement in the thermostability and protease resistance of the enzyme. PEGylated rSsoPox(2p) can be developed as a candidate for the prevention / treatment of OP-poisoning.

A cell-free enzymatic activity assay for the evaluation of HIV-1 drug resistance to protease inhibitors.

Due to their high frequency of genomic mutations, human retroviruses often develop resistance to antiretroviral drugs. The emergence of drug-resistant human immunodeficiency virus type 1 (HIV-1) is a significant obstacle to the effective long-term treatment of HIV infection. The development of a rapid and versatile drug-susceptibility assay would enable acquisition of phenotypic information and facilitate determination of the appropriate choice of antiretroviral agents. In this study, we developed a novel in vitro method, termed the Cell-free drug susceptibility assay (CFDSA), for monitoring phenotypic information regarding the drug resistance of HIV-1 protease (PR). The CFDSA utilizes a wheat germ cell-free protein production system to synthesize enzymatically active HIV-1 PRs directly from PCR products amplified from HIV-1 molecular clones or clinical isolates in a rapid one-step procedure. Enzymatic activity of PRs can be readily measured by AlphaScreen (Amplified Luminescent Proximity Homogeneous Assay Screen) in the presence or absence of clinically used protease inhibitors (PIs). CFDSA measurement of drug resistance was based on the fold resistance to the half-maximal inhibitory concentration (IC50) of various PIs. The CFDSA could serve as a non-infectious, rapid, accessible, and reliable alternative to infectious cell-based phenotypic assays for evaluation of PI-resistant HIV-1.

The Cystine Knot Is Responsible for the Exceptional Stability of the Insecticidal Spider Toxin ω-Hexatoxin-Hv1a.

The inhibitor cystine knot (ICK) is an unusual three-disulfide architecture in which one of the disulfide bonds bisects a loop formed by the two other disulfide bridges and the intervening sections of the protein backbone. Peptides containing an ICK motif are frequently considered to have high levels of thermal, chemical and enzymatic stability due to cross-bracing provided by the disulfide bonds. Experimental studies supporting this contention are rare, in particular for spider-venom toxins, which represent the largest diversity of ICK peptides. We used ω-hexatoxin-Hv1a (Hv1a), an insecticidal toxin from the deadly Australian funnel-web spider, as a model system to examine the contribution of the cystine knot to the stability of ICK peptides. We show that Hv1a is highly stable when subjected to temperatures up to 75 °C, pH values as low as 1, and various organic solvents. Moreover, Hv1a was highly resistant to digestion by proteinase K and when incubated in insect hemolymph and human plasma. We demonstrate that the ICK motif is essential for the remarkable stability of Hv1a, with the peptide’s stability being dramatically reduced when the disulfide bonds are eliminated. Thus, this study demonstrates that the ICK motif significantly enhances the chemical and thermal stability of spider-venom peptides and provides them with a high level of protease resistance. This study also provides guidance to the conditions under which Hv1a could be stored and deployed as a bioinsecticide.