Exploring Endolysin-Loaded Alginate-Chitosan Nanoparticles as Future Remedy for Staphylococcal Infections.

Endolysins are a novel class of antibacterials with proven efficacy in combating various bacterial infections, in vitro and in vivo. LysMR-5, an endolysin derived from phage MR-5, demonstrated high lytic activity in our laboratory against multidrug-resistant S. aureus (MRSA) and S. epidermidis strains. However, endolysin and proteins in general are associated with instability and short in vivo half-life, consequently limiting their usage as pharmaceutical preparation to treat bacterial infections. Nanoencapsulation of endolysins could help to achieve better therapeutic outcome, by protecting the proteins from degradation, providing sustained release, thus could increase their stability, shelf life, and therapeutic efficacy. Hence, in this study, the feasibility of alginate-chitosan nanoparticles (Alg-Chi NPs) to serve as drug delivery platform for LysMR-5 was evaluated. LysMR-5-loaded nanoparticles were prepared by calcium ion-induced pre-gelation of alginate core and its complexation with chitosan. The formation of nanoparticles was confirmed on the basis of DLS, zeta potential, and electron microscopy imaging. The LysMR-5-loaded nanoparticles presented a hydrodynamic diameter of 276.5 ± 42, a PDI of 0.342 ± 0.02, a zeta potential - 25 mV, and an entrapment efficiency of 62 ± 3.1%. The potential ionic interaction between alginate, chitosan, and LysMR-5 was investigated by FT-IR and SEM-EDX analysis. Using scanning electron microscopy (SEM) and transmission electron microscopy (TEM), nano-sized particles with characteristic morphology were seen. Different antibacterial assays and SDS-PAGE analysis showed no change in endolysin's structural integrity and bioactivity after entrapment. A direct antibacterial effect of blank Alg-Chi Nps, showing enhanced bactericidal activity upon LysMR-5 loading, was observed against S. aureus. At physiological pH (7.2), the release profile of LysMR-5 from Alg-Chi NPs showed a biphasic release and followed a non-Fickian release mechanism. The biocompatible nature as revealed by cytocompatibility and hemocompatibility studies endorsed their use as drug delivery system for in vivo studies. Collectively, these results demonstrate the potential of Alg-Chi NPs as nano-delivery vehicle for endolysin LysMR-5 and other therapeutic proteins for their use in various biomedical applications.

Targeted thrombolysis by using c-RGD-modified N,N,N-Trimethyl Chitosan nanoparticles loaded with lumbrokinase.

Lumbrokinase (LK) has strong fibrinolytic and thrombolytic activities, but it has a short half-life, can be easily inactivated, and may cause hemorrhage as a side effect. This study develops a potential thrombolytic therapy by fabricating N,N,N-Trimethyl Chitosan (TMC) nanoparticles modified with the cyclic Arg-Gly-Asp-Phe-Lys peptide (c-RGD) and loaded with LK (i.e. c-RGD-LK-NPs). The binding of c-RGD to platelet membrane GPIIb/IIIa receptors is expected to enable targeted delivery of the c-RGD-conjugated TMC to the thrombus. The synthesized c-RGD-LK-NPs had a mean particle size of 232.0 nm, zeta potential of 19.8 mV, entrapment efficiency of 52.7% ± 2.5%, and loading efficiency of 17.4% ± 0.65%. Transmission electron microscopy showed that they were generally spherical. The c-RGD-LK-NPs gave a cumulative in vitro LK release of 80.6% over 8 h, and the activity of LK was close to 80%, indicating that the nanoparticles protected the activity of LK. In vitro blood clot lysis assays were carried out and in vivo thrombolysis effect was tested in Sprague-Dawley rats carotid artery thrombus model. In all cases, the c-RGD-LK-NPs showed superior performance compared with the free LK and the unmodified TMC nanoparticles loaded with LK. The c-RGD-LK-NPs reagent is expected to be potentially useful in treating thromboembolic diseases.

Determination of the substrate specificity of tripeptidyl-peptidase I using combinatorial peptide libraries and development of improved fluorogenic substrates.

Classical late-infantile neuronal ceroid lipofuscinosis is a fatal neurodegenerative disease caused by mutations in CLN2, the gene encoding the lysosomal protease tripeptidyl-peptidase I (TPP I). The natural substrates for TPP I and the pathophysiological processes associated with lysosomal storage and disease progression are not well understood. Detailed characterization of TPP I substrate specificity should provide insights into these issues and also aid in the development of improved clinical and biochemical assays. To this end, we constructed fluorogenic and standard combinatorial peptide libraries and analyzed them using fluorescence and mass spectrometry-based activity assays. The fluorogenic group 7-amino-4-carbamoylmethylcoumarin was incorporated into a series of 7-amino-4-carbamoylmethylcoumarin tripeptide libraries using a design strategy that allowed systematic evaluation of the P1, P2, and P3 positions. TPP I digestion of these substrates liberates the fluorescence group and results in a large increase in fluorescence that can be used to calculate kinetic parameters and to derive the substrate specificity constant kcat/KM. In addition, we implemented a mass spectrometry-based assay to measure the hydrolysis of individual peptides in peptide pools and thus expand the scope of the analysis. Nonfluorogenic tetrapeptide and pentapeptide libraries were synthesized and analyzed to evaluate P1' and P2' residues. Together, this analysis allowed us to predict the relative specificity of TPP I toward a wide range of potential biological substrates. In addition, we evaluated a variety of new fluorogenic peptides with a P3 Arg residue, and we demonstrated their superiority compared with the widely used substrate Ala-Ala-Phe-AMC for selectively measuring TPP I activity in biological specimens.

Progress in the discovery of BACE inhibitors.

Dementia caused by Alzheimer's disease is a large medical burden on society in the developed world. Current treatments are largely symptomatic, and there is an urgent need for therapies which can interrupt or reverse the progression of disease. A number of strategies for intervention are being actively pursued; among the most promising is the inhibition of beta-secretase, or BACE. BACE is the enzyme responsible for N-terminal cleavage of the Alzheimer's precursor protein leading to the production of the beta-amyloid peptide. This cascade ultimately leads to the formation of amyloid plaques, one of the hallmark lesions of the disease. It is expected that inhibitors of BACE may therefore serve as an effective disease-modifing therapy for the treatment of AD. This concept has received significant attention by both academics and the pharmaceutical industry. This review focuses on a discussion of the reported structure-activity relationships for inhibitors of this important therapeutic target.

Toward protein-cleaving catalytic drugs: artificial protease selective for myoglobin.

A protein-cleaving catalyst highly selective for a disease-related protein can be used as a catalytic drug. As the first protein-cleaving catalyst selective for a protein substrate, a catalyst for myoglobin (Mb) was designed by attaching the Cu(II) or Co(III) complex of cyclen to a binding site searched by a combinatorial method using peptide nucleic acid monomers as building units. Various linkers were inserted between the catalytic Co(III) center and the binding site of the Mb-cleaving catalyst. Kinetic data revealed catalytic turnover of the Mb cleavage by the Cu(II) or Co(III) complex. MALDI-TOF MS revealed cleavage of the polypeptide backbone of Mb at selected positions. N-Terminal sequencing of the cleavage products identified the cleavage site and provided evidence for the hydrolytic nature of the Mb cleavage. Various chelating ligands were tested as the ligand for the Co(III) center of the Mb-cleaving catalyst. Among the nine chelating ligands examined, only cyclen and its triaza-monooxo analogue manifested catalytic activity.

Conservation of L and 3C proteinase activities across distantly related aphthoviruses.

The foot-and-mouth disease virus (FMDV) leader (L) proteinase is an important virulence determinant in FMDV infections. It possesses two distinct catalytic activities: (i) C-terminal processing at the L/VP4 junction; and (ii) induction of the cleavage of translation initiation factor eIF4G, an event that inhibits cap-dependent translation in infected cells. The only other member of the Aphthovirus genus, equine rhinitis A virus (ERAV), also encodes an L protein, but this shares only 32% amino acid identity with its FMDV counterpart. Another more distantly related picornavirus, equine rhinitis B virus (ERBV), which is not classified as an aphthovirus, also encodes an L protein. Using in vitro transcription and translation analysis, we have shown that both ERAV and ERBV L proteins have C-terminal processing activity. Furthermore, expression of ERAV L, but not ERBV L, in BHK-21 cells resulted in the efficient inhibition of cap-dependent translation in these cells. We have shown that the ERAV and FMDV L proteinases induce cleavage of eIF4GI at very similar or identical positions. Interestingly, ERAV 3C also induces eIF4GI cleavage and again produces distinct products that co-migrate with those induced by FMDV 3C. The ERBV L proteinase does not induce eIF4GI cleavage, consistent with its inability to shut down cap-dependent translation. We have also shown that another unique feature of FMDV L, the stimulation of enterovirus internal ribosome entry site (IRES) activity, is also shared by the ERAV L proteinase but not by ERBV L. The functional conservation of the divergent ERAV and FMDV proteinases indicates the likelihood of a similar and important role for these enzymes in the pathogenesis of infections caused by these distantly related aphthoviruses.

Effective artificial proteases synthesized by covering silica gel with aldehyde and various other organic groups.

Organic artificial proteases with broad substrate specificity were synthesized by covering the surface of silica gel with aldehyde and the functional groups present in amino acids. The artificial proteases hydrolyzed ovalbumin, albumin, hemoglobin, gamma-globulin with half-lives as short as 50 min at 25 degrees C or 7 min at 50 degrees C.

Enzymes from extremophiles.

The industrial application of enzymes that can withstand harsh conditions has greatly increased over the past decade. This is mainly a result of the discovery of novel enzymes from extremophilic microorganisms. Recent advances in the study of extremozymes point to the acceleration of this trend. In particular, enzymes from thermophilic organisms have found the most practical commercial use to date because of their overall inherent stability. This has also led to a greater understanding of stability factors involved in adaptation of these enzymes to their unusual environments.

Inhibición mixta de la endopeptidasa neutra y de la enzima convertidora por el compuesto S21402 (RB105) en un modelo experimental de infarto de miocardio / Inhibition of both angiotensin converting enzyme and neutral endopeptidase by S21402 in rats with experimental myocardial infarction

El vaso constrictor, angiotensina II y el péptido natriurético auricular (PNA) tienen funciones opuestas en el desarrollo de la insuficiencia cardíaca congestiva realizada en un modelo de insuficiencia cardíaca (ICC) en ratas. Este modelo de insuficiencia cardíaaca se caracteriza por una retención de sal a pesar del aumento de la concentración plasmática de PNA, indicando la ausencia de respuestas renales al PNA. S21402 (RB105), N-2S, 3R-(2-mercaptomethyl-1-oxo-3-phenyl butyl)L-alanine]}, es un inhibidor mixto que inhibe la enzima convertidora de angiotensina (ECA; Ki=4,2ñ0,5nM) y la endopeptidasa neutra (NEP;Ki=1,7ñ0,3nM). La inhibición de la NEP potencializa el PNA endógeno, y la inhibición de la ECA bloquea la producción de angiotensina II. La inhibición de las dos peptidasas permite una potencialización más completa de la bradicinina (BK). La inducción de la insuficiencia cardíaca en ratas por ligadura de las coronarias produjo el aumento de las concentraciones plasmáticas basales de PNA, de cGMP y de la renina, las cuales están relacionadas con el tamaño del infarto (moderados y severos). El PNA, el cGMP y la BK urinarias aumentan en las ratas con ICC en relación al tamaño del infarto. El S21402 (RB105) (25 mg.kg-1 i.v.) indujo un descenso de la presión arterial y un aumento de la natriuresis en función de la severidad del infarto. El compuesto S21402 (RB105) indujo un aumento de la renina plasmática a pesar del nivel basal elevado de la renina en los animales con ICC. El inhibidor mixto no produjo aumento detectable del PNA plasmático. Sin embargo, el cGMP plasmático fue aumentado por el compuesto S21402 (RB105) en función de la severidad del infarto. Las excreciones urinarias del PNA, del cGMP y de la BK fueron también aumentadas por el inhibidor mixto en función de la severidad del infarto. Independientemente del tamaño del infarto, el S21402 (RB105) es capaz de inducir natriuresis en un modelo caracterizado por la desensibilidad a la acción del PNA. La inhibición de ambas peptidasas (MEP,ECA) bloquea también la producción de angiotensina II. Esta combinación de efectos renales y vasculares podría tener un interés terapéutico de la insuficiencia cardíaca congestiva

Modelling, synthesis and biological activity of a BLV proteinase, made of (only) 116 amino acids.

Bovine leukaemia virus (BLV) is the aetiological agent of Leukosis enzootica bovis [Viral Oncology (1980), G. Klein (Ed.) Raven Press, New York, pp. 231-238], a widely spread disease in cattle. BLV is reported as the animal model of human T-cell leukaemia virus (HLTV) which is the causative agent of adult T-cell leukaemia and tropical spastic paraparesis. Like the viruses themselves, the two retroviral proteinases (PR) are very closely related [Virology 142 (1985) 357-377]. BLV and HTLV-I PR are reported as putative proteins made of 126 [J. Virol. 57 (1986) 826-832] and 125 [FEBS Lett. 293 (1991) 106-110] amino acids, respectively (long sequences), belonging to the aspartyl proteinase family [Nature 329 (1987) 351-354], with the aid of molecular modelling, we show that BLV and HTLV-I proteinases made of only 116 and 115 amino acids, respectively (short sequences), display three-dimensional structures similar to that observed for other retroviral aspartyl proteinases. The models are based on three-dimensional structures of Rous sarcoma virus (RSV PR) and the human immunodeficiency virus (HIV-1 PR). We used solid phase peptide synthesis to produce the putative proteolytic enzyme of BLV (116 amino acids). In this study, we show that the folded synthetic protease accurately hydrolyzes a decapeptide corresponding to the sequence of the Matrice-Capside (MA/CA) cleavage site of the gag polyprotein. In addition, the proteolytic activity is inhibited by a statine ((4S,3S)-4-amino-3-hydroxyl-6-methylheptanoic acid) containing an analogous sequence.

Solid phase synthesis of the proteinase of bovine leukemia virus. Comparison of its specificity to that of HIV-2 proteinase.

The 126-residue proteinase (PR) of bovine leukemia virus (BLV) was synthesized by solid-phase peptide synthesis and its activity was shown using various oligopeptide substrates representing cleavage sites in BLV, human T-cell leukemia virus type 1 (HTLV-1), murine leukemia virus (MuLV) and human immunodeficiency virus type 1 (HIV-1). The specificity of the BLV PR was also compared to that of chemically synthesized human immunodeficiency virus type 2 (HIV-2) PR. Many of the peptides were cleaved at the expected site, however, 6 out of 15 were hydrolyzed only by one of the PRs. Furthermore, one BLV peptide was processed differently by the two enzymes. These results, together with the relative activities and the lack of inhibition of BLV PR by two HIV-1 PR inhibitors, suggest that the BLV PR specificity is substantially different from that of HIV PRs.

Chemical synthesis of a biotinylated derivative of the simian immunodeficiency virus protease. Purification by avidin affinity chromatography and autocatalytic activation.

The protease from simian immunodeficiency virus (SIV) was chemically synthesized by automated solid-phase technology as an NH2-terminally extended derivative, capped with biotin. Biotin-linker-(SIV protease (1-99)): the linker segment, Gly-Gly-Asp-Arg-Gly-Phe-Ala-Ala, corresponds to the amino acid sequence preceding that of the protease in the SIV gag/pol precursor polyprotein. Accordingly, the Ala-Pro bond joining the octapeptide linker to the protease constitutes a site naturally cleaved by the protease during viral maturation. This strategy for synthesis was designed to facilitate purification of the biotinylated protein derivative from a complex mixture of reaction products by avidin/agarose-affinity chromatography and to provide the means for autocatalytic removal of the biotin-linker segment. As anticipated, folding of the full-length construct leads to activation of the enzyme and excision of the desired 99-residue SIV protease (overall yield, approximately). The specificity of the synthetic SIV protease toward a number of well characterized protein substrates was the same as observed for the nearly identical enzyme from human immunodeficiency virus type 2 (HIV-2 protease) and distinct from that of the more disparate HIV-1 protease. The same functional ordering with respect to the human retroviral proteases was reflected in Ki values observed with a number of protease inhibitors. Thus, the folded synthetic SIV protease shows patterns of specificity and susceptibility to inhibition that are in accord with what would be expected based upon its degree of structural similarity to proteases from HIV-1 and HIV-2.

Conserved folding in retroviral proteases: crystal structure of a synthetic HIV-1 protease.

The rational design of drugs that can inhibit the action of viral proteases depends on obtaining accurate structures of these enzymes. The crystal structure of chemically synthesized HIV-1 protease has been determined at 2.8 angstrom resolution (R factor of 0.184) with the use of a model based on the Rous sarcoma virus protease structure. In this enzymatically active protein, the cysteines were replaced by alpha-amino-n-butyric acid, a nongenetically coded amino acid. This structure, in which all 99 amino acids were located, differs in several important details from that reported previously by others. The interface between the identical subunits forming the active protease dimer is composed of four well-ordered beta strands from both the amino and carboxyl termini and residues 86 to 94 have a helical conformation. The observed arrangement of the dimer interface suggests possible designs for dimerization inhibitors.

Genetic locus, primary structure, and chemical synthesis of human immunodeficiency virus protease.

The genetic locus and primary structure of the human immunodeficiency virus (HIV) protease was determined by comparing the data of protein analyses with the published data of the gene analysis. The complete sequence of HIV-1 and HIV-2 protease was synthesized by solid-phase peptide synthesis. The synthetic protease was capable of accurately cleaving synthetic peptide substrates corresponding to known cleavage sites in gag polyproteins of HIV-1, HIV-2, and murine leukemia virus. The chemical synthesis of protease confirms the DNA sequence and provides a means of rapidly producing active protease in substantial quantities for biochemical and physical studies.

Chemical synthesis and enzymatic activity of a 99-residue peptide with a sequence proposed for the human immunodeficiency virus protease.

Retroviral proteins, including those from the human immunodeficiency virus (HIV), are synthesized as polyprotein precursors that require proteolytic cleavage to yield the mature viral proteins. A 99-residue polypeptide, encoded by the 5' end of the pol gene, has been proposed as the processing protease of HIV. The chemical synthesis of the 99-residue peptide was carried out by the solid-phase method, and the isolated product was found to exhibit specific proteolytic activity upon folding under reducing conditions. Upon size-exclusion chromatography, enzymatic activity was eluted at a point consistent with a dimeric molecular size. Specificity was demonstrated by the cleavage of the natural substrate HIV gag p55 into gag p24 and gag p17, as well as cleavage of small peptide substrates representing processing sites of HIV fusion proteins. The proteolytic action of the synthetic product could be inhibited by pepstatin, an aspartic protease inhibitor.