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Maksimenko A.V. Medical Research Archives, vol. 6, issue 1, January 2018 issue Page 1 of 13

REVIEW ARTICLE Results and Achievements in the Engineering of Pharmacological Enzymes for Clinical Application Alexander V. Maksimenko

Authors’ affiliations: National Medical Research Center for Cardiology, 15A, 3rd Cherepkovskaya str., 121552 Moscow, Russia Tel. 7-495-414-6025; Fax: 7-499-726-3116; Correspondence address: E – mail: [email protected] Conflicts of interest: none.

Abstract Research into modified enzyme derivatives and their development have expanded considerably modern arsenal of biopharmaceuticals. The effectiveness of enzyme drugs in vivo and their prospectiveness in clinical practice were confirmed by biomedical studies. Several directions have formed in the biocatalyst research, such as antibody-drug interactions, construction of polyenzyme nanoensembles producing extra- and intracellular effects, and enzyme modification for targeted delivery to the damaged area. Modified enzyme derivatives have been became the effective agents against injurious action of oxidative stress in vivo. Research promotion is highly noticeable for covalent bienzyme superoxide dismutase-chondroitin sulfate-catalase conjugate. The obtained data contribute the investigation of salable conjugates produced by methods of chemical and biological synthesis. A broad research front and collective biomedical effort increase the probability of successful breakthroughs in the control over different types of damage to biological tissues.

Keywords: vascular injuries, oxidative stress, inflammation, enzymes, modification, targeted drug delivery, nanoconjugates.

Introduction has revealed functional groups of amino acid residues on enzyme surface which could be Progress in medicine implies the use of modified to obtain derivatives with high advanced diagnostic technologies, stability for subsequent use in clinical therapeutic techniques and highly effective practice. This review concerns with water- new-generation drugs. A breakthrough in the soluble modified protein compounds for development of new therapeutic agents was medical purposes. provided by molecular construction on the basis of proteins and other high-molecular- The development of approaches to protein weight compounds. Examination of proteins modification was initiated within the concept as specifically folded macromolecular chains of targeted drug delivery. Expansion of the

Copyright 2018 KEI Journals. All Rights Reserved http://journals.ke-i.org/index.php/mra Maksimenko A.V. Medical Research Archives, vol. 6, issue 1, January 2018 issue Page 2 of 13 research led to interdisciplinary studies of was modified with p-chloromercuribenzoate biological conjugates for medical use.17, 19, 21 (PCMB) or poly(2-hydroxyethyl methacrylate) (pHEMA), which resulted in Methods for Bioconjugate Production obvious decrease or complete loss of the Bioconjugates have been obtained using enzyme activity. However, the protein various methods, such as chemical activity can be restored to different extents modification, site-specific mutagenesis, their by reductive reagents leading to dissociation combination, and construction of therapeutic of the modifiers (PCMB or pHEMA) to nanosystems. With accumulation of recover the enzyme activity. This strategy modified protein derivatives a group of provides an efficient control over enzyme antibody-drug conjugates has formed. activity at different levels of site-specific conjugation of small molecules and Further developments of this group were 41 provided particularly by GlycoConnect, a polymers. technique consisting of a two-stage process: Bovine pancreatic ribonuclease conjugated enzymatic remodeling of the N-glycan at with hydrophilic poly[N-(2-hydroxypropyl) Asp-297 in an IgG antibody and attachment methacrylamide] (classic or star-like) of an azide, followed by ligation of the produced antitumor effect in nude mice desired pharmacological agent using copper- 40 bearing various human tumors after 10 daily free click chemistry. The resulting intravenous or intraperitoneal doses of 2.5 conjugates with monoclonal antibodies and/or 1 mg/kg, respectively.39 demonstrate high stability and efficiency, promising their predominant therapeutic index. Galactose-conjugated antibodies are Effects of Low- and High-Molecular used in regionally specific and systemic Weight Modifiers on Enzymes 33 immunotherapy. Conjugation did not The perspectives of unifying small interfere with two antibody functions in molecules with biological drugs are addition to antigen binding: complement- hampered by the necessity of simultaneous mediated cytotoxicity and antibody- analytical characterization and regulatory dependent cell-mediated cytotoxicity. considerations for the resulting antibody- Conjugation of egg-white proteins and drug conjugates.6 The following aspects lysozyme by covalent attachment to remain open to discussion: identity galactomannan via Maillard reaction parameters for test and quality control potentiated antioxidant activity of ovalbumin samples, efficiency of the conjugates and (by increasing its lipid affinity) and 31 their heterogeneity, restriction for additives, antimicrobial activity of lysozyme. The reference standards, definitions for various prospects of biomedical investigation of a types of stability, etc. The developments in given protein conjugate are strongly the antibody-drug conjugate technologies determined by the retained activities of its depend on the use of short linker molecules, constituents. Therefore, a methodology for the number of drug molecules (between reactivation of conjugate components has 41 three and seven) attached to the antibody, been proposed. Site-directed mutagenesis and the safety of manufacturing.42 was used to generate mutant pyrophosphates Conjugation with synthetic nanocarriers can with a substituted Cys residue for the be facilitated by using polyethylene glycol specific Lys-148 site which is within a (PEG) functional nanostructures coupled to a conserved sequence near the active site and bispecific antibody.8 Dual specificity of exposed to the surface of the protein for these antibodies to methoxy PEG epitopes chemical reaction. The mutated Cys residue and cancer targets, such as epidermal growth

Copyright 2018 KEI Journals. All Rights Reserved http://journals.ke-i.org/index.php/mra Maksimenko A.V. Medical Research Archives, vol. 6, issue 1, January 2018 issue Page 3 of 13 factor receptor provide better accumulation particles are densely covered as a shell by of the conjugate compared with linear polymer chain and practically "fenced off" methoxy PEG. Various modifying from water environment and the conjugate- techniques have been employed to improve forming particles possess more friable pharmacological characteristics of structures in which protein molecules are conjugated enzymes: half-life in the practically exposed to the solvent. Covalent circulation, accumulation in the damaged binding of Cu,Zn-superoxide dismutase area, “grafting” useful therapeutic activities, (SOD) with low-molecular-weight heparin and to eliminate their drawbacks: low enhanced thermostability, acid and alkali stability in vivo, high inhibitability and resistance, and anti-trypsin stability of the immunogenicity, etc. Methoxypolyethylene enzyme.45 Recombinant arginine deiminase glycol-p-nitrophenyl carbonate (MW 5 kDa) (rADI) was conjugated with the C terminus was used for modification of lysozyme with of heparin-binding hemagglutinin adhesion preservation of its activity (77% its original protein via succinimidyl 3-(2- enzyme activity), molecular structure, pyridyldithio)propionate (SPDP), and a resistance to proteolysis and stability at recombinant fusion protein was produced to 50ºС.5 Overexpression of hyaluronan deliver this enzyme to rADI-resistant human synthase 3 promoted the growth of cancer breast adenocarcinoma cells.43 Increased pancreatic tumor with abundant extracellular uptake of the conjugate restored the hyaluronan accumulation.13 Treatment with sensitivity of MCF-7 cells to rADI PEGylated human recombinant treatment. Similar conjugates can be used as hyaluronidase inhibited these processes antitumor drugs with an intercellular (reduced adhesion of epithelial cells) and mechanism of action independent of suppressed tumor growth. PEG coupling argininosuccinate synthetase expression. In based on microbial transglutaminase has order to improve intravenous delivery of L- been proposed to increase molecular asparaginase the enzyme was covalently homogeneity of the resulting conjugate by linked to fatty acids having different chain 1 specific interaction with one or two Gln lengths (C12, C16 and C22). The asparaginase residues of a given protein.28 A targeted drug bioconjugates showed higher resistance to delivery system for cytostatics is based on a proteolysis, higher stability at different pH combination of the drug carrier Zn- and prolonged plasma half-life compared porphyrin-cyclodextrin and immunoglo- with native enzyme, which offers good bulins forming supramolecular coordination biomedical prospects for this strategy. complexes.10 Therapeutic superiority of the coordination assembly nanosystem over An interesting method based on chemical those of building blocks used for modification with 3-maleimidopropionic construction of the system was shown in a acid (MPA) has been suggested to produce an in vivo conjugate between serum albumin mouse model of human C32 carcinoma. 44 Bovine serum albumin (BSA) was and inhibitory peptide with rapid and conjugated to poly(N-isopropylacrylamide- irreversible conjugation of the peptide to co-acrylic acid) by using water-soluble serum albumin thiol groups at a 1:1 molar carbodiimide for application as a novel ratio. After intravenous injection into rats the immunogenic system in vaccine conjugate exhibited a remarkably extended technology.3 Two types of conjugate in vivo half-life. This lays a basis for particles formed depending on the mass ratio conjugation of efficient, safe and prolonged of albumin and polymer carrier: the protein peptide inhibitors to endogenous proteins in molecules in the structure of conjugate vivo. The approach may decrease the cost

Copyright 2018 KEI Journals. All Rights Reserved http://journals.ke-i.org/index.php/mra Maksimenko A.V. Medical Research Archives, vol. 6, issue 1, January 2018 issue Page 4 of 13 and improve compliance of the treatment low-molecular-weight compounds may and patient’s quality of life.44 provide additional interactions potentiating therapeutic effects of the resulting The great diversity of chemical and gene derivative. Site-directed mutagenesis offers engineering techniques for enzyme optimization of amino acid chain in the modification led to a wide range of protein protein molecule, and nanoparticles allow a pharmacological agents. Covalent binding of dose-regulating release of therapeutic agent, proteins with polymers and small-size thus improving its biomedical parameters. molecules allows production of conjugates So far, there are no versatile methods to with high stability5,17,21,40, expanded 3, 13, 28 achieve these goals, and the effects produced retention in the body/circulation and by modified enzymes are tested empirically. therapeutic efficiency in a wide range of The contribution of computer modeling to pathologies. The first-level effects, i.e., those the development of general principles of revealed at the initial stages of a consecutive enzyme drug construction has considerably research, manifest themselves due to increased in recent years. Experimental multicontact interaction between protein evaluation of modified enzymes is molecule and modifier. After modification associated with stabilization (which with high-molecular-weight compounds an generally results from the modification) or enzyme obtains polymeric sheathing which inactivation of the enzyme caused by increases its stability. Modification with modifiers (Figure 1).

Figure 1. A schematic drawing for irreversible conformational changes in bovine testicular hyaluronidase after insertion of hexasaccharides (trimers) of chondroitin sulfate. Insertion of chondroitin sulfate trimers (cs4 and cs6) in active center area of the enzyme (their location is shown before and after insertion) results in relocation of E-149 and D-147 amino acid residues responsible for catalysis at the periphery of the enzyme molecule, deformation of its active structure and inactivation.

Calculational studies in silico have bovine testicular hyaluronidase (Figure 2) demonstrated that the structure of native modified by polymeric chondroitin sulfate

Figure 2. Tree-dimensional structure of native bovine testicular hyaluronidase. Locations of lysine, arginine, glutamic and aspartic amino acid residues are indicated. Six lysine residues of the first-level availability are stained green/dark.

(Figure 3) becomes highly stable and structure, the centers that bind with a shielded from inhibitors.24 Depending on the modifier can be divided according to several molecular topography of the protein levels of availability to the modifying agent.

CHS

Figure 3. 3D model of bovine testicular hyaluronidase covalently modified by two chains of chondroitin sulfate.

The enzyme structure is stabilized and shielded by chondroitin sulfate (CHS). The conjugate results from profound modification of the biocatalyst at 19 surface lysine residues.

New strategies have suggested accurate and superoxide dismutase (SOD), catalase reversible modulation of enzyme activity at (CAT) and glutathione peroxidase17, 22 various stages of site-specific41 and in vivo renders them into potential drugs for (in the body)44 conjugation with reliable treatment of cardiovascular diseases activity preservation of the resulting associated with vascular wall inflammation, therapeutic agent. The prospects for the endothelial dysfunction and oxidative development of a modified protein agent are stress.38 Oxidative stress has a largely determined by its therapeutic pathophysiological role in systolic and activity. diastolic heart failure29 and acts as a key mechanistic mediator in hypertension.2 Enzyme Derivatives Preventing Oxidative Reduction of oxidative stress can be useful Stress in the therapy of ST-elevation myocardial infarction.4 Numerous pathologies are initiated and accompanied by oxidative stress, which Antioxidant effects of SOD and CAT were urges an extensive research into antioxidants potentiated by folic acid modification with and their clinical application. High carbodiimide activation. This increased effectiveness of the antioxidant enzymes activity and uptake of these enzymes by activated macrophages.14 Modification of

SOD with low-molecular-weight heparin coin the term antioxidant stress which enhanced its heat stability and pH indicates the condition when a decrease in resistance.45 Treatment of diabetic rats with physiological concentration of active oxygen SOD-polymer conjugate improved their species with a parallel intrinsic increase in antioxidant status, which demonstrates the antioxidant content impairs systemic advantages of enzyme modification for intracellular signaling and regulation of vital clinical application.26 These approaches are processes. Realization of these effects at the aimed at production of modified derivatives entire body level in the norm and pathology based on one type of antioxidant enzyme. requires further investigations. Nano-size supramolecular antioxidant The specific nanocarrier PACkET was used ensembles were suggested for prevention for endothelium-targeted delivery of SOD and reduction of oxidative stress.35 They and CAT.7 Catalase-loaded PACkET serve to deliver antioxidant enzymes, exert protects endothelial cells against killing by their own activity and reduce the area of H2O2 and alleviates pulmonary edema and antioxidant interactions. Combinations of leukocyte infiltration in a mouse model of biomolecules in these ensembles provide endotoxin-induced lung injury. SOD-loaded their mutifunctionality, rapid effect and PACkET mitigates cytokine-induced targeting at the damaged area in order to endothelial pro-inflammatory activation and achieve high therapeutic effect. endotoxin-induced lung inflammation. A Nanoparticles containing antioxidants have nanozyme produced by electrostatic shown promise as high-performance coupling of SOD1 with the cationic block therapeutic agents for attenuating oxidative copolymer poly(L-lysine)-poly(ethylene- stress with potential applications in treating glycol) followed by covalent cross-linking of and preventing neurodegenerative the complexes with 3,3′-dithiobis(sulfo- conditions.36 This approach demonstrates a succinimidylpropionate) sodium salt reduced new development in antioxidant therapy and uveitis in a rabbit model.11 The nanozyme prevention of diseases associated with can be regarded as a potential therapeutic oxidative stress and offers new prospects for agent for the treatment of ocular high-definition medicine. inflammatory disorders. Generally, nanocarriers proved to be effective in Interestingly, IgG of patients with multiple experimental therapy with the use of sclerosis display CAT and SOD activities as individual enzymes or their combinations. compensatory response to decreased activity of intercellular antioxidant enzymes.12 It Combined application of antioxidant should be stressed that the effective dose enzymes is more effective than their range for antioxidant therapy is crucially individual use. These combinations produce important, as evidenced by the finding that at greater antioxidant effect, remaining safe high doses the antioxidants slow down the pharmacological agents. For instance, when initiation of DNA synthesis at G0/G1 phase used together SOD and CAT neutralize and block G1 phase without genotoxic superoxide radical and detoxify hydrogen effects in cultured human endometrial peroxide to molecular oxygen and water mesenchymal cells.16 Genotoxic effect on (scheme) proliferating cells allowed the authors to

.- .- + SOD О2 + О2 + 2Н > Н2О2 + О2 CAT Н2О2 + Н2О2 > 2 Н2О + О2 In sum : .- + SOD / CAT 4 О2 + 4 Н > 2 Н2О + 3 О2 S c h e m e

Conjugation of SOD to CAT via the was observed after preventive and endothelial glycocalyx glycosaminoglycan therapeutic administration of SOD-CHS- chondroitin sulfate (CHS) generates SOD- CAT conjugate.20 These findings enhance CHS-CAT bienzyme derivative with substantially the frontiers for medical extracellular activity.25 The conjugate application of enzyme derivatives similar to displays SOD and CAT activities, and CHS SOD-CHS-CAT conjugate. promotes its affinity for potential atherosclerotic lesion foci/zones in the The interest into production of multienzyme vascular wall. Supramolecular structure of ensembles has its history. Trypsin and the conjugate allows one to regard it as a chymotrypsin (molecular ratio 1:1) were conjugated using N-succinimidyl pyridyl nanoparticle. These particles acquire unique 34 physical, chemical and biological properties dithiopropionate as a cross-linking reagent, resulting from quantum mechanical which increased its resistance to interactions. Thus, the characteristics of the trypsinolysis. Enolase and phosphoglycerate mutase were conjugated using a similar conjugate were determined by its size. In 32 contrast to individual SOD and CAT, SOD- technique with glutaraldehyde. SOD-CAT CHS-CAT inhibits platelet aggregation conjugate obtained with glutaraldehyde as a induced by ADP, serotonin or thrombin cross-lining agent displayed protective 25 activity in an isolated rat heart with receptor agonist peptide (TRAP). Both 27 enzyme activities and the acquired ischemia-reperfusion model. Hemoglobin nanostructure contributed to this process. conjugates with SOD and CAT obtained by Indeed, regarding platelet aggregation the employing dicarboxymethylated poly- conjugate displayed the properties of a ethylene glycol were designed to protect nanoparticle. It should be noted the high hemoglobin against free radicals upon cell transplantation with oxygen support by antithrombotic action of conjugate: its effect 30 was produced at doses two orders of proteins. The resulting conjugate had a magnitude lower than those for native SOD high molecular weight (appr. 1000 kDa) and and CAT and an order magnitude lower high SOD (70 %) and CAT (90%) remained than for their CHS-modified derivatives activities. Gene and protein engineering injected individually or in combination. technologies have been employed to produce Moreover, covalent conjugation of these protein ensembles which can be internalized enzymes provided simultaneous presence of by cells. A tri-functional protein with SOD, SOD and CAT activities in the lesion, which CAT and cell-permeable activities was obtained by coexpression of joined/fused was impossible with the use of their 15 mixtures.23 Higher survival rate in rats with genes. A recombinant chimeric protein lipopolysaccharide-induced endotoxic shock with peroxidase and SOD activities

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