Current Medicinal Chemistry - Volume 26, Issue 3, 2019

During the last two decades, the production of pharmaceutical proteins in plants evolved from proof of concept to established technology adopted by several biotechnological companies. This progress is particularly based on intensive research starting stable genetic transformation and moving to transient expression. Due to its advantages in yield and speed of protein production transient expression platforms became the leading plant-based manufacturing technology. Current transient expression methods rely on Agrobacteriummediated delivery of expression vectors into plant cells. In recent years, great advances have been made in the improvement of expression vectors, host cell engineering as well as in the development of commercial manufacturing processes. Several GMP-certified large-scale production facilities exist around the world to utilize agroinfiltration method. A number of pharmaceutical proteins produced by transient expression are currently in clinical development. The great potential of transient expression platform in respect to rapid response to emerging pandemics was demonstrated by the production of experimental ZMapp antibodies against Ebola virus as well as influenza vaccines. This review is focused on current design, status and future perspectives of plant transient expression system for the production of biopharmaceutical proteins.

Background: A cost-effective plant platform for therapeutic monoclonal antibody production is both flexible and scalable. Plant cells have mechanisms for protein synthesis and posttranslational modification, including glycosylation, similar to those in animal cells. However, plants produce less complex and diverse Asn-attached glycans compared to animal cells and contain plant-specific residues. Nevertheless, plant-made antibodies (PMAbs) could be advantageous compared to those produced in animal cells due to the absence of a risk of contamination from nucleic acids or proteins of animal origin. Objective: In this review, the various platforms of PMAbs production are described, and the widely used transient expression system based on Agrobacterium-mediated delivery of genetic material into plant cells is discussed in detail. Results: We examined the features of and approaches to humanizing the Asn-linked glycan of PMAbs. The prospects for PMAbs in the prevention and treatment of human infectious diseases have been illustrated by promising results with PMAbs against human immunodeficiency virus, rotavirus infection, human respiratory syncytial virus, rabies, anthrax and Ebola virus. The pre-clinical and clinical trials of PMAbs against different types of cancer, including lymphoma and breast cancer, are addressed. Conclusion: PMAb biosafety assessments in patients suggest that it has no side effects, although this does not completely remove concerns about the potential immunogenicity of some plant glycans in humans. Several PMAbs at various developmental stages have been proposed. Promise for the clinical use of PMAbs is aimed at the treatment of viral and bacterial infections as well as in anti-cancer treatment.

Monoclonal antibodies (mAbs) are an important class of therapeutic agents approved for the therapy of many types of malignancies. However, in certain cases applications of conventional mAbs have several limitations in anticancer immunotherapy. These limitations include insufficient efficacy and adverse effects. The antigen-binding fragments of antibodies have a considerable potential to overcome the disadvantages of conventional mAbs, such as poor penetration into solid tumors and Fc-mediated bystander activation of the immune system. Fragments of antibodies retain antigen specificity and part of functional properties of conventional mAbs and at the same time have much better penetration into the tumors and a greatly reduced level of adverse effects. Recent advantages in antibody engineering allowed to produce different types of antibody fragments with improved structure and properties for efficient elimination of tumor cells. These molecules opened up new perspectives for anticancer therapy. Here, we will overview the structural features of the various types of antibody fragments and their applications for anticancer therapy as separate molecules and as part of complex conjugates or structures. Mechanisms of antitumor action of antibody fragments as well as their advantages and disadvantages for clinical application will be discussed in this review.

Current advances in cancer treatment are based on the recent discoveries of molecular mechanisms of tumour maintenance. It was shown that heat shock proteins (HSPs) play a crucial role in the development of immune response against tumours. Thus, HSPs represent multifunctional agents not only with chaperone functions, but also possessing immunomodulatory properties. These properties are exploited for the development of HSP-based anticancer vaccines aimed to induce cytotoxic responses against tumours. To date, a number of strategies have been suggested to facilitate HSP-based vaccine production and to increase its effectiveness. The present review focuses on the current trend for the development of HSPbased vaccines aimed at inducing strong immunological tumour-specific responses against cancer cells of distinct etiology and localization.

Background: Amino acids are essential components in various biochemical pathways. The deprivation of certain amino acids is an antimetabolite strategy for the treatment of amino acid-dependent cancers which exploits the compromised metabolism of malignant cells. Several studies have focused on the development and preclinical and clinical evaluation of amino acid degrading enzymes, namely L-asparaginase, L-methionine γ-lyase, L-arginine deiminase, L-lysine α-oxidase. Further research into cancer cell metabolism may therefore define possible targets for controlling tumor growth. Objective: The purpose of this review was to summarize recent progress in the relationship between amino acids metabolism and cancer therapy, with a particular focus on Lasparagine, L-methionine, L-arginine and L-lysine degrading enzymes and their formulations, which have been successfully used in the treatment of several types of cancer. Methods: We carried out a structured search among literature regarding to amino acid degrading enzymes. The main aspects of search were in vitro and in vivo studies, clinical trials concerning application of these enzymes in oncology. Results: Most published research are on the subject of L-asparaginase properties and it’s use for cancer treatment. L-arginine deiminase has shown promising results in a phase II trial in advanced melanoma and hepatocellular carcinoma. Other enzymes, in particular Lmethionine γ-lyase and L-lysine α-oxidase, were effective in vitro and in vivo. Conclusion: The findings of this review revealed that therapy based on amino acid depletion may have the potential application for cancer treatment but further clinical investigations are required to provide the efficacy and safety of these agents.

The main function of proteases in any living organism is the cleavage of proteins resulting in the degradation of damaged, misfolded and potentially harmful proteins and therefore providing the cell with amino acids essential for the synthesis of new proteins. Besides this main function, proteases may play an important role as signal molecules and participate in numerous protein cascades to maintain the vital processes of an organism. Plant proteases are no exception to this rule. Moreover, in contrast to humanencoded enzymes, many plant proteases possess exceptional features such as higher stability, unique substrate specificity and a wide pH range for enzymatic activity. These valuable features make plant-derived proteolytic enzymes suitable for many biomedical applications, and furthermore, the plants can serve as factories for protein production. Plant proteases are already applied in the treatment of several pathological conditions in the human organism. Some of the enzymes possess antitumour, antibacterial and antifungal activity. The collagenolytic activity of plant proteases determines important medical applications such as the healing of wounds and burn debridement. Plant proteases may affect blood coagulation processes and can be applied in the treatment of digestive disorders. The present review summarizes recent advances and possible applications for plant proteases in biomedicine, and proposes further development of plant-derived proteolytic enzymes in the biotechnology and pharmaceutical industries.

Nowadays, enzymatic therapy is a very promising line of treatment for many different diseases. There is a group of disorders and conditions, caused by fibrotic and scar processes and associated with the excessive accumulation of collagen that needs to be catabolized to normalize the connective tissue content. The human body normally synthesizes special extracellular enzymes, matrix metalloproteases (MMPs) by itself. These enzymes can cleave components of extracellular matrix (ECM) and different types of collagen and thus maintain the balance of the connective tissue components. MMPs are multifunctional enzymes and are involved in a variety of organism processes. However, under pathological conditions, the function of MMPs is not sufficient, and these enzymes fail to deal with disease. Thus, medical intervention is required. Enzymatic therapy is a very effective way of treating such collagen-associated conditions. It involves the application of exogenous collagenolytic enzymes that catabolize excessive collagen at the affected site and lead to the successful elimination of disease. Such collagenolytic enzymes are synthesized by many organisms: bacteria, animals (especially marine organisms), plants and fungi. The most studied and commercially available are collagenases from Clostridium histolyticum and from the pancreas of the crab Paralithodes camtschatica, due to their ability to effectively hydrolyse human collagen without affecting other tissues, and their wide pH ranges of collagenolytic activity. In the present review, we summarize not only the data concerning existing collagenase-based medications and their applications in different collagen-related diseases and conditions, but we also propose collagenases from different sources for their potential application in enzymatic therapy.

Collagen and collagen-based materials have been successfully used in medicine for over 50 years. The number of scientific articles about the role of collagen in the construction of scaffolds for tissue engineering has risen precipitously in recent years. The review contains materials about historic and modern applications of collagen in medicine such as soluble collagen injections, solid constructs reconstructed from solution, and decellularized collagen matrices. The analysis of published data proves the efficacy of collagen material in the treatment of chronic wounds, burns, venous and diabetic ulcers, in plastic, reconstructive and general surgery, urology, proctology, gynecology, ophthalmology, otolaryngology, neurosurgery, dentistry, cardiovascular and bone and cartilage surgery, as well as in cosmetology. Further development of collagenoplasty requires addressing the problems of allergic complications, improvement of structure and maximizing therapeutic effects against pathological processes.

Corneal epithelial disorders take pride of place in modern ophthalmology. Defects of corneal epithelium are commonly accompanied by blurry vision, photophobia and tearing. Since cornea is the most densely innervated tissue of organisms, its disruption leads to development of a severe pain syndrome. Mild corneal erosions commonly undergo quick spontaneous recovery. Suppression of corneal wound healing due to various pathological causes results in development of severe recurrent erosions and persistent corneal defects. These pathological events can in turn lead to corneal scarring, opacification, and ulceration of cornea, and ultimately to the permanent vision impairment. The etiology of the underlying corneal diseases that commonly involves inflammatory, neurotrophic and systemic factors, should be considered for treating such defects. Therefore, the research focus has been shifted to establish therapeutics based on proteins and peptides. Due to varied mechanisms of action, proteinbased pharmaceuticals can be involved in the protection of corneal surface, mimicking tear components, stimulation of corneal wound healing, regeneration of corneal innervation, suppressing oxidative stress, inflammation and neovascularization. The active components can be naturally occurring (blood- or tear-derived) or be created de novo and optimized in order to achieve the level of activity required. Such pharmaceuticals are characterized by low toxicity and absence of systemic side-effects due to their low absorption into the bloodstream, if administrated topically. This review summarizes existing data on protein-based drugs for treatment of corneal epithelial defects that are currently under preclinical development or testing in clinical trials, or approved for medical use.

Specific peptide molecules classified as hormones, neuropeptides and cytokines are involved in intercellular signaling regulating various physiological processes in all organs and tissues. This justifies the peptidergic signaling as an attractive pharmacological target. Recently, a protein mimetic of a peptide hormone has been identified in Escherichia coli suggesting the potential use of specific bacterial proteins as a new type of peptide-like drugs. We review the scientific rational and technological approaches leading to the identification of the E. coli caseinolytic protease B (ClpB) homologue protein as a conformational mimetic of α-melanocyte-stimulating hormone (α-MSH), a melanocortin peptide critically involved in the regulation of energy homeostasis in humans and animals. Theoretical and experimental backgrounds for the validation of bacterial ClpB as a potential drug are discussed based on the known E. coli ClpB amino acid sequence homology with α-MSH. Using in silico analysis, we show that other protein sources containing similar to E. coli ClpB α-MSH-like epitopes with potential biological activity may exist in Enterobacteriaceae and in some Brassicaceae. Thus, the original approach leading to the identification of E. coli ClpB as an α-MSH mimetic protein can be applied for the identification of mimetic proteins of other peptide hormones and development of a new type of peptide-like protein-based drugs.

In the course of studying human mucin MUC1, the attitude towards this molecule has been changing time and again. Initially, the list of presumable functions of MUC1 was restricted to protecting and lubricating epithelium. To date, it is assumed to play an important role in cell signaling as well as in all stages of oncogenesis, from malignant cell transformation to tumor dissemination. The story of MUC1 is full of hopes and disappointments. However, the scientific interest to MUC1 has never waned, and the more profoundly it has been investigated, the clearer its hidden potential turned to be disclosed. The therapeutic potential of mucin MUC1 has already been noted by various scientific groups at the early stages of research. Over forty years ago, the first insights into MUC1 functions became a strong ground for considering this molecule as potential target for anticancer therapy. Therefore, this direction of research has always been of particular interest and practical importance. More than 200 papers on MUC1 were published in 2016; the majority of them are dedicated to MUC1-related anticancer diagnostics and therapeutics. Here we review the history of MUC1 studies from the very first attempts to reveal its functions to the ongoing renaissance.

Myofibroblast activation is a critical process in the pathogenesis of tissue fibrosis accounting for 45% of all deaths. No effective therapies are available for the treatment of fibrotic diseases. We focus our mini-review on recent data showing that cardiotonic steroids (CTS) that are known as potent inhibitors of Na⁺,K⁺-ATPase affect myofibroblast differentiation in a cell type-specific manner. In cultured human lung fibroblasts (HLF), epithelial cells, and cancer-associated fibroblasts, CTS blocked myofibroblast differentiation triggered by profibrotic cytokine TGF-β. In contrast, in the absence of TGF-β, CTS augmented myofibroblast differentiation of cultured cardiac fibroblasts. The cell type-specific action of CTS in myofibroblast differentiation is consistent with data obtained in in vivo studies. Thus, infusion of ouabain via osmotic mini-pumps attenuated the development of lung fibrosis in bleomycintreated mice, whereas marinobufagenin stimulated renal and cardiac fibrosis in rats with experimental renal injury. In TGF-β-treated HLF, suppression of myofibroblast differentiation by ouabain is mediated by elevation of the [Na+]i/[K+]i ratio and is accompanied by upregulation of cyclooxygenase COX-2 and downregulation of TGF-β receptor TGFBR2. Augmented expression of COX-2 is abolished by inhibition of Na+/Ca2+ exchanger, suggesting a key role of [Ca2+]i-mediated signaling. What is the relative impact in tissue fibrosis of [Na+]i,[K+]iindependent signaling documented in several types of CTS-treated cells? Do the different conformational transitions of Na⁺,K⁺-ATPase α1 subunit in the presence of ouabain and marinobufagenin contribute to their distinct involvement in myofibroblast differentiation? Additional experiments should be done to answer these questions and to develop novel pharmacological approaches for the treatment of fibrosis-related disorders.