Mitochondrial VDAC1: A Key Gatekeeper as Potential Therapeutic Target

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Mitochondria are the key source of ATP that fuels cellular functions, and they are also central in cellular signaling, cell division and apoptosis. Dysfunction of mitochondria has been implicated in a wide range of diseases, including neurodegenerative and cardiac diseases, and various types of cancer. One of the key proteins that regulate mitochondrial function is the voltage-dependent anion channel 1 (VDAC1), the most abundant protein on the outer membrane of mitochondria. VDAC1 is the gatekeeper for the passages of metabolites, nucleotides, and ions; it plays a crucial role in regulating apoptosis due to its interaction with apoptotic and anti-apoptotic proteins, namely members of the Bcl-2 family of proteins and hexokinase. Therefore, regulation of VDAC1 is crucial not only for metabolic functions of mitochondria, but also for cell survival. In fact, multiple lines of evidence have confirmed the involvement of VDAC1 in several diseases. Consequently, modulation or dysregulation of VDAC1 function can potentially attenuate or exacerbate pathophysiological conditions. Understanding the role of VDAC1 in health and disease could lead to selective protection of cells in different tissues and diverse diseases. The purpose of this review is to discuss the role of VDAC1 in the pathogenesis of diseases and as a potentially effective target for therapeutic management of various pathologies.

The VDACs are the most abundant protein in the OMM with a molecular weight of approximately 32 kD. Under normal physiological conditions, VDACs function in tandem with the IMM adenine nucleotide translocase (ANT), via the mitochondrial creatine kinase (mCK) in the IMS. The conformational states of VDAC are voltage-dependent and exhibit different selectivity and permeability for small ions, showing a preference for anions in the open state and for cations in the closed state. Therefore, they are considered the principal sites for the exchange of metabolites and small solutes between the IMS and the cytosol. The efficient transfer of energy metabolites across mitochondria depends on the interaction between VDAC, mCK, and ANT, ANT/mCK/VDAC, which is fostered by physiological [Ca2+].

Highly Efficient Photolabile Protecting Groups with Intramolecular Energy Transfer

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The light sensitivity of photolabile protecting groups of the nitrophenylpropoxycarbonyl (NPPOC) type was enhanced by up to a factor of 25. This was achieved by covalently linking the protecting group to a sensitizer with a much better absorptivity and the ability to transfer its electronic excitation to the protecting group (see scheme).


Single Chain Variable Fragment (scFv) Molecules

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Single chain variable fragments (scFvs) are generated by joining together the variable heavy and light chain of a monoclonal antibody (mAb) via a peptide linker. They offer some advantages over the parental mAb such as low molecular weight, heterologous production, multimeric form, and multivalency. The scFvs were produced against more than 50 antigens till date using 10 different plant species as the expression system. There were considerable improvements in the expression and purification strategies of scFv in the last 24 years. With the growing demand of scFv in therapeutic and diagnostic fields, its biosynthesis needs to be increased. The easiness in development, maintenance, and multiplication of transgenic plants make them an attractive expression platform for scFv production. 

The conventional antibody consists of two heavy chains and two light chains connected with disulfide bonds. The antibody structure can be divided into a constant Fc domain (crystallizable fragment domain) and the Fab fragment (antibody binding fragment) contains the Fv domains (variable fragment domains) at the end of both the arms. In humans, the antibody synthesized is usually glycosylated in the Fc region, which stabilizes the antibody and is necessary for the antibody-dependent immune responses. Enzymatic cleavage of antibody at the N-terminal side of the inter-heavy chain disulfide bridges results in the formation of Fc and Fab fragments. There are two variable regions in a Fab fragment interact with the antigen and each of these units represent the smallest functional antigen-binding domain.

The scFv can be generated by amplifying the variable regions of the Fab fragment from the mAb and by linking it together with a flexible peptide linker (usually (GGGGS)3). Advances in molecular techniques further improved the prospect of engineering scFv to improve its specificity, avidity, affinity, and half-life. Multimerization of the variable domains using, the linker, the tetramerization domain of a native protein like p53, the leucine zippers ,or the C-terminal fragment of C4-binding protein improved the affinity of the scFv to a great extent. The immunogenicity generated by the Fc portion of the antibody is absent in the conventional scFv molecule. The scFv expressing together with the Fc region of IgG (scFv-Fc) is found to be beneficiary with its “effector” functions in many reports. Antibody fragments of therapeutic and diagnostic importance have been expressed in mammalian systems plants and in prokaryotes. An extensive review of the biotechnological applications of antibody fragments has been given earlier. Plant expressed antibodies are also used in studying the basic metabolism of plants in terms of disease resistance against a pathogen, protein–protein interactions or the specific role of an endogenous protein in a metabolic process by selectively modulating its activity.

Advantages of Antibody Fragments and Biopharming

Expression of mAbs in heterologous production systems is precarious as the biological activity of the resultant molecule is dependent on several post-translational modifications. Biosynthesis of conventional antibody molecules (150 kDa), through the mammalian expression system and transgenic animals is highly expensive and time-consuming. The scFvs are smaller in size (~ 30 kDa) with less post-translational modifications. They show a similar specificity and affinity of the parental antibody against the antigen. Due to the smaller size, scFvs show a rapid blood clearance (a useful property in the radiotherapy and other diagnostic applications) and better tissue penetration (which has greater impact when they are used as therapeutics) than the full length mAbs. Due to the rapid blood clearance, the in vivo availability of scFv is low compared to the mAbs, which is considered as the major drawback of scFv. In order to increase the bioavailability of scFv in the bloodstream, the diabodies (~ 55 k Da), triabodies (~ 85 kDa) and tetrabodies (~ 120 kDa) were developed by manipulating the length of the linker peptide in between the VH and VL domains or by using the methods which facilitate the multimerization of scFv molecules. Tandem dimerization of the scFv generated from two different antibody sources can also create a bispecific antibody by the variation in the arrangement of the VH and VL sequences and was proved to be an effective methods to detect specific cell types with fluorescence-tagged scFv dimers. Further to that, the multivalency generated by combining the coding sequences of more than one scFv together with the multimerization is used to link different moieties (one may be targeting a cell surface receptor on a cancer cell and other bound to an anti-cancer drug) which facilitate the targeted delivery of the therapeutics to its site of action.

2′-Fluoro-c-di-GMP as an oral vaccine adjuvant

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Bis-(3′–5′)-cyclic dimeric 2′-deoxy-2′-fluoroguanosine monophosphate (2′-F-c-di-GMP) was synthesized through the modified H-phosphonate chemistry. Oral immunization of C57BL/6 mice with Helicobacter pylori cell-free sonicate extract adjuvanted with 2′-F-c-di-GMP led to the production of antigen-specific antibodies in feces and sera, and lowered bacterial counts in the stomach upon post-vaccination infections in immunized mice. Similarly, oral vaccination of BALB/c mice with flagillin proteins from Clostridium difficile and Listeria monocytogenes adjuvanted with 2′-F-c-di-GMP led to production of antigen-specific antibodies both systemically and mucosally. The adjuvanticity of 2′-F-c-di-GMP is associated with the enhanced induction of interferon γ. These results demonstrated the excellent oral adjuvanticity of 2′-F-c-di-GMP. (RSC Adv., 2019, 9, 41481–41489.)

Phage-Displayed Peptides for Targeting Tyrosine Kinase Membrane Receptors in Cancer Therapy

Receptor tyrosine kinases (RTKs) regulate critical physiological processes, such as cell growth, survival, motility, and metabolism. Abnormal activation of RTKs and relative downstream signaling is implicated in cancer pathogenesis. Phage display allows the rapid selection of peptide ligands of membrane receptors. These peptides can target in vitro and in vivo tumor cells and represent a novel therapeutic approach for cancer therapy. Further, they are more convenient compared to antibodies, being less expensive and non-immunogenic. In this review, we describe the state-of-the-art of phage display for development of peptide ligands of tyrosine kinase membrane receptors and discuss their potential applications for tumor-targeted therapy. The Evolution of Phage Display

Phage display represents a useful technique for studying protein–protein interactions that regulate the biological processes. Bacteriophages, which are viruses infecting bacteria, can express recombinant peptides on their surface coat following the cloning of random short DNA sequences within their genome. The phage display technique was first described in 1985 by George P. Smith, who demonstrated the expression of a foreign insert on a filamentous phage surface following its cloning in frame with the minor coat protein pIII. In the same year, George Pieczenik patented the production of random peptide libraries for phage display (US Patent, 5,866,363). In 1988, the selection of phage ligands of target proteins was improved by using a process called “biopanning”, which significantly reduced antibody requirements compared to the original procedure published in 1985. In the 1990s, combinatorial phage libraries containing 40 million 6-mer peptides or 20 million 15-mer peptides were built. As predicted by Smith, the use of these libraries allowed an effective investigation of the specific affinity binding to antibody epitopes, receptors, or other proteins using simple recombinant DNA methods. What Smith did not imagine was the wide number of applications of his invention in various biomedical fields. The phage display technology was further developed and improved by the following research teams: G. Winter and J. McCafferty of the Medical Research Council, Laboratory of Molecular Biology; R. Lerner and C. Barbas of the Scripps Research Institute; F. Breitling and S. Dübel of the German Cancer Research Center. All these researchers pursued the creation of phage-displayed combinatorial antibodies libraries, which were further improved by several other laboratories in the following years. As recognition of the phage display contribution to scientific advances in chemistry and pharmaceutics, George P. Smith and Sir Gregory P. Winter received the 2018 Nobel Prize in Chemistry “for phage visualization of peptides and antibodies”. More recently, the phage display has been useful for the mapping of antibody binding epitope and the screening of combinatorial peptide libraries in drugs discovery. A timeline of phage display development is shown below. (Viruses . 2021 Apr 9;13(4):649.)

Tumor penetrating peptides for improved drug delivery

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In vivo screening of phage libraries in tumor-bearing mice has been used to identify peptides that direct phage homing to a tumor. The power of in vivo phage screening is illustrated by the recent discovery of peptides with unique tumor-penetrating properties. These peptides activate an endocytic transport pathway related to but distinct from macropinocytosis. They do so through a complex process that involves binding to a primary, tumor-specific receptor, followed by a proteolytic cleavage, and binding to a second receptor. The second receptor, neuropilin-1 (or neuropilin-2) activates the transport pathway. This trans-tissue pathway, dubbed the C-end Rule (CendR) pathway, mediates the extravasation transport through extravascular tumor tissue of payloads ranging from small molecule drugs to nanoparticles. The CendR technology provides a solution to a major problem in tumor therapy, poor penetration of drugs into tumors. Targeted delivery with tumor-penetrating peptides has been shown to specifically increase the accumulation of drugs, antibodies and nanotherapeutics in experimental tumors in vivo, and in human tumors ex vivo. Remarkably the payload does not have to be coupled to the peptide; the peptide activates a bulk transport system that sweeps along a drug present in the blood. Treatment studies in mice have shown improved anti-tumor efficacy and less damage to normal tissues with drugs ranging from traditional chemotherapeutics to antibodies, and to nanoparticle drugs.

The iRGD peptide homes to tumors and accumulates in them through a 3-step process (Fig. 1): First, the integrin-binding RGD sequence motif binds to αvβ3 and αvβ5 integrins, which are specifically expressed in tumor endothelial cells. Other cells in tumors also express these integrins, which is likely to be important for the spreading of the peptide within tumor tissue, but the vascular endothelium is the gateway to the tumor for the peptide. Second, a protease cleavage event activates the CendR motif (R/KXXR/K). This protease(s) has not been identified, but is likely a furin or furin-like enzyme because the CendR motif is a preferred recognition motif for these proteases. In principle, any protease that cuts after a basic residue can activate iRGD. We have used trypsin and urokinase in vitro for this purpose. The protease cleavage requires the integrin binding; a peptide that has the CendR motif but does not bind to integrins (CRG cheap mexican pharmacy neurontin EKGPDC) is not activated. The requirement for integrin binding limits the activation of iRGD to tumors. Third, the CendR motif binds to neuropilin-1 (NRP-1) or neuropilin-2 (NRP-2), and the interaction activates an endocytotic/exocytotic transport pathway named the CendR pathway. This pathway is responsible for the enhanced transport of drugs into tumors triggered by iRGD.

Using an in vivo screening procedure designed to probe tumor lymphatic vessels, we identified a peptide that specifically accumulated in tumor lymphatics and not in normal lymphatics. We now know that this peptide, LyP-1, primarily accumulates in a myeloid cell/macrophage in tumors, when intravenously injected into tumor-bearing mice. Some of these cells incorporate into tumor lymphatics, causing LyP-1 accumulation in the endothelium of these vessels. Endothelial cells of tumor blood vessels and tumor cells also bind LyP-1, but much less of the peptide accumulates in these cells than in tumor macrophages. The macrophages are particularly abundant in hypoxic areas of tumors, which are low on blood vessels but contain abundant, albeit dysfunctional lymphatic vasculature. Remarkably, the phage carrying the LyP-1 peptide reaches these areas within minutes of systemic injection. The ability of this peptide to reach poorly vascularized parts of tumors remained a mystery for several years, until we discovered another peptide with similar tumor-penetrating properties, and set out to uncover the underlying mechanism. (Adv Drug Deliv Rev. 2017 Feb; 110-111: 3–12.)

Synthesis and Pharmacological Effects of Diosgenin–Betulinic Acid Conjugates

The target diosgenin–betulinic acid conjugates are reported to investigate their ability to enhance and modify the pharmacological effects of their components. The detailed synthetic procedure that includes copper(I)-catalyzed Huisgen 1,3-dipolar cycloaddition (click reaction), and palladium-catalyzed debenzylation by hydrogenolysis is described together with the results of cytotoxicity screening tests. Palladium-catalyzed debenzylation reaction of benzyl ester intermediates was the key step in this synthetic procedure due to the simultaneous presence of a 1,4-disubstituted 1,2,3-triazole ring in the molecule that was a competing coordination site for the palladium catalyst. High pressure (130 kPa) palladium-catalyzed procedure represented a successful synthetic step yielding the required products. The conjugate 7 showed selective cytotoxicity in human T-lymphoblastic leukemia (CEM) cancer cells (IC50 = 6.5 ± 1.1 µM), in contrast to the conjugate  8 showing no cytotoxicity, and diosgenin (1), an adaptogen, for which a potential to be active on central nervous system was calculated in silico. In addition, 5 showed medium multifarious cytotoxicity in human T-lymphoblastic leukemia (CEM), human cervical cancer (HeLa), and human colon cancer (HCT 116). Betulinic acid (2) and the intermediates 3 and 4 showed no cytotoxicity in the tested cancer cell lines. The experimental data obtained are supplemented by and compared with the in silico calculated physico-chemical and absorption, distribution, metabolism, and excretion (ADME) parameters of these compounds.

Diosgenin, (3β,25R)-spirost-5-en-3-ol, is a steroid sapogenin part of the saponin dioscin found in the tubers of Dioscorea zingiberensis C. H. Wright or Trigonella foenum-graecum L. and in numbers of legumes. Diosgenin is a widely used precursor in the synthesis of sexual hormones, peroral contraceptives and other steroids in the pharmaceutical industry. It is an adaptogen, displaying non-steroidogenic activity along with other beneficial effects. Diosgenin is unable to bind metal ions, and therefore, the change made from more traditional cholesterol/cholesterylamine system to diosgenin could influence the overall conformation of the bivalent structures, modifying the metal ions chelating properties. Saponins are always species formed from an aglycone and several monosaccharide units, the presence of which increases the solubility of saponins in natural aqueous media. Diosgenin is not metabolized in the human body, and it is considered to represent a safe natural drug. It has also been investigated for treating hyperglycemia, hypercholesterolemia, hypertriacylglycerolemia, and Alzheimer’s disease.
Betulinic acid, 3β-hydroxylup-20(29)-en-28-oic acid, is a pharmacologically perspective triterpenoid plant product with a broad spectrum of effects, e.g., antitumor, anti-HIV, cytostatic, and anti-inflammatory. It can be obtained from the bark of Betula pendula Roth, widely distributed in Europe, and from a number of subtropical and tropical plants. (Molecules. 2020 Aug; 25(15): 3546.)

Mitocanic Di- and Triterpenoid Rhodamine B Conjugates

The combination of the “correct” triterpenoid, the “correct” spacer and rhodamine B (RhoB) seems to be decisive for the ability of the conjugate to accumulate in mitochondria. So far, several triterpenoid rhodamine B conjugates have been prepared and screened for their cytotoxic activity. To obtain cytotoxic compounds with EC50 values in a low nano-molar range combined with good tumor/non-tumor selectivity, the Rho B unit has to be attached via an amine spacer to the terpenoid skeleton. To avoid spirolactamization, secondary amines have to be used. First results indicate that a homopiperazinyl spacer is superior to a piperazinyl spacer. Hybrids derived from maslinic acid or tormentic acid are superior to those from oleanolic, ursolic, glycyrrhetinic or euscaphic acid. Thus, a tormentic acid-derived RhoB conjugate 32, holding a homopiperazinyl spacer can be regarded, at present, as the most promising candidate for further biological studies.

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Mitochondrial membranes of malignant cells hold an increased membrane potential compared to non–malignant cells. This effect fosters the accumulation of cationic molecules, hence inducing high selectivity for mitocans holding a (more or less) lipophilic cation such as a rhodamine scaffold. The same effect applies for triphenylphosphonium cations and to a small extent for quaternary ammonium ions, zwitterionic N-oxides and triterpenes substituted with BODIPYs or a safirinium moiety [67].
To date, hybrid molecules have been prepared from oleanolic acid (OA, Figure 2), ursolic acid (UA), glycyrrhetinic acid (GA), betulinic acid (BA), maslinic acid (MA), augustic acid (AU), 11-keto-β-boswellic acid (KBA), asiatic acid (AA), tormentic acid (TA) and euscaphic acid (EA).
OA-derived RhoB conjugates appear to be superior to analog UA-derived compounds in the majority of cases with respect to their cytotoxicity. Although AKBA-derived derivatives have good cytotoxicity properties, they were found to be less cytotoxic compared to other triterpene carboxylic acid derivatives, but they often showed better tumor cell/non-tumor cell selectivity. So far, the best cytotoxicity properties have been found for MA-, EA- and TA-derived derivatives. These allowed the transition to compounds of nano-molar activity, while many other triterpene carboxylic acid derivatives were cytotoxic only on a micro-molar concentration range. MA- derived derivatives seem to be approximately equivalent to EA-derived compounds. They are currently only surpassed in many tumor cell lines only by the analogous derivatives from TA. From results available so far, it can be concluded that compounds holding a homopiperazinyl spacer are superior to those with a piperazinyl spacer. This underlines the importance of the spacer for obtaining good cytotoxicity properties. (Molecules. 2020 Nov; 25(22): 5443.)