Unified Methodology for the Primary Preclinical In Vivo Screening of New Anticoagulant Pharmaceutical Agents from Hematophagous Organisms.

The development of novel anticoagulants requires a comprehensive investigational approach that is capable of characterizing different aspects of antithrombotic activity. The necessary experiments include both in vitro assays and studies on animal models. The required in vivo approaches include the assessment of pharmacokinetic and pharmacodynamic profiles and studies of hemorrhagic and antithrombotic effects. Comparison of anticoagulants with different mechanisms of action and administration types requires unification of the experiment scheme and its adaptation to existing laboratory conditions. The rodent thrombosis models in combination with the assessment of hemostasis parameters and hematological analysis are the classic methods for conducting preclinical studies. We report an approach for the comparative study of the activity of different anticoagulants in vivo, including the investigation of pharmacodynamics and the assessment of hemorrhagic effects (tail-cut bleeding model) and pathological thrombus formation (inferior vena cava stenosis model of venous thrombosis). The reproducibility and uniformity of our set of experiments were illustrated on unfractionated heparin and dabigatran etexilate (the most common pharmaceuticals in antithrombic therapy) as comparator drugs and an experimental drug variegin from the tick Amblyomma variegatum. Variegin is notorious since it is a potential analogue of bivalirudin (Angiomax, Novartis AG, Basel, Switzerland), which is now being actively introduced into antithrombotic therapy.

Intramolecular Hydrogen Bonding in N6-Substituted 2-Chloroadenosines: Evidence from NMR Spectroscopy.

Two forms were found in the NMR spectra of N6-substituted 2-chloroadenosines. The proportion of the mini-form was 11-32% of the main form. It was characterized by a separate set of signals in COSY, 15N-HMBC and other NMR spectra. We assumed that the mini-form arises due to the formation of an intramolecular hydrogen bond between the N7 atom of purine and the N6-CH proton of the substituent. The 1H,15N-HMBC spectrum confirmed the presence of a hydrogen bond in the mini-form of the nucleoside and its absence in the main form. Compounds incapable of forming such a hydrogen bond were synthesized. In these compounds, either the N7 atom of the purine or the N6-CH proton of the substituent was absent. The mini-form was not found in the NMR spectra of these nucleosides, confirming the importance of the intramolecular hydrogen bond in its formation.

Enzymatic Synthesis of 2-Chloropurine Arabinonucleosides with Chiral Amino Acid Amides at the C6 Position and an Evaluation of Antiproliferative Activity In Vitro.

A number of purine arabinosides containing chiral amino acid amides at the C6 position of the purine were synthesized using a transglycosylation reaction with recombinant E. coli nucleoside phosphorylases. Arsenolysis of 2-chloropurine ribosides with chiral amino acid amides at C6 was used for the enzymatic synthesis, and the reaction equilibrium shifted towards the synthesis of arabinonucleosides. The synthesized nucleosides were shown to be resistant to the action of E. coli adenosine deaminase. The antiproliferative activity of the synthesized nucleosides was studied on human acute myeloid leukemia cell line U937. Among all the compounds, the serine derivative exhibited an activity level (IC50 = 16 µM) close to that of Nelarabine (IC50 = 3 µM) and was evaluated as active.

Chaperone-mediated production of active homodimer human bone morphogenetic protein - 2 in E. coli.

Human bone morphogenetic protein 2 (hBMP-2) plays a leading role in the process of osteogenesis and is one of the key components of osteoplastic materials, ensuring their high osteoinduction. In order to obtain a homodimeric form hBMP-2 using the E. coli expression system, a number of problems associated with refolding in vitro and purification from monomer and oligomeric forms must be solved. The developed method for co-expression of the target protein with chaperone proteins makes it possible to obtain the biologically active homodimeric form of hBMP-2 in vivo. Purification with simple ion-exchange sorbents without the use of denaturing reagents affecting the structure of the protein molecule provides a chromatographic purity of the product of at least 97%. The expressed hBMP-2 was identified by Western blotting and the LC-ESI-TOF mass spectrometry confirmed its molecular weight of 26052.72 Da. Circular dichroism spectroscopy showed that recombinant hBMP-2 has a native secondary structure.

Heparin-Induced Changes of Vascular Endothelial Growth Factor (VEGF165) Structure.

Vascular endothelial growth factor-A (VEGF-A), a secreted homodimeric glycoprotein, is a critical regulator of angiogenesis in normal and pathological states. The binding of heparin (HE) to VEGF165 (the major form of VEGF-A) modulates the angiogenesis-related cascade, but the mechanism of the observed changes at the structural level is still insufficiently explored. In the present study, we examined the effect of HE on the structural and physicochemical properties of recombinant human VEGF165 (rhVEGF165). The HE binding results in an increase of hydrophobic surface exposure in rhVEGF165 without changes in its secondary structure. Differential scanning calorimetry measurements for intact and HE-bound rhVEGF165 reveals the absence of any pronounced thermally induced transitions in the protein in the temperature range from 20 to 100 °C. The apolar area increase during the heparin binding explains the pronounced HE-induced oligomerization/aggregation of rhVEGF165, as studied by chemical glutaraldehyde cross-linking and dynamic light scattering. Molecular modeling and docking techniques were used to model the full structure of dimeric VEGF165 and to reveal putative molecular mechanisms underlying the function of the VEGF165/HE system. In general, the results obtained can be a basis for explaining the modulating effect of HE on the biological activity of VEGF-A.

Rational Mutagenesis in the Lid Domain of Ribokinase from E. coli Results in an Order of Magnitude Increase in Activity towards D-arabinose.

Development of efficient approaches for the production of medically important nucleosides is a highly relevant challenge for biotechnology. In particular, cascade synthesis of arabinosides would allow relatively easy production of various cytostatic and antiviral drugs. However, the biocatalyst necessary for this approach, ribokinase from Escherichia coli (EcoRK), has a very low activity towards D-arabinose, making the synthesis using the state-of-art native enzyme technologically unfeasible. Here, we report the results of our enzyme design project, dedicated to engineering a mutant form of EcoRK with elevated activity towards arabinose. Analysis of the active site structure has allowed us to hypothesize the reasons behind the low EcoRK activity towards arabinose and select feasible mutations. Enzyme assay and kinetic studies have shown that the A98G mutation has caused a large 15-fold increase in kcat and 1.5-fold decrease in KM for arabinose phosphorylation. As a proof of concept, we have performed the cascade synthesis of 2-chloroadenine arabinoside utilizing the A98G mutant with 10-fold lower amount of enzyme compared to the wild type without any loss of synthesis efficiency. Our results are valuable both for the development of new technologies of synthesis of modified nucleosides and providing insight into the structural reasons behind EcoRK substrate specificity.

Synthesis of 2-chloropurine ribosides with chiral amino acid amides at C6 and their evaluation as A1 adenosine receptor agonists.

A series of purine ribonucleosides bearing chiral amino acid amides at the C6 position of 2-chloropurine was synthesized. Molecular docking of the synthesized analogs of 2-chloroadenosine by their affinity for A1 adenosine receptors (A1ARs) was conducted. The investigation of A1AR stimulating activity of synthesized nucleosides was carried out in a model of an isolated mouse atrium. We have shown that derivatives with tyrosine, valine, and serine residues exhibit the properties of A1AR partial agonists. Animal experiments in the open field test have shown that these compounds have different profiles of psychoactive action. These nucleosides have an ophthalmic hypotensive effect and reduce intraocular pressure in a manner slightly inferior to that of timolol and brimonidine. The synthesized nucleosides can be the basis for further design and synthesis of new A1AR agonists.

Comparative Analysis of Enzymatic Transglycosylation Using E. coli Nucleoside Phosphorylases: A Synthetic Concept for the Preparation of Purine Modified 2'-Deoxyribonucleosides from Ribonucleosides

A comparative analysis of the transglycosylation conditions catalyzed by E. coli nucleoside phosphorylases, leading to the formation of 2'-deoxynucleosides, was performed. We demonstrated that maximal yields of 2'-deoxynucleosides, especially modified, can be achieved under small excess of glycosyl-donor (7-methyl-2'-deoxyguanosine, thymidine) and a 4-fold lack of phosphate. A phosphate concentration less than equimolar one allows using only a slight excess of the carbohydrate residue donor nucleoside to increase the reaction's output. A three-step methodology was elaborated for the preparative synthesis of purine-modified 2'-deoxyribonucleosides, starting from the corresponding ribonucleosides.

The comparative analysis of the properties and structures of purine nucleoside phosphorylases from thermophilic bacterium Thermus thermophilus HB27.

Two recombinant purine nucleoside phosphorylases from thermophilic bacterium Thermus thermophilus HB27 encoded by genes TT_C1070 (TthPNPI) and TT_C0194 (TthPNPII) were purified and characterized. The comparative analysis of their sequences, molecular weight, enzymes specificity and kinetics of the catalyzed reaction were realized. As a result, it was determined that the TthPNPI is specific to guanosine while the TthPNPII to adenosine. According to the results of the size exclusion chromatography and SAXS study both enzymes are hexameric molecules. Based on the sequence alignment with homologous purine nucleoside phosphorylases (PNPs), Asn was identified as a purine base recognizing residue in the active site of TthPNPI and Asp in TthPNPII. The three-dimensional structure of TthPNPII was solved at 2.5 Å resolution by molecular replacement method using crystals grown in microgravity. Position of phosphate in the active site cavity is located. The possible arrangement of adenosine and guanosine in TthPNPII active site cavity is considered using superposition with the structures of homologous trimeric and hexameric PNPs complexed with corresponding substrates. The peculiarities of oligomeric structure of TthPNPII in comparison with homologous PNPs are described. It is shown that two trimeric molecules of TthPNPII in the asymmetric part of the unit cell are connected by three two-fold axis into a hexamer with 32-point symmetry. This type of hexameric structure of PNP is found for the first time. The interface area between the subunits in trimeric molecule and between the trimers in TthPNPII hexamer is described.Communicated by Ramaswamy H. Sarma.

Multi-Enzymatic Cascades in the Synthesis of Modified Nucleosides: Comparison of the Thermophilic and Mesophilic Pathways.

A comparative study of the possibilities of using ribokinase → phosphopentomutase → nucleoside phosphorylase cascades in the synthesis of modified nucleosides was carried out. Recombinant phosphopentomutase from Thermus thermophilus HB27 was obtained for the first time: a strain producing a soluble form of the enzyme was created, and a method for its isolation and chromatographic purification was developed. It was shown that cascade syntheses of modified nucleosides can be carried out both by the mesophilic and thermophilic routes from D-pentoses: ribose, 2-deoxyribose, arabinose, xylose, and 2-deoxy-2-fluoroarabinose. The efficiency of 2-chloradenine nucleoside synthesis decreases in the following order: Rib (92), dRib (74), Ara (66), F-Ara (8), and Xyl (2%) in 30 min for mesophilic enzymes. For thermophilic enzymes: Rib (76), dRib (62), Ara (32), F-Ara (<1), and Xyl (2%) in 30 min. Upon incubation of the reaction mixtures for a day, the amounts of 2-chloroadenine riboside (thermophilic cascade), 2-deoxyribosides (both cascades), and arabinoside (mesophilic cascade) decreased roughly by half. The conversion of the base to 2-fluoroarabinosides and xylosides continued to increase in both cases and reached 20-40%. Four nucleosides were quantitatively produced by a cascade of enzymes from D-ribose and D-arabinose. The ribosides of 8-azaguanine (thermophilic cascade) and allopurinol (mesophilic cascade) were synthesized. For the first time, D-arabinosides of 2-chloro-6-methoxypurine and 2-fluoro-6-methoxypurine were synthesized using the mesophilic cascade. Despite the relatively small difference in temperatures when performing the cascade reactions (50 and 80 °C), the rate of product formation in the reactions with Escherichia coli enzymes was significantly higher. E. coli enzymes also provided a higher content of the target products in the reaction mixture. Therefore, they are more appropriate for use in the polyenzymatic synthesis of modified nucleosides.

Radical Dehalogenation and Purine Nucleoside Phosphorylase E. coli: How Does an Admixture of 2',3'-Anhydroinosine Hinder 2-fluoro-cordycepin Synthesis.

During the preparative synthesis of 2-fluorocordycepin from 2-fluoroadenosine and 3'-deoxyinosine catalyzed by E. coli purine nucleoside phosphorylase, a slowdown of the reaction and decrease of yield down to 5% were encountered. An unknown nucleoside was found in the reaction mixture and its structure was established. This nucleoside is formed from the admixture of 2',3'-anhydroinosine, a byproduct in the preparation of 3-'deoxyinosine. Moreover, 2',3'-anhydroinosine forms during radical dehalogenation of 9-(2',5'-di-O-acetyl-3'-bromo- -3'-deoxyxylofuranosyl)hypoxanthine, a precursor of 3'-deoxyinosine in chemical synthesis. The products of 2',3'-anhydroinosine hydrolysis inhibit the formation of 1-phospho-3-deoxyribose during the synthesis of 2-fluorocordycepin. The progress of 2',3'-anhydroinosine hydrolysis was investigated. The reactions were performed in D2O instead of H2O; this allowed accumulating intermediate substances in sufficient quantities. Two intermediates were isolated and their structures were confirmed by mass and NMR spectroscopy. A mechanism of 2',3'-anhydroinosine hydrolysis in D2O is fully determined for the first time.

Screening of the Promising Direct Thrombin Inhibitors from Haematophagous Organisms. Part I: Recombinant Analogues and Their Antithrombotic Activity In Vitro.

The success in treatment of venous thromboembolism and acute coronary syndromes using direct thrombin inhibitors has stimulated research aimed at finding a new anticoagulant from haematophagous organisms. This study deals with the comparison between hirudin-1 from Hirudomedicinalis(desirudin), being the first-known and most well-studied natural anticoagulant, along with recombinant analogs of haemadin from the leech Haemadipsa sylvestris, variegin from the tick Amblyomma variegatum, and anophelin from Anopheles albimanus. These polypeptides were chosen due to their high specificity and affinity for thrombin, as well as their distinctive inhibitory mechanisms. We have developed a universal scheme for the biotechnological production of these recombinant peptides as pharmaceutical substances. The anticoagulant activities of these peptides were compared using the thrombin amidolytic activity assay and prolongation of coagulation time (thrombin time, prothrombin time, and activated partial thromboplastin time) in mouse and human plasma. The preliminary results obtained suggest haemadin as the closest analog of recombinant hirudin-1, the active substance of the medicinal product Iprivask (Aventis Pharmaceuticals, USA) for the prevention of deep venous thrombosis in patients undergoing elective hip or knee replacement surgery. In contrast, variegin can be regarded as a natural analog of bivalirudin (Angiomax, The Medicines Company), a synthetic hirudin-1 derivative certified for the treatment of patients undergoing percutaneous coronary intervention and of patients with unstable angina pectoris after percutaneous transluminal coronary angioplasty.

Anion exchange resins in phosphate form as versatile carriers for the reactions catalyzed by nucleoside phosphorylases.

In the present work, we suggested anion exchange resins in the phosphate form as a source of phosphate, one of the substrates of the phosphorolysis of uridine, thymidine, and 1-(ß-ᴅ-arabinofuranosyl)uracil (Ara-U) catalyzed by recombinant E. coli uridine (UP) and thymidine (TP) phosphorylases. α-ᴅ-Pentofuranose-1-phosphates (PF-1Pis) obtained by phosphorolysis were used in the enzymatic synthesis of nucleosides. It was found that phosphorolysis of uridine, thymidine, and Ara-U in the presence of Dowex® 1X8 (phosphate; Dowex-nPi) proceeded smoothly in the presence of magnesium cations in water at 20-50 °C for 54-96 h giving rise to quantitative formation of the corresponding pyrimidine bases and PF-1Pis. The resulting PF-1Pis can be used in three routes: (1) preparation of barium salts of PF-1Pis, (2) synthesis of nucleosides by reacting the crude PF-1Pi with an heterocyclic base, and (3) synthesis of nucleosides by reacting the ionically bound PF-1Pi to the resin with an heterocyclic base. These three approaches were tested in the synthesis of nelarabine, kinetin riboside, and cladribine with good to excellent yields (52-93%).

Use of nucleoside phosphorylases for the preparation of 5-modified pyrimidine ribonucleosides.

Enzymatic transglycosylation, a transfer of the carbohydrate moiety from one heterocyclic base to another, is catalyzed by nucleoside phosphorylases (NPs) and is being actively developed and applied for the synthesis of biologically important nucleosides. Here, we report an efficient one-step synthesis of 5-substitited pyrimidine ribonucleosides starting from 7-methylguanosine hydroiodide in the presence of nucleoside phosphorylases (NPs).

An explanation for the narrow carbohydrate substrate specificity of adenine phosphoribosyltransferase from Thermus thermophilus from the model of the enzyme, substrate, and magnesium cation cofactor complex.

Communicated by Ramaswamy H. Sarma.

Enzymatic synthesis of novel purine nucleosides bearing a chiral benzoxazine fragment.

A series of ribo- and deoxyribonucleosides bearing 2-aminopurine as a nucleobase with 7,8-difluoro- 3,4-dihydro-3-methyl-2H-[1,4]benzoxazine (conjugated directly or through an aminohexanoyl spacer) was synthesized using an enzymatic transglycosylation reaction. Nucleosides 3-6 were resistant to deamination under action of adenosine deaminase (ADA) Escherichia coli and ADA from calf intestine. The antiviral activity of the modified nucleosides was evaluated against herpes simplex virus type 1 (HSV-1, strain L2). It has been shown that at sub-toxic concentrations, nucleoside (S)-4-[2-amino-9-(ß-D-ribofuranosyl)-purin-6-yl]-7,8-difluoro-3,4-dihydro-3-methyl-2H-[1,4]benzoxazine exhibit significant antiviral activity (SI > 32) on the model of HSV-1 in vitro, including an acyclovir-resistant virus strain (HSV-1, strain L2/R).

Synthesis of Cytokinins via Enzymatic Arsenolysis of Purine Nucleosides.

This unit describes an effective method for the preparation of natural cytokinins and their synthetic derivatives based on enzymatic cleavage of the N-glycosidic bond of N6 -substituted adenosine or O6 -substituted inosine derivatives in the presence of purine nucleoside phosphorylase (PNP) and Na2 HAsO4 . The arsenolysis reaction is irreversible due to the hydrolysis of the resulting α-D-ribose-1-arsenate. As a result, the desired products are formed in near-quantitative yields, as indicated by high-performance liquid chromatography (HPLC) analysis, and can easily be isolated. In the strategy used here, the ribose residue acts as a protective group. © 2018 by John Wiley & Sons, Inc.

Crystal structure of Escherichia coli purine nucleoside phosphorylase complexed with acyclovir.

Escherichia coli purine nucleoside phosphorylase (PNP), which catalyzes the reversible phosphorolysis of purine ribonucleosides, belongs to the family I hexameric PNPs. Owing to their key role in the purine salvage pathway, PNPs are attractive targets for drug design against some pathogens. Acyclovir (ACV) is an acyclic derivative of the PNP substrate guanosine and is used as an antiviral drug for the treatment of some human viral infections. The crystalline complex of E. coli PNP with acyclovir was prepared by co-crystallization in microgravity using counter-diffusion through a gel layer in a capillary. The structure of the E. coli PNP-ACV complex was solved at 2.32 Šresolution using the molecular-replacement method. The ACV molecule is observed in two conformations and sulfate ions were located in both the nucleoside-binding and phosphate-binding pockets of the enzyme. A comparison with the complexes of other hexameric and trimeric PNPs with ACV shows the similarity in acyclovir binding by these enzymes.

Crystal structure of Escherichia coli purine nucleoside phosphorylase in complex with 7-deazahypoxanthine.

Purine nucleoside phosphorylases (EC 2.4.2.1; PNPs) reversibly catalyze the phosphorolytic cleavage of glycosidic bonds in purine nucleosides to generate ribose 1-phosphate and a free purine base, and are key enzymes in the salvage pathway of purine biosynthesis. They also catalyze the transfer of pentosyl groups between purine bases (the transglycosylation reaction) and are widely used for the synthesis of biologically important analogues of natural nucleosides, including a number of anticancer and antiviral drugs. Potent inhibitors of PNPs are used in chemotherapeutic applications. The detailed study of the binding of purine bases and their derivatives in the active site of PNPs is of particular interest in order to understand the mechanism of enzyme action and for the development of new enzyme inhibitors. Here, it is shown that 7-deazahypoxanthine (7DHX) is a noncompetitive inhibitor of the phosphorolysis of inosine by recombinant Escherichia coli PNP (EcPNP) with an inhibition constant Ki of 0.13 mM. A crystal of EcPNP in complex with 7DHX was obtained in microgravity by the counter-diffusion technique and the three-dimensional structure of the EcPNP-7DHX complex was solved by molecular replacement at 2.51 Šresolution using an X-ray data set collected at the SPring-8 synchrotron-radiation facility, Japan. The crystals belonged to space group P6122, with unit-cell parameters a = b = 120.370, c = 238.971 Å, and contained three subunits of the hexameric enzyme molecule in the asymmetric unit. The 7DHX molecule was located with full occupancy in the active site of each of the three crystallographically independent enzyme subunits. The position of 7DHX overlapped with the positions occupied by purine bases in similar PNP complexes. However, the orientation of the 7DHX molecule differs from those of other bases: it is rotated by ∼180° relative to other bases. The peculiarities of the arrangement of 7DHX in the EcPNP active site are discussed.

Pilot production of the recombinant peptide toxin of Heteractis crispa as a potential analgesic by intein-mediated technology.

APHC3 is an analgesic polypeptide that was found in the sea anemone (Heteractis crispa), and contains 56 amino acid residues. This polypeptide is of interest for the development of medications for diseases, associated with inflammatory or neuropathological processes, as well as its use as an analgesic. This work presents an innovative biotechnological method for APHC3 production. We have constructed a recombinant plasmid intended for biosynthesizing the fusion protein consisting of a chitin-binding domain, DnaB mini-intein from Synechocystis sp. capable of undergoing pH-dependent self-cleavage, and the target peptide. In the process of biosynthesis the fusion protein aggregates and forms the inclusion bodies that are welcomed since APHC3 is a cytotoxic peptide. The target peptide recovery process developed by us involves 3 chromatographic steps. The method developed by us enables to produce 940 mg of the recombinant APHC3 from 100 g of the inclusion bodies. The method is straightforward to implement and scale up. The recombinant APHC3 activity and effectiveness as an analgesic was proved by animal testing.