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.

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.

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.

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.

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.

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).

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.

Thermophilic phosphoribosyltransferases Thermus thermophilus HB27 in nucleotide synthesis.

Phosphoribosyltransferases are the tools that allow the synthesis of nucleotide analogues using multi-enzymatic cascades. The recombinant adenine phosphoribosyltransferase (TthAPRT) and hypoxanthine phosphoribosyltransferase (TthHPRT) from Thermus thermophilus HB27 were expressed in E.coli strains and purified by chromatographic methods with yields of 10-13 mg per liter of culture. The activity dependence of TthAPRT and TthHPRT on different factors was investigated along with the substrate specificity towards different heterocyclic bases. The kinetic parameters for TthHPRT with natural substrates were determined. Two nucleotides were synthesized: 9-(ß-D-ribofuranosyl)-2-chloroadenine 5'-monophosphate (2-Сl-AMP) using TthAPRT and 1-(ß-D-ribofuranosyl)pyrazolo[3,4-d]pyrimidine-4-one 5'-monophosphate (Allop-MP) using TthНPRT.

Crystal structure of recombinant phosphoribosylpyrophosphate synthetase 2 from Thermus thermophilus HB27 complexed with ADP and sulfate ions.

Phosphoribosylpyrophosphate synthetase (PRPPS) from the thermophilic bacterial strain Thermus thermophilus HB27 catalyzes the synthesis of phosphoribosylpyrophosphate from ribose 5-phosphate and ATP, and belongs to the class I PRPPSs. The three-dimensional structure of the recombinant enzyme was solved at 2.2 Šresolution using crystals grown in microgravity from protein solution containing ATP, magnesium and sulfate ions. An ADP molecule was located in the active site of each subunit of the hexameric enzyme molecule and sulfate ions were located in both the active and allosteric sites. It was found that the catalytic loop that restricts the active-site area and is usually missing from the electron-density map of class I PRPPSs adopts different conformations in three independent subunits in T. thermophilus PRPPS. A closed conformation of the active site was found in one of subunits where the highly ordered catalytic ß-hairpin delivers the Lys and Arg residues that are essential for activity directly to the ADP molecule, which occupies the ATP-binding site. A comparison of the conformations of the catalytic loop in the three independent subunits reveals a possible mode of transition from the open to the closed state of the active site during the course of the catalyzed reaction.

Comparative analysis of the effectiveness of C-terminal cleavage intein-based constructs in producing a recombinant analog of anophelin, an anticoagulant from Anopheles albimanus.

Production of small recombinant peptides by expressing them as fusion proteins, with subsequent proteolytic or chemical cleavage of the latter, is a widespread approach in modern biotechnology. An alternative method is to produce such peptides as self-cleaving fusion proteins with inteins. To date, only a small proportion of known inteins have been used for this purpose, and analysis of other inteins for the ability to cleave off the target polypeptide can significantly expand the range of intein-based transgenic constructs available to researchers. Most interesting in practical terms are С-terminal cleavage constructs for producing target polypeptides without an N-terminal methionine residue. We prepared two new such constructs with mini-inteins GyrA from Mycobacterium xenopi and RIR1 from Methanobacterium thermoautotrophicum. Together with the previous construct based on the artificial mini-intein derived from Synechocystis sp. DnaB intein, they were used to produce a recombinant analog of anophelin, the naturally occurring thrombin inhibitor from the mosquito Anopheles albimanus. The effectiveness of the constructs with Ssp DnaB and Mth RIR1 proved to be relatively low because of spontaneous fusion protein cleavage during the producer strain culturing in the former case and a low degree of its cleavage upon purification in the latter case. The most effective Mxe GyrA construct was used to develop a semipreparative procedure for producing recombinant anophelin, with its yield reaching 91 ± 2 mg protein per liter of culture medium. As determined by an amidolytic assay, the antithrombin activity and K i of recombinant anophelin were 3362.8 ATU/mg and 87 ± 3 рМ, respectively.