New Synthetic Thrombin Inhibitors: Molecular Design and Experimental Verification

BACKGROUND: The development of new anticoagulants is an important goal for the improvement of thromboses treatments. OBJECTIVES: The design, synthesis and experimental testing of new safe and effective small molecule direct thrombin inhibitors for intravenous administration. METHODS: Computer-aided molecular design of new thrombin inhibitors was performed using our original docking program SOL, which is based on the genetic algorithm of global energy minimization in the framework of a Merck Molecular Force Field. This program takes into account the effects of solvent. The designed molecules with the best scoring functions (calculated binding energies) were synthesized and their thrombin inhibitory activity evaluated experimentally in vitro using a chromogenic substrate in a buffer system and using a thrombin generation test in isolated plasma and in vivo using the newly developed model of hemodilution-induced hypercoagulation in rats. The acute toxicities of the most promising new thrombin inhibitors were evaluated in mice, and their stabilities in aqueous solutions were measured. RESULTS: New compounds that are both effective direct thrombin inhibitors (the best K(I) was <1 nM) and strong anticoagulants in plasma (an IC(50) in the thrombin generation assay of approximately 100 nM) were discovered. These compounds contain one of the following new residues as the basic fragment: isothiuronium, 4-aminopyridinium, or 2-aminothiazolinium. LD(50) values for the best new inhibitors ranged from 166.7 to >1111.1 mg/kg. A plasma-substituting solution supplemented with one of the new inhibitors prevented hypercoagulation in the rat model of hemodilution-induced hypercoagulation. Activities of the best new inhibitors in physiological saline (1 µM solutions) were stable after sterilization by autoclaving, and the inhibitors remained stable at long-term storage over more than 1.5 years at room temperature and at 4°C. CONCLUSIONS: The high efficacy, stability and low acute toxicity reveal that the inhibitors that were developed may be promising for potential medical applications

Phylogeographic structure of the Common hamster (Cricetus cricetus L.): Late Pleistocene connections between Caucasus and Western European populations.

The Common hamster (Cricetus cricetus) is one of the most endangered mammals in Western and Central Europe. Its genetic diversity in Russia and Kazakhstan was investigated for the first time. The analysis of sequences of an mtDNA control region and cytochrome b gene revealed at least three phylogenetic lineages. Most of the species range (approximately 3 million km2), including central Russia, Crimea, the Ural region, and northern Kazakhstan), is inhabited by a single, well-supported phylogroup, E0. Phylogroup E1, previously reported from southeastern Poland and western Ukraine, was first described from Russia (Bryansk Province). E0 and E1 are sister lineages but both are monophyletic and separated by considerable genetic distance. Hamsters inhabiting Ciscaucasia represent a separate, distant phylogenetic lineage, named "Caucasus". It is sister to the North phylogroup from Western Europe and the contemporary phylogeography for this species is discussed considering new data. These data enabled us to develop a new hypothesis to propose that in the Late Pleistocene, the continuous range of the Common hamster in the northern Mediterranean extended from the central and southern parts of modern France to the Caucasus; however, its distribution was subsequently interrupted, likely because of climate change

Phylogenetic relations and range history of jerboas of the Allactaginae subfamily (Dipodidae, Rodentia)

Five-toed jerboas of the subfamily Allactaginae comprise several complex taxa occurring over a wide distribution range covering a large part of the Eurasian arid belt. In this study, we employed current methods of molecular phylogenetics based on 15 nuclear genes and the mitochondrial gene cytb to revise relations and systematics within Allactaginae. We also applied species distribution modelling projected on paleo-environmental data to reconstruct the geographic patterns of speciation in Allactaginae. We elucidated the intergeneric relationships within this subfamily and clarifed interspecies relations within the genus Scarturus. Moreover, our results demonstrate the species status of S. caprimulgaoutline the currently understudied diversity within Orientallactaga, Allactaga, and Pygeretmusand improve the divergence estimates of these taxa. Based on our results from modelling of geographic range fragmentation in allactagines, we suggest the dating and location of speciation events and present hypotheses regarding general habitat niche conservatism in small mammals

List of samples localities.

<p>Point numbers correspond to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0187527#pone.0187527.g001" target="_blank">Fig 1</a>.</p

Sample distribution map.

<p>Bryansk Province in Western Russia is indicated by a square, localities from the Ciscaucasian area by triangles, and the others by circles. In the breakout: Nalchik City is shown by dark blue, the Nalchik surrounds (restricted by 25-km radius) by light blue, Kislovodsk by red, and other localities by yellow.</p

Divergence time (kya) between the Common hamsters phylogenetic lineages as evaluated based on suggested (1) <i>Tscherskia triton and (Cricetulus migratorius + Allocricetulus eversmanni + Cricetus cricetus)</i> clade and (2) Pannonia and North-type lineages separation time.

<p>Divergence time (kya) between the Common hamsters phylogenetic lineages as evaluated based on suggested (1) <i>Tscherskia triton and (Cricetulus migratorius + Allocricetulus eversmanni + Cricetus cricetus)</i> clade and (2) Pannonia and North-type lineages separation time.</p

The hypothetical ranges of the Common hamster hyplogroups: Vertical hatching–Late Pleistocene Northern Mediterranean; horizontal hatching–south-western LGM refugium; filled with colors–modern (mid-20th century).

<p>Note: Atlantic ocean, Mediterranean, Black and Azov sea max. regression, Caspian sea max. transgression coast line shown with the dashed line.</p

Cladogram resulting from Bayesian phylogenetic analysis of haplotypes of concatenated sequences of <i>cytb</i> gene and control region for hamsters within the investigated area.

<p>Support values are given if they exceeded 0.5 for nodes that included three or more haplotypes. For GenBank accession numbers see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0187527#pone.0187527.t002" target="_blank">Table 2</a>.</p

Phylogram resulting from Bayesian phylogenetic analysis of 904 bp cyt<i>b</i> gene fragment haplotypes.

<p>Support values are given if they exceeded 0.5 for nodes that included three or more haplotypes.</p