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

Designing Stable Bacillus anthracis Antigens with a View to Recombinant Anthrax Vaccine Development

Anthrax is a disease caused by Bacillus anthracis that affects mammals, including humans. Recombinant B. anthracis protective antigen (rPA) is the most common basis for modern anthrax vaccine candidates. However, this protein is characterised by low stability due to proteolysis and deamidation. Here, for the first time, two modification variants leading to full-size rPA stabilisation have been implemented simultaneously, through deamidation-prone asparagine residues substitution and by inactivation of proteolysis sites. Obtained modified rPA (rPA83m) has been demonstrated to be stable in various temperature conditions. Additionally, rPA1+2 containing PA domains I and II and rPA3+4 containing domains III and IV, including the same modifications, have been shown to be stable as well. These antigens can serve as the basis for a vaccine, since the protective properties of PA can be attributed to individual PA domains. The stability of each of three modified anthrax antigens has been considerably improved in compositions with tobacco mosaic virus-based spherical particles (SPs). rPA1+2/rPA3+4/rPA83m in compositions with SPs have maintained their antigenic specificity even after 40 days of incubation at +37 &deg;C. Considering previously proven adjuvant properties and safety of SPs, their compositions with rPA83m/rPA1+2/rPA3+4 in any combinations might be suitable as a basis for new-generation anthrax vaccines

Gene Polymorphism of Biotransformation Enzymes and Ciprofloxacin Pharmacokinetics in Pediatric Patients with Cystic Fibrosis

(1) Background: Ciprofloxacin (CPF) is widely used for the treatment of cystic fibrosis, including pediatric patients, but its pharmacokinetics is poorly studied in this population. Optimal CPF dosing in pediatric patients may be affected by gene polymorphism of the enzymes involved in its biotransformation. (2) Materials and Methods: a two-center prospective non-randomized study of CPF pharmacokinetics with sequential enrollment of patients (n-33, mean age 9.03 years, male-33.36%), over a period from 2016 to 2021. All patients received tablets of the original CPF drug Cyprobay&reg; at a dose of 16.5 mg/kg to 28.80 mg/kg. Blood sampling schedule: 0 (before taking the drug), 1.5 h; 3.0 h; 4.5 h; 6.0 h; 7.5 h after the first dosing. CPF serum concentrations were analyzed by high performance liquid chromatography mass spectrometry. The genotype of biotransformation enzymes was studied using total DNA isolated from whole blood leukocytes by the standard method. (4) Results: a possible relationship between the CA genotype of the CYP2C9 gene (c.1075A &gt; C), the GG genotype of the CYP2D6*4 gene (1846G &gt; A), the AG genotype of the GSTP1 gene (c.313A &gt; G), the GCLC* genotype 7/7 and the CPF concentration in plasma (increased value of the area under the concentration&ndash;time curve) was established. Conclusions: Gene polymorphism of biotransformation enzymes may affect ciprofloxacin pharmacokinetics in children

Two approaches for the stabilization of Bacillus anthracis recombinant protective antigen

Anthrax is a zoonotic disease caused by the gram-positive spore-forming bacteria Bacillus anthracis. There is a need for safe, highly effective, long-term storage vaccine formulations for mass vaccination. However, the development of new subunit vaccines based on recombinant protective antigen (rPA) faces the problem of vaccine antigen instability. Here, the potential of simultaneous application of two different approaches to stabilize rPA was demonstrated. Firstly, we employed spherical particles (SPs) obtained from the tobacco mosaic virus (TMV). Previously, we had reported that SPs can serve as an adjuvant and platform for antigen presentation. In the current work, SPs were shown to increase the stability of the full-size rPA without loss of its antigenic properties. The second direction was site-specific mutagenesis of asparagine residues to avoid deamidation that causes partial protein degradation. The modified recombinant protein comprising the PA immunogenic domains 3 and 4 (rPA3 + 4) was stable during storage at 4 and 25°C. rPA3 + 4 interacts with antibodies to rPA83 both individually and as a part of a complex with SPs. The results obtained can underpin the development of a recombinant vaccine with a full-size modified rPA (with similar amino acid substitutions that stabilize the protein) and SPs

Novel Universal Recombinant Rotavirus A Vaccine Candidate: Evaluation of Immunological Properties

Rotavirus infection is a leading cause of severe dehydrating gastroenteritis in children under 5 years of age. Although rotavirus-associated mortality has decreased considerably because of the introduction of the worldwide rotavirus vaccination, the global burden of rotavirus-associated gastroenteritis remains high. Current vaccines have a number of disadvantages; therefore, there is a need for innovative approaches in rotavirus vaccine development. In the current study, a universal recombinant rotavirus antigen (URRA) for a novel recombinant vaccine candidate against rotavirus A was obtained and characterised. This antigen included sequences of the VP8* subunit of rotavirus spike protein VP4. For the URRA, for the first time, two approaches were implemented simultaneously—the application of a highly conserved neutralising epitope and the use of the consensus of the extended protein’s fragment. The recognition of URRA by antisera to patient-derived field rotavirus isolates was proven. Plant virus-based spherical particles (SPs), a novel, effective and safe adjuvant, considerably enhanced the immunogenicity of the URRA in a mouse model. Given these facts, a URRA + SPs vaccine candidate is regarded as a prospective basis for a universal vaccine against rotavirus

Magnetic resonance imaging for diagnosing a rare disease: incontinentia pigmenti (Bloch–Sulzberger syndrome) on the example of a clinical case

Incontinentia pigmenti, also known as Bloch–Sulzberger syndrome, is a rare hereditary disease characterized by typical skin rashes and involvement of other organs and systems. Magnetic resonance imaging stands as the primary method for visualizing the structural pathology of the brain and predicting neurological manifestations in an affected child. Diagnosing incontinentia pigmenti predominantly falls within the domain of dermatologists; verification is performed by molecular genetic analysis of the IKBKG gene. This study involved magnetic resonance imaging of the brain in a patient with skin rashes, characteristic of Bloch–Sulzberger syndrome, and deletion in the IKBKG gene, where numerous foci of ischemia, hemorrhages, and lesions of the tracts were detected. Magnetic resonance imaging of the brain in patients with Bloch–Sulzberger syndrome is used to evaluate the severity of damage to the brain substance, which makes it possible to explain the cause of neurological symptoms and correct habilitation, as well as predict the development of the child

Proteasome functioning in breast cancer: connection with clinical-pathological factors.

Breast cancer is one of four oncology diseases that are most widespread in the world. Moreover, breast cancer is one of leading causes of cancer-related deaths in female population within economically developed regions of the world. So far, detection of new mechanisms of breast cancer development is very important for discovery of novel areas in which therapy approaches may be elaborated. The objective of the present study is to investigate involvement of proteasomes, which cleave up to 90% of cellular proteins and regulate numerous cellular processes, in mechanisms of breast cancer development. Proteasome characteristics in 106 patient breast carcinomas and adjacent tissues, as well as relationships of detected proteasome parameters with clinical-pathological factors, were investigated. Proteasome chymotrypsin-like activity was evaluated by hydrolysis of fluorogenic peptide Suc-LLVY-AMC. The expression of proteasome subunits was studied by Western-blotting and immunohistochemistry. The wide range of chymotrypsin-like activity in tumors was detected. Activity in tumors was higher if compared to adjacent tissues in 76 from 106 patients. Multiple analysis of generalized linear models discovered that in estrogen α-receptor absence, tumor growth was connected with the enhanced expression of proteasome immune subunit LMP2 and proteasome activator PA700 in tumor (at 95% confidence interval). Besides, by this analysis we detected some phenomena in adjacent tissue, which are important for tumor growth and progression of lymph node metastasis in estrogen α-receptor absence. These phenomena are related to the enhanced expression of activator PA700 and immune subunit LMP7. Thus, breast cancer development is connected with functioning of immune proteasome forms and activator PA700 in patients without estrogen α-receptors in tumor cells. These results could indicate a field for search of new therapy approaches for this category of patients, which has the worst prognosis of health recovery

Dependence of ChTL activity difference in tumor and adjacent tissue on ChTL activity in breast samples.

<p>(A) F(1) = 519.763; <i>p</i><0.001. (B) F(1) = 0.947; <i>p</i> = 0.333. Calculated at 95% confidence.</p