Biological evaluation of 9-thioansamitocin P3.

Highly cytotoxic maytansine derivatives are widely used in targeted tumor delivery. Structure-activity studies published earlier suggested the C9 carbinol to be a key element necessary to retain the potency. However, in 1984 a patent was published by Takeda in which the synthesis of 9-thioansamitocyn (AP3SH) was described and its activity in xenograft models was shown. In this article we summarize the results of an extended study of the anti-tumor properties of AP3SH. Like other maytansinoids, it induces apoptosis and arrests the cell cycle in the G2/M phase. It is metabolized in liver microsomes predominately by C3A4 isoform and doesn't inhibit any CYP isoforms except CYP3A4 (midazolam, IC50 7.84 µM). No hERG inhibition, CYP induction or mutagenicity in Ames tests were observed. AP3SH demonstrates high antiproliferative activity against 25 tumor cell lines and tumor growth inhibition in U937 xenograft model. Application of AP3SH as a cytotoxic payload in drug delivery system was demonstrated by us earlier.

The Suppressive Effect of Rebastinib on Triple-negative Breast Cancer Tumors Involves Multiple Mechanisms of Action.

BACKGROUND/AIM: High resistance of triple-negative breast cancer has prompted scientists to look for new targets susceptible to treatment. CDK16 has been suggested as a promising target whose inhibition can lead to tumor growth suppression. Rebastinib, a potent inhibitor of CDK16, has been reported to exhibit anti-tumor activity both in vitro and in vivo. MATERIALS AND METHODS: The anticancer activity of rebastinib was studied in vitro using cell proliferation, cell cycle arrest and cell apoptosis assays and in vivo in xenograft tumor models using MDA-MB-231 and MDA-MB-468-derived tumors. The safety and drug-like properties of rebastinib were assessed using a panel of Absorption, Distribution, Metabolism, and Excretion (ADME) assays, Ames tests, human Ether-a-go-go Related Gene (hERG) experiments and pharmacokinetic studies in mice and rats. RESULTS: Rebastinib demonstrates antitumor activity against breast cancer both in vitro and in vivo. However, the response of the tumor strongly depends on the type of triple-negative breast cancer. Rebastinib-induced cell cycle arrest was observed in G0/G1 phase suggesting a more complex mechanism than just CDK16 inhibition. ADME and PK studies confirmed the drug-like properties and reasonable safety of rebastinib. CONCLUSION: Our studies confirmed rebastinib to be a promising drug candidate for breast cancer treatment with high oral bioavailability and reasonable safety. Our data suggest that the mechanism of action of rebastinib is not limited to CDK16 inhibition but also involves other pathways. This does not diminish the importance of rebastinib as a drug candidate, but reveals the presence of several mechanisms, suggesting a wider scope of possible applications.

Prediction of ceftriaxone MIC in Neisseria gonorrhoeae using DNA microarray technology and regression analysis.

BACKGROUND: Decreased susceptibility of Neisseria gonorrhoeae to extended-spectrum cephalosporins is a major concern. Elucidation of the phenotypic and genetic characteristics of such isolates is a priority task. METHODS: We developed a method for predicting the N. gonorrhoeae ceftriaxone susceptibility level (MICcro) by identifying genetic determinants of resistance using low-density hydrogel microarrays and a regression equation. A training dataset, containing 5631 isolates from the Pathogenwatch database and 181 isolates obtained in the Russian Federation during 2018-19, was used to build a regression model. The regression equation was tested on 14 WHO reference strains. Ceftriaxone resistance determinants for the 448 evaluated clinical isolates collected in Russia were identified using microarray analysis, and MICcro values were calculated using the regression equation and compared with those measured by the serial dilution method. RESULTS: The regression equation for calculating MICcro values included 20 chromosomal resistance determinants. The greatest contributions to the increase in MICcro were shown to be PBP2: Ala-501→Pro, Ala-311→Val, Gly-545→Ser substitutions, Asp(345-346) insertion; and PorB: Gly-120→Arg substitution. The substitutions PBP2: Ala-501→Thr/Val, PorB: Gly-120→Asn/Asp/Lys and PBP1: Leu-421→Pro had weaker effects. For 94.4% of the isolates in the evaluation set, the predicted MICcro was within one doubling dilution of the experimentally determined MICcro. No ceftriaxone-resistant isolates were identified in the analysed samples from Russia, and no interpretative errors were detected in the MICcro calculations. CONCLUSIONS: The developed strategy for predicting ceftriaxone MIC can be used for the continuous surveillance of known and emerging resistant N. gonorrhoeae isolates.

Molecular Typing of Neisseria gonorrhoeae Clinical Isolates in Russia, 2018-2019: A Link Between penA Alleles and NG-MAST Types.

This work aimed to study penA gene polymorphisms in clinical isolates of Neisseria gonorrhoeae collected in Russia in 2018-2019 and the contribution of the penA allele type to susceptibility to ß-lactam antibiotics. A total of 182 isolates were analyzed. penA allele types were determined by sequencing, and the minimum inhibitory concentrations (MICs) of benzylpenicillin and ceftriaxone were measured. The influence of genetic factors on MICs was evaluated by regression analysis. All isolates were susceptible to ceftriaxone, and 40.1% of isolates were susceptible to penicillin. Eleven penA allele types were identified. The mosaic type XXXIV penA allele and the Gly120Lys substitution in PorB made the greatest contributions to increasing the ceftriaxone MIC; the presence of the blaTEM plasmid, Gly120Asp, Ala121Gly/Asn substitutions in PorB, and the adenine deletion in the promoter region of the mtrR gene caused an increase in the penicillin MIC. Among 61 NG-MAST types identified, the most frequent were types 228, 807, 9486, 1993, and 6226. A link between penA alleles and Neisseria gonorrhoeae multi-antigen sequence typing (NG-MAST) types was established. Resistance to two groups of ß-lactam antibiotics was associated with non-identical changes in penA alleles. To prevent the emergence of ceftriaxone resistance in Russia, NG-MAST genotyping must be supplemented with penA allele analysis.

Genetic diversity of Neisseria gonorrhoeae multi-antigen sequence types in Russia and Europe.

OBJECTIVES: The goal of this work was to assess the genetic diversity of Neisseria gonorrhoeae isolates in Russia and Europe and to compare the distribution of the N. gonorrhoeae multi-antigen sequencing types (NG-MAST) of Russian isolates with that of isolates from European countries. METHODS: NG-MAST typing was performed for 804 N. gonorrhoeae isolates collected in Russia in 2013-2018. For isolates from European countries, data from the https://pathogen.watch/collection/eurogasp2013 database were used. RESULTS: Among the isolates from Russia, 296 NG-MAST types were found. A maximum likelihood phylogenetic tree was constructed. Phylogenetic analysis revealed seven major genogroups uniting the most frequent Russian sequence types: G807, G1993, G9476, G14942, G1152, G9486, and G12531. CONCLUSIONS: The NG-MAST type distribution in Russia differed from that in European countries. Most of the Russian isolates had sequence types that were not found in Europe. Only 33% of the Russian isolates belonged to genogroups established for European countries, and the widespread European genogroup G1407 was represented by only nine isolates. Analysis of the Russian isolates belonging to phylogenetically close European genogroups indicated similarities in drug resistance, although no epidemically dangerous drug-resistant clones were found among the Russian isolates.

Resistance of Neisseria gonorrhoeae isolates to beta-lactam antibiotics (benzylpenicillin and ceftriaxone) in Russia, 2015-2017.

The goal of this work was to study the phenotypic susceptibility and resistance determinants of N. gonorrhoeae isolates to beta-lactam antimicrobials (benzylpenicillin and ceftriaxone). A total of 522 clinical isolates collected in Russia in 2015-2017 were analysed for susceptibility using the agar dilution method. DNA loci involved in antimicrobial resistance were identified using DNA microarray analysis and sequencing. Resistance to benzylpenicillin remained high, with 7.7% of isolates resistant (MICpen > 1 mg/L) and 47.5% of isolates showing intermediate susceptibility (MICpen = 0.12-1 mg/L). The most frequent resistance determinant (72.4% isolates) was the Asp345 insertion in penA, both as a single mutation and in combination with other mutations, particularly with the substitution Leu421Pro in ponA (39.0%). Mutations affecting the influx and efflux of drugs were also found, including amino acid substitutions in PorB (26.8% isolates) and delA in the promoter region of mtrR (22.8%). The accumulation of mutations in chromosomal genes (penA, pon, porA, and mtrR) led to a stepwise increase in MICpen to values characteristic of intermediate resistance. The presence of blaTEM plasmids was found in 25 isolates (4.8%), resulting in a strong increase in resistance to penicillin (MICpen > 16 mg/L) compared with the chromosomal mutations; 23 plasmids were of the African type with TEM-1 beta-lactamase, and two plasmids were of the Toronto/Rio type with TEM-135 beta-lactamase. Only three isolates were found with reduced susceptibility to ceftriaxone, with MICcef = 0.12-0.25 mg/L. Sequencing of penA did not reveal mutations associated with resistance to third-generation cephalosporins, and the gene structure was non-mosaic. The majority of isolates (21 of 25) carrying the blaTEM plasmid also contained the conjugative plasmid with tetM (resistance to tetracyclines), consistent with previously reported data that the presence of the conjugative plasmid facilitates the transfer of other plasmids associated with antimicrobial resistance.

Tetracycline resistance of Neisseria gonorrhoeae in Russia, 2015-2017.

The objective of this study was to estimate the tetracycline resistance level in the modern population of Neisseria gonorrhoeae in the Russian Federation, where this drug was removed from the treatment regimen for gonococcal infections in 2003. A total of 401 isolates collected between 2015 and 2017 were analyzed for genetic markers (chromosomal porB, rpsJ and mtrR gene mutations and the plasmid-located tetM gene) involved in tetracycline resistance. Antibiotic susceptibility testing revealed that 19% of the strains were tetracycline resistant (MIC > 1 mg/L) and that 10% of the strains had intermediate susceptibility (0.5 < MIC ≤ 1 mg/L). Various combinations of mutations identified in the rpsJ (Val57Met/Leu), porB (Gly120Lys/Asp/Asn/Thr and Ala121/Asp/Asn/Gly), and mtrR (-35 del A) genes resulted in MIC increases of up to 1.47 mg/L (geometric mean value). The presence of the tetM gene was detected in 29 strains, including 18 tetM genes of the American type and 11 of the Dutch type. The tetM gene was associated with a strong increase in resistance (MIC > 8 mg/L). One N. gonorrhoeae isolate was found to carry a defective tetM gene with an AG deletion at position 1239-1240, а new stop codon was introduced that caused a defect in TetM protein synthesis and decrease in the tetracycline resistance. Phylogenetic trees constructed using N. gonorrhoeae NG-MAST and tetM loci were compared. Complex relationship was observed between the N. gonorrhoeae sequence type and the tetM plasmid type. Partial recovery of N. gonorrhoeae tetracycline susceptibility was observed relative to the proportion of isolates with resistance detected ten years ago (75%). However, the current levels of tetracycline resistance still preclude the renewed use of these drugs for gonococcal infection therapy.