A phase 3, single-arm, open-label study to evaluate the safety, tolerability, and immunogenicity of a 15-valent pneumococcal conjugate vaccine, V114, in a 3+1 regimen in healthy infants in South Korea (PNEU-PED-KOR).

There is an ongoing burden of pneumococcal disease in children despite the use of pneumococcal conjugate vaccines (PCVs). This phase 3, open-label, single-arm, multisite, descriptive study was designed to evaluate the safety and immunogenicity of a 3 + 1 regimen of V114 (VAXNEUVANCE™), a 15-valent PCV, in South Korean infants and toddlers. Adverse events (AEs) were reported for 14 d following any vaccination, and throughout the study period for serious AEs. Serotype-specific immunoglobulin G (IgG) response rates (proportion of participants meeting an IgG threshold value of ≥0.35 µg/mL) and geometric mean concentrations (GMCs) for the 15 serotypes at 30 d postdose 3 (PD3) and at 30 d postdose 4 (PD4) were evaluated as endpoints. Healthy infants enrolled at 42-90 d after birth were vaccinated with V114 (N = 57). The most commonly reported AEs were those solicited in the trial. The majority of reported AEs were transient and of mild or moderate intensity. Few serious AEs were reported; none were vaccine related. No participants died nor discontinued the study vaccine because of an AE. V114 was immunogenic for all 15 serotypes contained in the vaccine, as assessed by IgG response rates at 30 d PD3 and IgG GMCs at 30 d PD3 and at 30 d PD4. V114 was well tolerated and immunogenic when administered as a 3 + 1 regimen in healthy South Korean infants and toddlers.

Despite the use of pneumococcal vaccines, the burden of pneumococcal disease in children persists. V114, a 15-valent pneumococcal conjugate vaccine, was immunogenic and well-tolerated in healthy South Korean infants and toddlers.

Health and economic burden associated with 15-valent pneumococcal conjugate vaccine serotypes in Korea and Hong Kong.

Use of pneumococcal conjugate vaccines (PCVs) has greatly reduced the incidence of invasive pneumococcal disease (IPD). V114 (VAXNEUVANCE™, Merck Sharp & Dohme Corp. a subsidiary of Merck & Co. Inc. Kenilworth, NJ, USA) is a 15-valent PCV currently approved in adults in the United States, containing the 13 serotypes in licensed PCV13 and 2 additional serotypes (22F and 33F) which are important contributors to residual pneumococcal disease. This study quantified the health and economic burden of IPD attributable to V114 serotypes in hypothetical birth cohorts from Korea and Hong Kong. A Markov model was used to estimate the case numbers and costs of IPD in unvaccinated birth cohorts over 20 years. The model was applied to 3 scenarios in Korea (pre-PCV7, pre-PCV13, and post-PCV13) and to 2 scenarios in Hong Kong (pre-PCV7 and post-PCV13). For Korea, the model predicted 62, 26, and 8 IPD cases attributable to V114 serotypes in the pre-PCV7, pre-PCV13, and post-PCV13 scenarios, respectively. Costs of V114-type IPD fell from $1.691 million pre-PCV7 to $.212 million post-PCV13. For Hong Kong, the model estimated 62 V114-associated IPD cases in the pre-PCV7 scenario and 46 in the post-PCV13 scenario. Costs attributed to all V114 serotypes were $2.322 million and $1.726 million in the pre-PCV7 and post-PCV13 periods, respectively. Vaccine-type serotypes are predicted to cause continuing morbidity and cost in Korea (19A) and Hong Kong (3 and 19A). New pediatric pneumococcal vaccines must continue to protect against serotypes in licensed vaccines to maintain disease reduction, while extending coverage to non-vaccine serotypes.

Drug-like molecules with anti-trypanothione synthetase activity identified by high throughput screening.

Trypanothione synthetase (TryS) catalyses the synthesis of N1,N8-bis(glutathionyl)spermidine (trypanothione), which is the main low molecular mass thiol supporting several redox functions in trypanosomatids. TryS attracts attention as molecular target for drug development against pathogens causing severe and fatal diseases in mammals. A drug discovery campaign aimed to identify and characterise new inhibitors of TryS with promising biological activity was conducted. A large compound library (n = 51,624), most of them bearing drug-like properties, was primarily screened against TryS from Trypanosoma brucei (TbTryS). With a true-hit rate of 0.056%, several of the TbTryS hits (IC50 from 1.2 to 36 µM) also targeted the homologue enzyme from Leishmania infantum and Trypanosoma cruzi (IC50 values from 2.6 to 40 µM). Calmidazolium chloride and Ebselen stand out for their multi-species anti-TryS activity at low µM concentrations (IC50 from 2.6 to 13.8 µM). The moieties carboxy piperidine amide and amide methyl thiazole phenyl were identified as novel TbTryS inhibitor scaffolds. Several of the TryS hits presented one-digit µM EC50 against T. cruzi and L. donovani amastigotes but proved cytotoxic against the human osteosarcoma and macrophage host cells (selectivity index ≤ 3). In contrast, seven hits showed a significantly higher selectivity against T. b. brucei (selectivity index from 11 to 182). Non-invasive redox assays confirmed that Ebselen, a multi-TryS inhibitor, induces an intracellular oxidative milieu in bloodstream T. b. brucei. Kinetic and mass spectrometry analysis revealed that Ebselen is a slow-binding inhibitor that modifies irreversible a highly conserved cysteine residue from the TryS's synthetase domain. The most potent TbTryS inhibitor (a singleton containing an adamantine moiety) exerted a non-covalent, non-competitive (with any of the substrates) inhibition of the enzyme. These data feed the drug discovery pipeline for trypanosomatids with novel and valuable information on chemical entities with drug potential.

Evaluation of Parameters Impacting Drug Susceptibility in Intracellular Trypanosoma cruzi Assay Protocols.

In order to understand the key parameters influencing drug susceptibility, different Trypanosoma cruzi assay protocols were evaluated using a comparative assay design. The assays compared in this study were an image-based intracellular T. cruzi assay quantified through an image-mining algorithm and an intracellular assay utilizing a ß-galactosidase-expressing T. cruzi strain. Thirty-one reference compounds known to exhibit activities against intracellular T. cruzi were used as benchmarks. Initial comparison using EC50 values from two assays showed a very poor correlation, with an R2 value of 0.005. Nitroheterocyclics and CYP51 inhibitors were inactive in an image-based assay, but were highly active in a colorimetric assay. In order to identify the differentiating factor, we synchronized the compound-parasite incubation times or the sequential cell and compound seeding schemes between assays, but the correlation remained low. A high correlation ( R2 = 0.86) was observed only after both compound incubation time and cell seeding were synchronized between assays. Further analysis of EC50 and maximum inhibition values showed that nitroheterocyclics and CYP51 inhibitors exhibit relatively large deviations in activity between experimental protocols routinely used for in vitro intracellular T. cruzi assays. These findings suggest that the factors mentioned are critical when designing an intracellular T. cruzi assay.

Antileishmanial Mechanism of Diamidines Involves Targeting Kinetoplasts.

Leishmaniasis is a disease caused by pathogenic Leishmania parasites; current treatments are toxic and expensive, and drug resistance has emerged. While pentamidine, a diamidine-type compound, is one of the treatments, its antileishmanial mechanism of action has not been investigated in depth. Here we tested several diamidines, including pentamidine and its analog DB75, against Leishmania donovani and elucidated their antileishmanial mechanisms. We identified three promising new antileishmanial diamidine compounds with 50% effective concentrations (EC50s) of 3.2, 3.4, and 4.5 µM, while pentamidine and DB75 exhibited EC50s of 1.46 and 20 µM, respectively. The most potent antileishmanial inhibitor, compound 1, showed strong DNA binding properties, with a shift in the melting temperature (ΔTm) of 24.2°C, whereas pentamidine had a ΔTm value of 2.1°C, and DB75 had a ΔTm value of 7.7°C. Additionally, DB75 localized in L. donovani kinetoplast DNA (kDNA) and mitochondria but not in nuclear DNA (nDNA). For 2 new diamidines, strong localization signals were observed in kDNA at 1 µM, and at higher concentrations, the signals also appeared in nuclei. All tested diamidines showed selective and dose-dependent inhibition of kDNA, but not nDNA, replication, likely by inhibiting L. donovani topoisomerase IB. Overall, these results suggest that diamidine antileishmanial compounds exert activity by accumulating toward and blocking replication of parasite kDNA.

Phenolic Constituents of Medicinal Plants with Activity against Trypanosoma brucei.

Neglected tropical diseases (NTDs) affect over one billion people all over the world. These diseases are classified as neglected because they impact populations in areas with poor financial conditions and hence do not attract sufficient research investment. Human African Trypanosomiasis (HAT or sleeping sickness), caused by the parasite Trypanosoma brucei, is one of the NTDs. The current therapeutic interventions for T. brucei infections often have toxic side effects or require hospitalization so that they are not available in the rural environments where HAT occurs. Furthermore, parasite resistance is increasing, so that there is an urgent need to identify novel lead compounds against this infection. Recognizing the wide structural diversity of natural products, we desired to explore and identify novel antitrypanosomal chemotypes from a collection of natural products obtained from plants. In this study, 440 pure compounds from various medicinal plants were tested against T. brucei by in a screening using whole cell in vitro assays. As the result, twenty-two phenolic compounds exhibited potent activity against cultures of T. brucei. Among them, eight compounds-4, 7, 11, 14, 15, 18, 20, and 21-showed inhibitory activity against T. brucei, with IC50 values below 5 µM, ranging from 0.52 to 4.70 µM. Based on these results, we attempt to establish some general trends with respect to structure-activity relationships, which indicate that further investigation and optimization of these derivatives might enable the preparation of potentially useful compounds for treating HAT.

In Vitro and In Vivo Investigation of the Inhibition of Trypanosoma brucei Cell Growth by Lipophilic Bisphosphonates.

We report the results of a screen of a library of 925 potential prenyl synthase inhibitors against Trypanosoma brucei farnesyl diphosphate synthase (TbFPPS) and against T. brucei, the causative agent of human African trypanosomiasis. The most potent compounds were lipophilic analogs of the bone resorption drug zoledronate, some of which had submicromolar to low micromolar activity against bloodstream form T. brucei and selectivity indices of up to ∼ 300. We evaluated the effects of two such inhibitors on survival and parasitemia in a T. brucei mouse model of infection and found that survival increased by up to 16 days. We also investigated the binding of three lipophilic bisphosphonates to an expressed TbFPPS using crystallography and investigated the thermodynamics of binding using isothermal titration calorimetry.

Oxa, Thia, Heterocycle, and Carborane Analogues of SQ109: Bacterial and Protozoal Cell Growth Inhibitors.

We synthesized a library of 48 analogs of the Mycobacterium tuberculosis cell growth inhibitor SQ109 in which the ethylene diamine linker was replaced by oxa-, thia- or heterocyclic species, and in some cases, the adamantyl group was replaced by a 1,2-carborane or the N-geranyl group by another hydrophobic species. Compounds were tested against Mycobacterium tuberculosis (H37Rv and/or Erdman), Mycobacterium smegmatis, Bacillus subtilis, Escherichia coli, Saccharomyces cerevisiae, Trypanosoma brucei and two human cell lines (human embryonic kidney, HEK293T, and the hepatocellular carcinoma, HepG2). Most potent activity was found against T. brucei, the causative agent of human African trypanosomiasis, and involved targeting of the mitochondrial membrane potential with 15 SQ109 analogs being more active than was SQ109 in cell growth inhibition, having IC50 values as low as 12 nM (5.5 ng/mL) and a selectivity index of ~300.

In Vitro and in Vivo Activity of Multitarget Inhibitors against Trypanosoma brucei.

We tested a series of amidine and related compounds against Trypanosoma brucei. The most active compound was a biphenyldiamidine that had an EC 50 of 7.7 nM against bloodstream-form parasites. There was little toxicity against two human cell lines with CC 50 > 100 µM. There was also good in vivo activity in a mouse model of infection with 100% survival at 3 mg/kg i.p. The most potent lead blocked replication of kinetoplast DNA (k-DNA), but not nuclear DNA, in the parasite. Some compounds also inhibited the enzyme farnesyl diphosphate synthase (FPPS), and some were uncouplers of oxidative phosphorylation. We developed a computational model for T. brucei cell growth inhibition (R (2) = 0.76) using DNA ΔT m values for inhibitor binding combined with T. brucei FPPS IC 50 values. Overall, the results suggest that it may be possible to develop multitarget drug leads against T. brucei that act by inhibiting both k-DNA replication and isoprenoid biosynthesis.

Synthesis and biological evaluation of 2,3-dihydroimidazo[1,2-a]benzimidazole derivatives against Leishmania donovani and Trypanosoma cruzi.

A high-throughput (HTS) and high-content screening (HCS) campaign of a commercial library identified 2,3-dihydroimidazo[1,2-a]benzimidazole analogues as a novel class of anti-parasitic agents. A series of synthetic derivatives were evaluated for their in vitro anti-leishmanial and anti-trypanosomal activities against Leishmania donovani and Trypanosoma cruzi, which have been known as the causative parasites for visceral leishmaniasis and Chagas disease, respectively. In the case of Leishmania, the compounds were tested in both intracellular amastigote and extracellular promastigote assays. Compounds 4 and 24 showed promising anti-leishmanial activity against intracellular L. donovani (3.05 and 5.29 µM, respectively) and anti-trypanosomal activity against T. cruzi (1.10 and 2.10 µM, respectively) without serious cytotoxicity toward THP-1 and U2OS cell lines.

Secondary metabolites from Vietnamese marine invertebrates with activity against Trypanosoma brucei and T. cruzi.

Marine-derived natural products from invertebrates comprise an extremely diverse and promising source of the compounds from a wide variety of structural classes. This study describes the discovery of five marine natural products with activity against Trypanosoma species by natural product library screening using whole cell in vitro assays. We investigated the anti-trypanosomal activity of the extracts from the soft corals and echinoderms living in Vietnamese seas. Of the samples screened, the methanolic extracts of several marine organisms exhibited potent activities against cultures of Trypanosoma brucei and T. cruzi (EC50 < 5.0 µg/mL). Among the compounds isolated from these extracts, laevigatol B (1) from Lobophytum crassum and L. laevigatum, (24S)-ergost-4-ene-3-one (2) from Sinularia dissecta, astropectenol A (3) from Astropecten polyacanthus, and cholest-8-ene-3ß,5α,6ß,7α-tetraol (4) from Diadema savignyi showed inhibitory activity against T. brucei with EC50 values ranging from 1.57 ± 0.14 to 14.6 ± 1.36 µM, relative to the positive control, pentamidine (EC50 = 0.015 ± 0.003 µM). Laevigatol B (1) and 5α-cholest-8(14)-ene-3ß,7α-diol (5) exhibited also significant inhibitory effects on T. cruzi. The cytotoxic activity of the pure compounds on mammalian cells was also assessed and found to be insignificant in all cases. This is the first report on the inhibitory effects of marine organisms collected in Vietnamese seas against Trypanosoma species responsible for neglected tropical diseases.

An image-based algorithm for precise and accurate high throughput assessment of drug activity against the human parasite Trypanosoma cruzi.

We present a customized high content (image-based) and high throughput screening algorithm for the quantification of Trypanosoma cruzi infection in host cells. Based solely on DNA staining and single-channel images, the algorithm precisely segments and identifies the nuclei and cytoplasm of mammalian host cells as well as the intracellular parasites infecting the cells. The algorithm outputs statistical parameters including the total number of cells, number of infected cells and the total number of parasites per image, the average number of parasites per infected cell, and the infection ratio (defined as the number of infected cells divided by the total number of cells). Accurate and precise estimation of these parameters allow for both quantification of compound activity against parasites, as well as the compound cytotoxicity, thus eliminating the need for an additional toxicity-assay, hereby reducing screening costs significantly. We validate the performance of the algorithm using two known drugs against T.cruzi: Benznidazole and Nifurtimox. Also, we have checked the performance of the cell detection with manual inspection of the images. Finally, from the titration of the two compounds, we confirm that the algorithm provides the expected half maximal effective concentration (EC50) of the anti-T. cruzi activity.

An image-based high-content screening assay for compounds targeting intracellular Leishmania donovani amastigotes in human macrophages.

Leishmaniasis is a tropical disease threatening 350 million people from endemic regions. The available drugs for treatment are inadequate, with limitations such as serious side effects, parasite resistance or high cost. Driven by this need for new drugs, we developed a high-content, high-throughput image-based screening assay targeting the intracellular amastigote stage of different species of Leishmania in infected human macrophages. The in vitro infection protocol was adapted to a 384-well-plate format, enabling acquisition of a large amount of readouts by automated confocal microscopy. The reading method was based on DNA staining and required the development of a customized algorithm to analyze the images, which enabled the use of non-modified parasites. The automated analysis generated parameters used to quantify compound activity, including infection ratio as well as the number of intracellular amastigote parasites and yielded cytotoxicity information based on the number of host cells. Comparison of this assay with one that used the promastigote form to screen 26,500 compounds showed that 50% of the hits selected against the intracellular amastigote were not selected in the promastigote screening. These data corroborate the idea that the intracellular amastigote form of the parasite is the most appropriate to be used in primary screening assay for Leishmania.

Antileishmanial high-throughput drug screening reveals drug candidates with new scaffolds.

Drugs currently available for leishmaniasis treatment often show parasite resistance, highly toxic side effects and prohibitive costs commonly incompatible with patients from the tropical endemic countries. In this sense, there is an urgent need for new drugs as a treatment solution for this neglected disease. Here we show the development and implementation of an automated high-throughput viability screening assay for the discovery of new drugs against Leishmania. Assay validation was done with Leishmania promastigote forms, including the screening of 4,000 compounds with known pharmacological properties. In an attempt to find new compounds with leishmanicidal properties, 26,500 structurally diverse chemical compounds were screened. A cut-off of 70% growth inhibition in the primary screening led to the identification of 567 active compounds. Cellular toxicity and selectivity were responsible for the exclusion of 78% of the pre-selected compounds. The activity of the remaining 124 compounds was confirmed against the intramacrophagic amastigote form of the parasite. In vitro microsomal stability and cytochrome P450 (CYP) inhibition of the two most active compounds from this screening effort were assessed to obtain preliminary information on their metabolism in the host. The HTS approach employed here resulted in the discovery of two new antileishmanial compounds, bringing promising candidates to the leishmaniasis drug discovery pipeline.