Antileishmanial Mechanism of Diamidines Involves Targeting Kinetoplasts

Yang, Gyongseon,Choi, Gahee,No, Joo Hwan American Society for Microbiology 2016 Antimicrobial Agents and Chemotherapy Vol.60 No.11

<B>ABSTRACT</B><P>Leishmaniasis is a disease caused by pathogenicLeishmaniaparasites; 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, againstLeishmania donovaniand 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 (Δ<I>Tm</I>) of 24.2°C, whereas pentamidine had a Δ<I>Tm</I>value of 2.1°C, and DB75 had a Δ<I>Tm</I>value of 7.7°C. Additionally, DB75 localized inL. donovanikinetoplast 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 inhibitingL. donovanitopoisomerase IB. Overall, these results suggest that diamidine antileishmanial compounds exert activity by accumulating toward and blocking replication of parasite kDNA.</P>

Evaluation of Parameters Impacting Drug Susceptibility in Intracellular <i>Trypanosoma cruzi</i> Assay Protocols

Yang, Gyongseon,Lee, Nakyung,Ioset, Jean-Robert,No, Joo Hwan SAGE Publications 2017 SLAS discovery Vol.22 No.2

<P><B>초록</B></P><P>약물 감수성에 영향을 미치는 주요 매개변수를 이해하기 위해, 비교 분석법 설계를 통해 다양한 <I>크루스파동편모충</I> 분석법 프로토콜을 평가했습니다. 이 연구에서 비교된 분석법은 이미지 마이닝 알고리즘을 통해 정량화된 이미지 기반 세포내 <I>크루스파동편모충</I> 분석법과 β-갈락토시다아제 발현 <I>크루스파동편모충</I> 균주를 이용한 세포내 분석법이었습니다. 세포내 <I>크루스파동편모충</I>에 대해 활성을 나타내는 것으로 알려진 서른 한 개의 표준물질이 기준 화합물로 사용되었습니다. 두 가지 분석법을 통해 얻은 EC<SUB>50</SUB> 값을 사용한 초기 비교에서는 <I>R</I><SUP>2</SUP> 값이 0.005로 상관관계가 매우 낮은 것으로 나타났습니다. 질소헤테로고리(Nitroheterocyclics) 및 CYP51 억제제는 이미지 기반 분석법에서 불활성을 보였으나, 비색(Colorimetric) 분석법에서는 활성도가 높게 나타났습니다. 차별화된 요소를 확인하기 위해, 화합물 기생충 배양 시간을 일치시키거나 분석법 간 세포 및 화합물의 순차적 파종 방식을 일치시켰으나, 상관관계는 여전히 낮았습니다. 분석법 간 화합물 배양 시간과 세포 파종을 모두 일치시킨 후에만 높은 상관관계(<I>R</I><SUP>2</SUP> = 0.86)가 관찰되었습니다. EC<SUB>50</SUB> 및 최대 억제 값을 추가로 분석한 결과, 질소헤테로 고리 및 CYP51 억제제 활성은 시험관내 세포내 <I>크루스파동편모충</I> 분석법에 일반적으로 사용된 실험 프로토콜 간에 상대적으로 큰 편차를 보이는 것으로 나타났습니다. 이러한 결과는 언급된 요소가 세포내 <I>크루스파동편모충</I> 분석법 설계 시 중요하다는 것을 시사합니다.</P>

Trypanosoma brucei Inhibitory Activities of Vietnamese Medicinal Plants

Ya Nan Sun,Seo Young Yang,Hong Xu Li,Xiang Dong Su,Gyongseon Yang,Joo Hwan No,Jong Seong Kang,Young Ho Kim 충남대학교 약학대학 의약품개발연구소 2016 藥學論文集 Vol.31 No.-

A large number of extracts from the medicinal plants growing in Vietnam were screened for their in vitro antitrypanosomal activity toward Trypanosoma brucei. Among them, eighty-seven extracts showed the significant antitrypanosomal activity. Especially, the extracts from Neocinnamomum delavayi, Mallotus cuneatus, and Mallotus api exhibited the highest inhibitory activity against T. brucei (IC50: 0.16, 0.49 and <0.05 μg/mL). Cephalotaxus mannii, Pittosporum floribundum, Cleistanthus aff. annamensis, Wendlangia tinctoria subsp. barbatus, Maesa parvifolia, Alpinia malaccensis, Glycosmis scandens, Mallotus yunnanensis, and Maesa sp also presented potent T. brucei inhibitory activity, with IC50 value ranging from 1.14 to 4.98 μg/mL. Based on these results of screening study, the extracts from N. delavayi, M. cuneatus, M. api, C. mannii, P. floribundum, C. aff. annamensis, W. tinctoria subsp. barbatus, M. parvifolia, A. malaccensis, G. scandens, M. yunnanensis, and Mallotus sp. might be the rich source of antitrypanosomal components. Therefore, further phytochemistry and pharmacological study of these medicinal plants should be researched and developed.

Antileishmanial High-Throughput Drug Screening Reveals Drug Candidates with New Scaffolds

Siqueira-Neto, Jair L.,Song, Ok-Ryul,Oh, Hyunrim,Sohn, Jeong-Hun,Yang, Gyongseon,Nam, Jiyoun,Jang, Jiyeon,Cechetto, Jonathan,Lee, Chang Bok,Moon, Seunghyun,Genovesio, Auguste,Chatelain, Eric,Christoph Public Library of Science 2010 PLoS neglected tropical diseases Vol.4 No.5

<▼1><P>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 <I>Leishmania</I>. Assay validation was done with <I>Leishmania</I> 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. <I>In vitro</I> 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.</P></▼1><▼2><P><B>Author Summary</B></P><P>Every year, more than 2 million people worldwide suffer from leishmaniasis, a neglected tropical disease present in 88 countries. The disease is caused by the single-celled protozoan parasite species of the genus <I>Leishmania</I>, which is transmitted to humans by the bite of the sandfly. The disease manifests itself in a broad range of symptoms, and its most virulent form, named visceral leishmaniasis, is lethal if not treated. Most of the few available treatments for leishmaniasis were developed decades ago and are often toxic, sometimes even leading to the patient's death. Furthermore, the parasite is developing resistance to available drugs, making the discovery and development of new antileishmanials an urgent need. To tackle this problem, the authors of this study employed the use of high-throughput technologies to screen a large library of small, synthetic molecules for their ability to interfere with the viability of <I>Leishmania</I> parasites. This study resulted in the discovery of two novel compounds with leishmanicidal properties and promising drug-like properties, bringing new candidates to the leishmaniasis drug discovery pipeline.</P></▼2>

An Image-Based High-Content Screening Assay for Compounds Targeting Intracellular <i>Leishmania donovani</i> Amastigotes in Human Macrophages

Siqueira-Neto, Jair L.,Moon, Seunghyun,Jang, Jiyeon,Yang, Gyongseon,Lee, Changbok,Moon, Hong Kee,Chatelain, Eric,Genovesio, Auguste,Cechetto, Jonathan,Freitas-Junior, Lucio H. Public Library of Science 2012 PLoS neglected tropical diseases Vol.6 No.6

<▼1><P>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 <I>Leishmania</I> in infected human macrophages. The <I>in vitro</I> 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 <I>Leishmania</I>.</P></▼1><▼2><P><B>Author Summary</B></P><P>Leishmaniasis, one of the most neglected tropical diseases, affects over 2 million people each year. Visceral leishmaniasis (VL), also known as Kala-azar, is caused by the protozoan parasites <I>Leishmania donovani</I> and <I>Leishmania infantum</I> and is fatal if left untreated. Because existing treatments are often ineffective due to parasite resistance and/or toxicity new drugs are urgently needed. Leishmaniasis is transmitted to humans by the bite of an infected sandfly. In the insect vector, parasites exist as flagellated forms—promastigotes, which infect macrophage cells of the human host, where they differentiate to round forms known as amastigotes. Amastigotes and promastigotes are substantially different from a molecular perspective. Drug discovery for leishmaniasis has traditionally been complicated by the unavailability of validated drug targets and of relevant drug assays. Whole cell-based assays have been widely used, as they bypass the need for a validated target. However, they use the insect form of the parasite; indeed, the human form, the intracellular amastigote, is difficult to obtain in the laboratory in quantities compatible with drug screening. We describe here the technical advances that made it possible to adapt the intracellular amastigote form of <I>L. donovani</I> to a drug assay compatible with high-throughput screening.</P></▼2>