Stigmasterol as a potential biomarker for amphotericin B resistance in Leishmania donovani

2019 ◽  
Vol 75 (4) ◽  
pp. 942-950 ◽  
Author(s):  
Ruby Bansal ◽  
Shib Sankar Sen ◽  
Rohini Muthuswami ◽  
Rentala Madhubala
Keyword(s):  
Visceral Leishmaniasis ◽  
Amphotericin B ◽  
Resistant Strain ◽  
Drug Targets ◽  
Leishmania Donovani ◽  
New Drugs ◽  
Axenic Amastigotes ◽  
Potential Biomarker

Abstract Background Leishmania donovani, a protozoan parasite, is the primary causative agent for visceral leishmaniasis. Toxicity and increased resistance to existing drugs have led to an urgent need for identifying new drugs and drug targets. Understanding the risks and mechanisms of resistance is of great importance. Amphotericin B (AmB) is a polyene antimicrobial, the mainstay therapy for visceral leishmaniasis in several parts of India. Objectives In the present study, we established a line of AmB-resistant L. donovani promastigotes in vitro and demonstrated the molecular basis of resistance to AmB. Methods AmB-resistant promastigotes were generated and characterized to evaluate the mechanism of resistance to AmB. We examined the sterol composition of the promastigotes and the axenic amastigotes derived from the WT and AmB-resistant promastigotes. The role of the plant-like C-22 desaturase responsible for stigmasterol production was also evaluated in the AmB-resistant strain. Results The IC50 for resistant cells was four times higher than for the WT. AmB-resistant promastigotes showed an increase in the conversion of β-sitosterol into stigmasterol. The presence of higher amounts of stigmasterol in resistant promastigotes, as well as in axenic amastigotes, signifies its role in AmB resistance in Leishmania. The resistant strain showed reduced infectivity in vitro. Conclusions We have elucidated the mode of action and resistance mechanisms to the drug. However, further work is required to validate the potential role of stigmasterol in resistance and to help develop a diagnostic kit that can assist in diagnosing potentially resistant lines in the field.

scholarly journals Studies on the Antileishmanial Mechanism of Action of the Arylimidamide DB766: Azole Interactions and Role of CYP5122A1

2014 ◽  
Vol 58 (8) ◽  
pp. 4682-4689 ◽  
Author(s):  
Trupti Pandharkar ◽  
Xiaohua Zhu ◽  
Radhika Mathur ◽  
Jinmai Jiang ◽  
Thomas D. Schmittgen ◽  
...  
Keyword(s):  
Cytochrome P450 ◽  
Mechanism Of Action ◽  
Leishmania Donovani ◽  
Synergistic Activity ◽  
Wild Type ◽  
Content Type ◽  
Axenic Amastigotes ◽  
Intracellular Parasites

ABSTRACTArylimidamides (AIAs) are inspired by diamidine antimicrobials but show superior activity against intracellular parasites. The AIA DB766 {2,5-bis[2-(2-i-propoxy)-4-(2-pyridylimino)aminophenyl]furan hydrochloride} displays outstanding potency against intracellularLeishmaniaparasites and is effective in murine and hamster models of visceral leishmaniasis when given orally, but its mechanism of action is unknown. In this study, through the use of continuous DB766 pressure, we raisedLeishmania donovaniaxenic amastigotes that displayed 12-fold resistance to this compound. These DB766-resistant (DB766R) parasites were 2-fold more sensitive to miltefosine than wild-type organisms and were hypersensitive to the sterol 14α-demethylase (CYP51) inhibitors ketoconazole and posaconazole (2,000-fold more sensitive and over 12,000-fold more sensitive than the wild type, respectively). Western blot analysis of DB766R parasites indicated that while expression of CYP51 is slightly increased in these organisms, expression of CYP5122A1, a recently identified cytochrome P450 associated with ergosterol metabolism inLeishmania, is dramatically reduced in DB766R parasites.In vitrosusceptibility assays demonstrated that CYP5122A1 half-knockoutL. donovanipromastigotes were significantly less susceptible to DB766 and more susceptible to ketoconazole than their wild-type counterparts, consistent with observations in DB766R parasites. Further, DB766-posaconazole combinations displayed synergistic activity in both axenic and intracellularL. donovaniamastigotes. Taken together, these studies implicate CYP5122A1 in the antileishmanial action of the AIAs and suggest that DB766-azole combinations are potential candidates for the development of synergistic antileishmanial therapy.

scholarly journals Immunoadjuvant Chemotherapy of Visceral Leishmaniasis in Hamsters Using Amphotericin B-Encapsulated Nanoemulsion Template-Based Chitosan Nanocapsules

2013 ◽  
Vol 57 (4) ◽  
pp. 1714-1722 ◽  
Author(s):  
Shalini Asthana ◽  
Anil K. Jaiswal ◽  
Pramod K. Gupta ◽  
Vivek K. Pawar ◽  
Anuradha Dube ◽  
...  
Keyword(s):  
Visceral Leishmaniasis ◽  
Amphotericin B ◽  
Leishmania Donovani ◽  
Transforming Growth Factor ◽  
Treatment Options ◽  
Interleukin 12 ◽  
Necrosis Factor Alpha ◽  
Content Type

ABSTRACTThe accessible treatment options for life-threatening neglected visceral leishmaniasis (VL) disease have problems with efficacy, stability, adverse effects, and cost, making treatment a complex issue. Here we formulated nanometric amphotericin B (AmB)-encapsulated chitosan nanocapsules (CNC-AmB) using a polymer deposition technique mediated by nanoemulsion template fabrication. CNC-AmB exhibited good steric stabilityin vitro, where the chitosan content was found to be efficient at preventing destabilization in the presence of protein and Ca2+. A toxicity study on the model cell line J774A and erythrocytes revealed that CNC-AmB was less toxic than commercialized AmB formulations such as Fungizone and AmBisome. The results ofin vitro(macrophage-amastigote system; 50% inhibitory concentration [IC50], 0.19 ± 0.04 μg AmB/ml) andin vivo(Leishmania donovani-infected hamsters; 86.1% ± 2.08% parasite inhibition) experiments in conjunction with effective internalization by macrophages illustrated the efficacy of CNC-AmB at augmenting antileishmanial properties. Quantitative mRNA analysis by real-time PCR (RT-PCR) showed that the improved effect was synergized with the upregulation of tumor necrosis factor alpha (TNF-α), interleukin-12 (IL-12), and inducible nitric oxide synthase and with the downregulation of transforming growth factor β (TGF-β), IL-10, and IL-4. These research findings suggest that a cost-effective CNC-AmB immunoadjuvant chemotherapeutic delivery system could be a viable alternative to the current high-cost commercial lipid-based formulations.

scholarly journals In Vitro Evaluation of Antileishmanial Activity of Computationally Screened Compounds against Ascorbate Peroxidase To Combat Amphotericin B Drug Resistance

2017 ◽  
Vol 61 (7) ◽  
Author(s):  
Rani Mansuri ◽  
Ashish Kumar ◽  
Sindhuprava Rana ◽  
Bhavana Panthi ◽  
M. Yousuf Ansari ◽  
...  
Keyword(s):  
Amphotericin B ◽  
Ascorbate Peroxidase ◽  
Leishmania Donovani ◽  
Harmful Effect ◽  
Annexin V ◽  
Antileishmanial Activity ◽  
New Drugs ◽  
Content Type ◽  
Lipinski’S Rule

ABSTRACT In visceral leishmaniasis (VL), the host macrophages generate oxidative stress to destroy the pathogen, while Leishmania combats the harmful effect of radicals by redox homeostasis through its unique trypanothione cascade. Leishmania donovani ascorbate peroxidase (LdAPx) is a redox enzyme that regulates the trypanothione cascade and detoxifies the effect of H2O2. The absence of an LdAPx homologue in humans makes it an excellent drug target. In this study, the homology model of LdAPx was built, including heme, and diverse compounds were prefiltered (PAINS, ADMET, and Lipinski's rule of five) and thereafter screened against the LdAPx model. Compounds having good affinity in terms of the Glide XP (extra precision) score were clustered to select diverse compounds for experimental validation. A total of 26 cluster representatives were procured and tested on promastigote culture, yielding 12 compounds with good antileishmanial activity. Out of them, six compounds were safer on the BALB/c peritoneal macrophages and were also effective against disease-causing intracellular amastigotes. Three out of six compounds inhibited recombinant LdAPx in a noncompetitive manner and also demonstrated partial reversion of the resistance property in an amphotericin B (AmB)-resistant strain, which may be due to an increased level of reactive oxygen species (ROS) and decrease of glutathione (GSH) content. However, inhibition of LdAPx in resistant parasites enhanced annexin V staining and activation of metacaspase-like protease activity, which may help in DNA fragmentation and apoptosis-like cell death. Thus, the present study will help in the search for specific hits and templates of potential therapeutic interest and therefore may facilitate the development of new drugs for combination therapy against VL.

In-vitro evaluation of nano-liposomal formulation of Fluconazole and Amphotericin B against visceral leishmaniasis

2021 ◽  
pp. 4929-4933
Author(s):  
Atul Tripathi ◽  
Amber Vyas
Keyword(s):  
Visceral Leishmaniasis ◽  
Amphotericin B ◽  
Cellular Uptake ◽  
Leishmania Donovani ◽  
In Vitro Release ◽  
Physicochemical Characterization ◽  
Production Costs ◽  
Liposomal Formulation

Objectives: The aim of the present study was to compare the efficacy of a dual and single drug loaded nano-liposomal formulation of Amphotericin B and Fluconazole for the treatment of visceral leishmaniasis with plain drugs. Methods: We have formulated nano-liposomes (200-250 nm) from Amphotericin B and Fluconazole using dry film hydration method and have tested their efficacy on promastigotes and amastigotes of Leishmania donovani strain. Physicochemical characterization, entrapment study, stability study, in-vitro release study, in-vitro macrophagic uptake studies (Confocal microscopy) and in-vitro antileishmanial activity were evaluated for various formulations containing Amphotericin B and Fluconazole. Results: The in-vitro cellular uptake confocal studies revealed that NR-loaded AmpB + Flu nanoliposomes have enhanced cellular uptake of formulation. The in-vitro inhibition of promastigotes and amastigotes with liposome containing both Amphotericin B and Fluconazole was significantly more than with liposomes containing individual drugs. The IC50 and CC50 of AmpB + Flu nanoliposomes against promastigotes was found to be 3.308μg/mL and 73.48μg/mL respectively, while the IC50 against axenic and intramacrophagic amastigotes was found to be 3.412 and 3.7028μg/mL respectively. Conclusion: In conclusion, Liposomal formulation containing both Amphotericin B and Fluconazole had significantly greater efficacy than conventional combination and other formulation with individual drugs. Current dual drug loaded formulation may have a favourable safety profile, and if production costs are low, it may prove to be a feasible alternative to currently available therapy after in-vivo testing.

scholarly journals Hsa_circ_0005273 facilitates breast cancer tumorigenesis by regulating YAP1-hippo signaling pathway

2021 ◽  
Vol 40 (1) ◽  
Author(s):  
Xuehui Wang ◽  
Changle Ji ◽  
Jiashu Hu ◽  
Xiaochong Deng ◽  
Wenfang Zheng ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Signaling Pathway ◽  
Cell Lines ◽  
Luciferase Reporter ◽  
Circular Rnas ◽  
Hippo Signaling ◽  
Hippo Signaling Pathway ◽  
Potential Biomarker

Abstract Background Circular RNAs (circRNAs), a novel class of endogenous RNAs, have shown to participate in the development of breast cancer (BC). Hsa_circ_0005273 is a circRNA generated from several exons of PTK2. However, the potential functional role of hsa_circ_0005273 in BC remains largely unknown. Here we aim to evaluate the role of hsa_circ_0005273 in BC. Methods The expression level of hsa_circ_0005273 and miR-200a-3p were examined by RT-qPCR in BC tissues and cell lines. The effect of knocking down hsa_circ_0005273 in BC cell lines were evaluated by examinations of cell proliferation, migration and cell cycle. In addition, xenografts experiment in nude mice were performed to evaluate the effect of hsa_circ_0005273 in BC. RNA immunoprecipitation assay, RNA probe pull-down assay, luciferase reporter assay and fluorescence in situ hybridization were conducted to confirm the relationship between hsa_circ_0005273, miR-200a-3p and YAP1. Results Hsa_circ_0005273 is over-expressed in BC tissues and cell lines, whereas miR-200a-3p expression is repressed. Depletion of hsa_circ_0005273 inhibited the progression of BC cells in vitro and in vivo, while overexpression of hsa_circ_0005273 exhibited the opposite effect. Importantly, hsa_circ_0005273 upregulated YAP1 expression and inactivated Hippo pathway via sponging miR-200a-3p to promote BC progression. Conclusions Hsa_circ_0005273 regulates the miR-200a-3p/YAP1 axis and inactivates Hippo signaling pathway to promote BC progression, which may become a potential biomarker and therapeutic target.

scholarly journals Cathepsin L plays a key role in SARS-CoV-2 infection in humans and humanized mice and is a promising target for new drug development

2021 ◽  
Vol 6 (1) ◽  
Author(s):  
Miao-Miao Zhao ◽  
Wei-Li Yang ◽  
Fang-Yuan Yang ◽  
Li Zhang ◽  
Wei-Jin Huang ◽  
...  
Keyword(s):  
Drug Development ◽  
Cathepsin L ◽  
Human Cells ◽  
New Drugs ◽  
Disease Course ◽  
Promising Target ◽  
First Time

AbstractTo discover new drugs to combat COVID-19, an understanding of the molecular basis of SARS-CoV-2 infection is urgently needed. Here, for the first time, we report the crucial role of cathepsin L (CTSL) in patients with COVID-19. The circulating level of CTSL was elevated after SARS-CoV-2 infection and was positively correlated with disease course and severity. Correspondingly, SARS-CoV-2 pseudovirus infection increased CTSL expression in human cells in vitro and human ACE2 transgenic mice in vivo, while CTSL overexpression, in turn, enhanced pseudovirus infection in human cells. CTSL functionally cleaved the SARS-CoV-2 spike protein and enhanced virus entry, as evidenced by CTSL overexpression and knockdown in vitro and application of CTSL inhibitor drugs in vivo. Furthermore, amantadine, a licensed anti-influenza drug, significantly inhibited CTSL activity after SARS-CoV-2 pseudovirus infection and prevented infection both in vitro and in vivo. Therefore, CTSL is a promising target for new anti-COVID-19 drug development.

scholarly journals The Role of Collagen Triple Helix Repeat-Containing 1 Protein (CTHRC1) in Rheumatoid Arthritis

2021 ◽  
Vol 22 (5) ◽  
pp. 2426
Author(s):  
Askhat Myngbay ◽  
Limara Manarbek ◽  
Steve Ludbrook ◽  
Jeannette Kunz
Keyword(s):  
Rheumatoid Arthritis ◽  
Drug Targets ◽  
Triple Helix ◽  
Cancer Metastasis ◽  
Bone Erosion ◽  
Cartilage Destruction ◽  
Patient Treatment ◽  
Collagen Triple Helix ◽  
Potential Biomarker

Rheumatoid arthritis (RA) is a chronic autoimmune disease causing inflammation of joints, cartilage destruction and bone erosion. Biomarkers and new drug targets are actively sought and progressed to improve available options for patient treatment. The Collagen Triple Helix Repeat Containing 1 protein (CTHRC1) may have an important role as a biomarker for rheumatoid arthritis, as CTHRC1 protein concentration is significantly elevated in the peripheral blood of rheumatoid arthritis patients compared to osteoarthritis (OA) patients and healthy individuals. CTHRC1 is a secreted glycoprotein that promotes cell migration and has been implicated in arterial tissue-repair processes. Furthermore, high CTHRC1 expression is observed in many types of cancer and is associated with cancer metastasis to the bone and poor patient prognosis. However, the function of CTHRC1 in RA is still largely undefined. The aim of this review is to summarize recent findings on the role of CTHRC1 as a potential biomarker and pathogenic driver of RA progression. We will discuss emerging evidence linking CTHRC1 to the pathogenic behavior of fibroblast-like synoviocytes and to cartilage and bone erosion through modulation of the balance between bone resorption and repair.

Role of glycine-containing oligopeptides in blood cells aggregation stimulated by adrenaline

2017 ◽  
Author(s):  
М. Голубева
Keyword(s):  
Body Weight ◽  
Platelet Aggregation ◽  
Blood Cells ◽  
Regulatory Peptides ◽  
Pituitary Hormones ◽  
New Drugs ◽  
Male Rats ◽  
Current Interest

Введение. Изменение реологических свойств крови характерно для различных заболеваний, многие из которых связаны с нарушением реологии и, прежде всего, с изменением агрегации эритроцитов. Целью исследования было сравнение влияния малых регуляторных пептидов, являющихся фрагментами нейрогормонов, на агрегацию эритроцитов и тромбоцитов под действием адреналина в экспериментах in vitro. Материалы и методы. Эксперименты проводили на белых беспородных крысах-самцах, массой тела 180-200 г. Использовали пептиды, представляющие собой С-концевые фрагменты вазопрессина (Pro-Arg-Gly-NH2) и окситоцина (Pro-Leu-Gly-NH2). Результаты. Показано, что малые регуляторные пептиды, являющиеся продуктами протеолиза нейропептидов, оказывают существенное влияние на агрегатное состояние клеток крови. При сравнении влияния пептидов на агрегацию клеток крови, стимулированную адреналином, установлено, что фрагмент вазопрессина Pro-Arg-Gly-NH2 вызывал достоверное усиление агрегации как эритроцитов, так и тромбоцитов; тогда как фрагмент окситоцина Pro-Leu-Gly-NH2 ингибировал только агрегацию эритроцитов, не изменяя агрегации тромбоцитов. Заключение. Изучение путей поэтапного протеолиза пептидов может привести к разработке новых препаратов для направленной коррекции различных нарушений в организме, поэтому изучение эффектов С-концевых фрагментов гипофизарных гормонов на гемостаз является актуальным. Introduction. The changing of blood rheological properties is typical for various diseases; many of them are associated with rheology disorder and primarily with change of erythrocytes aggregation. The aim was to compare the effect of small regulatory peptides (fragments of neurohormones) on the aggregation of erythrocytes and platelets under adrenaline action in experiments in vitro. Materials and methods. Experiments were conducted on white outbred male rats, body weight 180-200 g. We used 2 peptides – C-terminal fragments of vasopressin (Pro-Arg-Gly-NH2) and oxytocin (Pro-Leu-Gly-NH2). Results. It was shown that small regulatory peptides (they are products of neuropeptides proteolysis) had a significant effect on blood cells aggregation. We compared the peptides effect on blood cells aggregation stimulated by adrenaline. It was found that vasopressin fragment Pro-Arg-Gly-NH2 significantly increased both erythrocytes and platelets aggregation, while oxytocin fragment Pro-Leu-Gly-NH2 inhibited only erythrocytes aggregation without changing of platelet aggregation. Conclusion. Investigation of phased peptides proteolysis may result in the development of new drugs for targeted correction of various disturbances. So it is of current interest to study the effects of C-terminal fragments of pituitary hormones on hemostasis.

scholarly journals Preclinical candidate for the treatment of visceral leishmaniasis that acts through proteasome inhibition

2019 ◽  
Vol 116 (19) ◽  
pp. 9318-9323 ◽  
Author(s):  
Susan Wyllie ◽  
Stephen Brand ◽  
Michael Thomas ◽  
Manu De Rycker ◽  
Chun-wa Chung ◽  
...  
Keyword(s):  
Visceral Leishmaniasis ◽  
Leishmania Donovani ◽  
Proteasome Inhibition ◽  
New Drugs ◽  
Resource Poor Setting ◽  
Drug Candidate ◽  
Proteasome Subunits ◽  
Current Therapies ◽  
Mouse Model Of Infection

Visceral leishmaniasis (VL), caused by the protozoan parasitesLeishmania donovaniandLeishmania infantum, is one of the major parasitic diseases worldwide. There is an urgent need for new drugs to treat VL, because current therapies are unfit for purpose in a resource-poor setting. Here, we describe the development of a preclinical drug candidate, GSK3494245/DDD01305143/compound 8, with potential to treat this neglected tropical disease. The compound series was discovered by repurposing hits from a screen against the related parasiteTrypanosoma cruzi. Subsequent optimization of the chemical series resulted in the development of a potent cidal compound with activity against a range of clinically relevantL. donovaniandL. infantumisolates. Compound 8 demonstrates promising pharmacokinetic properties and impressive in vivo efficacy in our mouse model of infection comparable with those of the current oral antileishmanial miltefosine. Detailed mode of action studies confirm that this compound acts principally by inhibition of the chymotrypsin-like activity catalyzed by the β5 subunit of theL. donovaniproteasome. High-resolution cryo-EM structures of apo and compound 8-boundLeishmania tarentolae20S proteasome reveal a previously undiscovered inhibitor site that lies between the β4 and β5 proteasome subunits. This induced pocket exploits β4 residues that are divergent between humans and kinetoplastid parasites and is consistent with all of our experimental and mutagenesis data. As a result of these comprehensive studies and due to a favorable developability and safety profile, compound 8 is being advanced toward human clinical trials.

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