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

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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Proteasome Activity Modulates Chromatin Modifications and RNA Polymerase II Phosphorylation To Enhance Glucocorticoid Receptor-Mediated Transcription

Molecular and Cellular Biology ◽

10.1128/mcb.02162-06 ◽

2007 ◽

Vol 27 (13) ◽

pp. 4891-4904 ◽

Cited By ~ 30

Author(s):

H. Karimi Kinyamu ◽

Trevor K. Archer

Keyword(s):

Glucocorticoid Receptor ◽

Rna Polymerase ◽

Rna Polymerase Ii ◽

Gene Transcription ◽

Proteasome Inhibition ◽

The Body ◽

26S Proteasome ◽

Pol Ii ◽

Proteasome Subunits

ABSTRACT The 26S proteasome modulates steroid hormone receptor-dependent gene transcription at least in part by regulating turnover and recycling of receptor/transcriptional DNA complexes, thereby ensuring continued hormone response. For the glucocorticoid receptor (GR), inhibition of proteasome-mediated proteolysis or RNA interference-mediated depletion of specific proteasome subunits results in an increase in gene expression. To facilitate transcription, proteasome inhibition alters at least two features associated with modification of chromatin architecture and gene transcription. First, proteasome inhibition increases trimethyl histone H3K4 levels with a corresponding accumulation of this modification on GR-regulated promoters in vivo. Secondly, global levels of phosphorylated RNA polymerase II (Pol II) increase, together with hormone-dependent association of the phosphorylated Pol II, with the promoter and the body of the activated gene. We propose that apart from modulating receptor turnover, the proteasome directly influences both the transcription machinery and chromatin structure, factors integral to nuclear receptor-regulated gene transcription.

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Novel Arylimidamides for Treatment of Visceral Leishmaniasis

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00250-10 ◽

2010 ◽

Vol 54 (6) ◽

pp. 2507-2516 ◽

Cited By ~ 44

Author(s):

Michael Zhuo Wang ◽

Xiaohua Zhu ◽

Anuradha Srivastava ◽

Qiang Liu ◽

J. Mark Sweat ◽

...

Keyword(s):

Visceral Leishmaniasis ◽

Leishmania Donovani ◽

Leishmania Major ◽

Oral Treatment ◽

Antileishmanial Activity ◽

Repeat Dose ◽

Lead Discovery ◽

Preclinical Development

ABSTRACT Arylimidamides (AIAs) represent a new class of molecules that exhibit potent antileishmanial activity (50% inhibitory concentration [IC50], <1 μM) against both Leishmania donovani axenic amastigotes and intracellular Leishmania, the causative agent for human visceral leishmaniasis (VL). A systematic lead discovery program was employed to characterize in vitro and in vivo antileishmanial activities, pharmacokinetics, mutagenicities, and toxicities of two novel AIAs, DB745 and DB766. They were exceptionally active (IC50 ≤ 0.12 μM) against intracellular L. donovani, Leishmania amazonensis, and Leishmania major and did not exhibit mutagenicity in an Ames screen. DB745 and DB766, given orally, produced a dose-dependent inhibition of liver parasitemia in two efficacy models, L. donovani-infected mice and hamsters. Most notably, DB766 (100 mg/kg of body weight/day for 5 days) reduced liver parasitemia in mice and hamsters by 71% and 89%, respectively. Marked reduction of parasitemia in the spleen (79%) and bone marrow (92%) of hamsters was also observed. Furthermore, these compounds distributed to target tissues (liver and spleen) and had a moderate oral bioavailability (up to 25%), a large volume of distribution, and an elimination half-life ranging from 1 to 2 days in mice. In a repeat-dose toxicity study of mice, there was no indication of liver or kidney toxicity for DB766 from serum chemistries, although mild hepatic cell eosinophilia, hypertrophy, and fatty changes were noted. These results demonstrated that arylimidamides are a promising class of molecules that possess good antileishmanial activity and desirable pharmacokinetics and should be considered for further preclinical development as an oral treatment for VL.

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Efficacy of Spironolactone Treatment in Murine Models of Cutaneous and Visceral Leishmaniasis

Frontiers in Pharmacology ◽

10.3389/fphar.2021.636265 ◽

2021 ◽

Vol 12 ◽

Author(s):

Valter Viana Andrade-Neto ◽

Juliana da Silva Pacheco ◽

Job Domingos Inácio ◽

Elmo Eduardo Almeida-Amaral ◽

Eduardo Caio Torres-Santos ◽

...

Keyword(s):

Visceral Leishmaniasis ◽

Cutaneous Lesion ◽

Parasite Burden ◽

Parasite Load ◽

New Drugs ◽

Drug Candidate ◽

Success Rates ◽

Meglumine Antimoniate ◽

Future Possibility

Translational studies involving the reuse and association of drugs are approaches that can result in higher success rates in the discovery and development of drugs for serious public health problems, including leishmaniasis. If we consider the number of pathogenic species in relation to therapeutic options, this arsenal is still small, and each drug possesses a disadvantage in terms of toxicity, efficacy, price, or treatment regimen. In the search for new drugs, we performed a drug screening of L. amazonensis promastigotes and intracellular amastigotes of fifty available drugs belonging to several classes according to their pharmacophoric group. Spironolactone, a potassium-sparing diuretic, proved to be the most promising drug candidate. After demonstrating the in vitro antileishmanial activity, we evaluated the efficacy on a murine experimental model with L. amazonensis and L. infantum. The treatment controlled the cutaneous lesion and reduced the parasite burden of L. amazonensis significantly, as effectively as meglumine antimoniate. The treatment of experimental visceral leishmaniasis was effective in reducing the parasite load on the main affected organs (spleen and liver) via high doses of spironolactone. The association between spironolactone and meglumine antimoniate promoted better control of the parasite load in the spleen and liver compared to the group treated with meglumine antimoniate alone. These results reveal a possible benefit of the concomitant use of spironolactone and meglumine antimoniate that should be studied more in depth for the future possibility of repositioning for leishmaniasis co-therapy.

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Tissue and host species-specific transcriptional changes in models of experimental visceral leishmaniasis

Wellcome Open Research ◽

10.12688/wellcomeopenres.14867.2 ◽

2019 ◽

Vol 3 ◽

pp. 135 ◽

Cited By ~ 3

Author(s):

Helen Ashwin ◽

Karin Seifert ◽

Sarah Forrester ◽

Najmeeyah Brown ◽

Sandy MacDonald ◽

...

Keyword(s):

Visceral Leishmaniasis ◽

Peripheral Blood ◽

Leishmania Donovani ◽

Systemic Disease ◽

Tissue Response ◽

New Drugs ◽

Immune Checkpoints ◽

Tissue Specific ◽

Transcriptomic Data ◽

Human Visceral Leishmaniasis

Background: Human visceral leishmaniasis, caused by infection with Leishmania donovani or L. infantum, is a potentially fatal disease affecting 50,000-90,000 people yearly in 75 disease endemic countries, with more than 20,000 deaths reported. Experimental models of infection play a major role in understanding parasite biology, host-pathogen interaction, disease pathogenesis, and parasite transmission. In addition, they have an essential role in the identification and pre-clinical evaluation of new drugs and vaccines. However, our understanding of these models remains fragmentary. Although the immune response to Leishmania donovani infection in mice has been extensively characterized, transcriptomic analysis capturing the tissue-specific evolution of disease has yet to be reported. Methods: We provide an analysis of the transcriptome of spleen, liver and peripheral blood of BALB/c mice infected with L. donovani. Where possible, we compare our data in murine experimental visceral leishmaniasis with transcriptomic data in the public domain obtained from the study of L. donovani-infected hamsters and patients with human visceral leishmaniasis. Digitised whole slide images showing the histopathology in spleen and liver are made available via a dedicated website, www.leishpathnet.org. Results: Our analysis confirms marked tissue-specific alterations in the transcriptome of infected mice over time and identifies previously unrecognized parallels and differences between murine, hamster and human responses to infection. We show commonality of interferon-regulated genes whilst confirming a greater activation of type 2 immune pathways in infected hamsters compared to mice. Cytokine genes and genes encoding immune checkpoints were markedly tissue specific and dynamic in their expression, and pathways focused on non-immune cells reflected tissue specific immunopathology. Our data also addresses the value of measuring peripheral blood transcriptomics as a potential window into underlying systemic disease. Conclusions: Our transcriptomic data, coupled with histopathologic analysis of the tissue response, provide an additional resource to underpin future mechanistic studies and to guide clinical research.

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Transforming Growth Factor β and Immunosuppression in Experimental Visceral Leishmaniasis

Infection and Immunity ◽

10.1128/iai.66.3.1233-1236.1998 ◽

1998 ◽

Vol 66 (3) ◽

pp. 1233-1236 ◽

Cited By ~ 48

Author(s):

Virmondes Rodrigues ◽

João Santana da Silva ◽

Antonio Campos-Neto

Keyword(s):

T Cells ◽

Growth Factor ◽

Visceral Leishmaniasis ◽

Leishmania Donovani ◽

Transforming Growth Factor ◽

Transforming Growth Factor Β ◽

Spleen Cells ◽

Immunohistochemical Studies

ABSTRACT Hamsters infected with Leishmania donovani develop a disease similar to human kala-azar. They present hypergammaglobulinemia, and their T cells do not respond to parasite antigens. This unresponsiveness has been primarily ascribed to defects in antigen-presenting cells (APCs), because these cells are unable to stimulate proliferation of parasite-specific T cells from immunized animals. In this study, we show that APCs (adherent spleen cells) fromL. donovani-infected hamsters produce high levels of the inhibitory cytokine transforming growth factor β (TGF-β). Immunohistochemical studies with an anti-TGF-β monoclonal antibody (MAb) showed that this cytokine is abundantly produced in vivo by the spleen cells of infected animals. In addition, high levels of TGF-β are produced in vitro by infected hamster cells, either spontaneously or after stimulation with parasite antigen or lipopolysaccharide. Furthermore, in vivo-infected adherent cells obtained from spleens ofL. donovani-infected hamsters caused profound inhibition of the in vitro antigen-induced proliferative response of lymph node cells from hamsters immunized with leishmanial antigens. Moreover, this inhibition was totally abrogated by the anti-TGF-β MAb. These results suggest that the immunosuppression observed in visceral leishmaniasis is, at least in part, due to the abundant production of TGF-β during the course of the infection.

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The Effect of Iodium 30c on Experimental Visceral Leishmaniasis

Homeopathy ◽

10.1055/s-0040-1713361 ◽

2020 ◽

Vol 109 (04) ◽

pp. 213-223

Author(s):

Jyoti Joshi ◽

Chetna Bandral ◽

Raj Kumar Manchanda ◽

Anil Khurana ◽

Debadatta Nayak ◽

...

Keyword(s):

T Cells ◽

Visceral Leishmaniasis ◽

Amphotericin B ◽

Cd8 T Cells ◽

Therapeutic Efficacy ◽

Leishmania Donovani ◽

Neglected Tropical Diseases ◽

Parasite Load ◽

Poor People

Abstract Background Leishmaniasis is one of several neglected tropical diseases that warrant serious attention. A disease of socio-economically poor people, it demands safer and cheaper drugs that help to overcome the limitations faced by the existing anti-leishmanials. Complementary or traditional medicines might be a good option, with an added advantage that resistance may not develop against these drugs. Thus, the present investigation was performed to evaluate the anti-leishmanial efficacy of an ultra-diluted homeopathic medicine (Iodium 30c) in experimental visceral leishmaniasis (VL). Methods Compliant with strict ethical standards in animal experimentation, the study was performed in-vivo in inbred BALB/c mice which were injected intravenously with 1 × 107 promastigotes of Leishmania donovani before (therapeutic) or after (prophylactic) treatment with Iodium 30c for 30 days. In other groups of mice (n = 6 per group), amphotericin B served as positive control, infected animals as the disease control, while the naïve controls included normal animals; animals receiving only Iodium 30c or Alcohol 30c served as sham controls. The anti-leishmanial efficacy was assessed by determining the hepatic parasite load and analysing percentages of CD4+ and CD8+ T cells. Biochemical analysis and histological studies were performed to check any toxicities. Results Iodium-treated animals showed a significantly reduced parasite load (to 1503 ± 39 Leishman Donovan Units, LDU) as compared with the infected controls (4489 ± 256 LDU) (p < 0.05): thus, the mean therapeutic efficacy of Iodium 30c was 66.5%. In addition, the population of CD4+ and CD8+ T cells was significantly increased (p < 0.05) after treatment. No toxicity was observed, as evidenced from biochemical and histopathological studies of the liver and kidneys. Efficacy of Iodium 30c prophylaxis was 58.3%, while the therapeutic efficacy of amphotericin B was 85.9%. Conclusion This original study has shown that Iodium 30c had significant impact in controlling parasite replication in experimental VL, though the effect was less than that using standard pharmaceutical treatment.

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Leishmania donovani p36(LACK) DNA Vaccine Is Highly Immunogenic but Not Protective against Experimental Visceral Leishmaniasis

Infection and Immunity ◽

10.1128/iai.69.8.4719-4725.2001 ◽

2001 ◽

Vol 69 (8) ◽

pp. 4719-4725 ◽

Cited By ~ 114

Author(s):

Peter C. Melby ◽

Jue Yang ◽

Weiguo Zhao ◽

Luis E. Perez ◽

Jun Cheng

Keyword(s):

T Cell ◽

Visceral Leishmaniasis ◽

Dna Vaccine ◽

Cell Response ◽

Leishmania Donovani ◽

T Cell Response ◽

Vaccine Antigen ◽

Interleukin 4 ◽

Th1 Response

ABSTRACT The acquisition of immunity following subclinical or resolved infection with the intracellular parasite Leishmania donovani suggests that vaccination could prevent visceral leishmaniasis (VL). The LACK (Leishmania homolog of receptors for activated C kinase) antigen is of interest as a vaccine candidate for the leishmaniases because of its immunopathogenic role in murine L. major infection. Immunization of mice with a truncated (24-kDa) version of the 36-kDa LACK antigen, delivered in either protein or DNA form, was found previously to protect against cutaneous L. major infection by redirecting the early T-cell response away from a pathogenic interleukin-4 (IL-4) response and toward a protective Th1 response. The amino acid sequence of theLeishmania p36(LACK) antigen is highly conserved, but the efficacy of this vaccine antigen in preventing disease caused by strains other than L. major has not been determined. We investigated the efficacy of a p36(LACK) DNA vaccine against VL because of the serious nature of this form of leishmaniasis and because it was unclear whether the LACK vaccine would be effective in a model where there was not a dominant pathogenic IL-4 response. We demonstrate here that although the LACK DNA vaccine induced a robust parasite-specific Th1 immune response (IFN-γ but not IL-4 production) and primed for an in vivo T-cell response to inoculated parasites, it did not induce protection against cutaneous or systemic L. donovanichallenge. Coadministration of IL-12 DNA with the vaccine did not enhance the strong vaccine-induced Th1 response or augment a protective effect.

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Nanotized Curcumin and Miltefosine, a Potential Combination for Treatment of Experimental Visceral Leishmaniasis

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01169-16 ◽

2016 ◽

Vol 61 (3) ◽

Cited By ~ 27

Author(s):

Brajendra Tiwari ◽

Richa Pahuja ◽

Pradeep Kumar ◽

Srikanta Kumar Rath ◽

Kailash Chand Gupta ◽

...

Keyword(s):

Visceral Leishmaniasis ◽

Leishmania Donovani ◽

Oral Drug ◽

Hamster Model ◽

Content Type ◽

Leishmanicidal Activity ◽

Traditional Indian Medicine ◽

Indian Medicine

ABSTRACT Leishmaniasis chemotherapy remains very challenging due to high cost of the drug and its associated toxicity and drug resistance, which develops over a period of time. Combination therapies (CT) are now in use to treat many diseases, such as cancer and malaria, since it is more effective and affordable than monotherapy. CT are believed to represent a new explorable strategy for leishmaniasis, a neglected tropical disease caused by the obligate intracellular parasite Leishmania. In the present study, we investigated the effect of a combination of a traditional Indian medicine (ayurveda), a natural product curcumin and miltefosine, the only oral drug for visceral leishmaniasis (VL) using a Leishmania donovani-hamster model. We developed an oral nanoparticle-based formulation of curcumin. Nanoformulation of curcumin alone exhibited significant leishmanicidal activity both in vitro and in vivo. In combination with miltefosine, it exhibited a synergistic effect on both promastigotes and amastigotes under in vitro conditions. The combination of these two agents also demonstrated increased in vivo leishmanicidal activity accompanied by increased production of toxic reactive oxygen/nitrogen metabolites and enhanced phagocytic activity. The combination also exhibited increased lymphocyte proliferation. The present study thus establishes the possible use of nanocurcumin as an adjunct to antileishmanial chemotherapy.

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Antileishmanial Efficacy and Pharmacokinetics of DB766-Azole Combinations

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01129-17 ◽

2017 ◽

Vol 62 (1) ◽

Cited By ~ 4

Author(s):

April C. Joice ◽

Sihyung Yang ◽

Abdelbasset A. Farahat ◽

Heidi Meeds ◽

Mei Feng ◽

...

Keyword(s):

Visceral Leishmaniasis ◽

Leishmania Donovani ◽

Treatment Strategies ◽

Parasite Burden ◽

Pharmacokinetic Analysis ◽

Content Type ◽

New Treatment ◽

Almost All

ABSTRACT Given the limitations of current antileishmanial drugs and the utility of oral combination therapy for other infections, developing an oral combination against visceral leishmaniasis should be a high priority. In vitro combination studies with DB766 and antifungal azoles against intracellular Leishmania donovani showed that posaconazole and ketoconazole, but not fluconazole, enhanced DB766 potency. Pharmacokinetic analysis of DB766-azole combinations in uninfected Swiss Webster mice revealed that DB766 exposure was increased by higher posaconazole and ketoconazole doses, while DB766 decreased ketoconazole exposure. In L. donovani-infected BALB/c mice, DB766-posaconazole combinations given orally for 5 days were more effective than DB766 or posaconazole alone. For example, 81% ± 1% (means ± standard errors) inhibition of liver parasite burden was observed for 37.5 mg/kg of body weight DB766 plus 15 mg/kg posaconazole, while 37.5 mg/kg DB766 and 15 mg/kg posaconazole administered as monotherapy gave 40% ± 5% and 21% ± 3% inhibition, respectively. Combination index (CI) analysis indicated that synergy or moderate synergy was observed in six of nine combined dose groups, while the other three were nearly additive. Liver concentrations of DB766 and posaconazole increased in almost all combination groups compared to monotherapy groups, although many increases were not statistically significant. For DB766-ketoconazole combinations evaluated in this model, two were antagonistic, one displayed synergy, and one was nearly additive. These data indicate that the efficacy of DB766-posaconazole and DB766-ketoconazole combinations in vivo is influenced in part by the pharmacokinetics of the combination, and that the former combination deserves further consideration in developing new treatment strategies against visceral leishmaniasis.

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Discovery of Safe and Orally Effective 4-Aminoquinaldine Analogues as Apoptotic Inducers with Activity against Experimental Visceral Leishmaniasis

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00700-11 ◽

2011 ◽

Vol 56 (1) ◽

pp. 432-445 ◽

Cited By ~ 17

Author(s):

Partha Palit ◽

Abhijit Hazra ◽

Arindam Maity ◽

R. S. K. Vijayan ◽

Prabu Manoharan ◽

...

Keyword(s):

Visceral Leishmaniasis ◽

Oral Delivery ◽

Leishmania Donovani ◽

Reactive Oxygen Species Generation ◽

Oral Route ◽

Apoptotic Pathway ◽

Content Type ◽

Lead Compounds

ABSTRACTNovel antileishmanials are urgently required to overcome emergence of drug resistance, cytotoxic effects, and difficulties in oral delivery. Toward this, we investigated a series of novel 4-aminoquinaldine derivatives, a new class of molecules, as potential antileishmanials. 4-Aminoquinaldine derivatives presented inhibitory effects onL. donovanipromastigotes and amastigotes (50% inhibitory concentration range, 0.94 to 127 μM). Of these, PP-9 and PP-10 were the most effectivein vitroand demonstrated strong efficaciesin vivothrough the intraperitoneal route. They were also found to be effective against both sodium antimony gluconate-sensitive and -resistantLeishmania donovanistrains in BALB/c mice when treated orally, resulting in more than 95% protection. Investigation of their mode of action revealed that killing by PP-10 involved moderate inhibition of dihydrofolate reductase and elicitation of the apoptotic cascade. Our studies implicate that PP-10 augments reactive oxygen species generation, evidenced from decreased glutathione levels and increased lipid peroxidation. Subsequent disruption ofLeishmaniapromastigote mitochondrial membrane potential and activation of cytosolic proteases initiated the apoptotic pathway, resulting in DNA fragmentation and parasite death. Our results demonstrate that PP-9 and PP-10 are promising lead compounds with the potential for treating visceral leishmaniasis (VL) through the oral route.

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