Synergy of the antiretroviral protease inhibitor indinavir and chloroquine against malaria parasites in vitro and in vivo

ABSTRACTEx vivoassay systems provide a powerful approach to studying human malaria parasite biology and to testing antimalarials. For rodent malaria parasites, short-termin vitroculture andex vivoantimalarial susceptibility assays are relatively cumbersome, relying onin vivopassage for synchronization, since ring-stage parasites are an essential starting material. Here, we describe a new approach based on the enrichment of ring-stagePlasmodium berghei,P. yoelii, andP. vinckei vinckeiusing a single-step Percoll gradient. Importantly, we demonstrate that the enriched ring-stage parasites develop synchronously regardless of the parasite strain or species used. Using a flow cytometry assay with Hoechst and ethidium or MitoTracker dye, we show that parasite development is easily and rapidly monitored. Finally, we demonstrate that this approach can be used to screen antimalarial drugs.

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Mammalian deubiquitinating enzyme inhibitors display in vitro and in vivo activity against malaria parasites and potentiate artemisinin action

10.1101/2020.08.13.249425 ◽

2020 ◽

Author(s):

Nelson V. Simwela ◽

Katie R. Hughes ◽

Michael T. Rennie ◽

Michael P. Barrett ◽

Andrew P. Waters

Keyword(s):

Anticancer Agents ◽

Drug Targets ◽

Reverse Genetics ◽

Enzyme Inhibitors ◽

Artemisinin Resistance ◽

Drug Repurposing ◽

Antimalarial Drugs ◽

Malaria Parasites

AbstractCurrent malaria control efforts rely significantly on artemisinin combinational therapies which have played massive roles in alleviating the global burden of the disease. Emergence of resistance to artemisinins is therefore, not just alarming but requires immediate intervention points such as development of new antimalarial drugs or improvement of the current drugs through adjuvant or combination therapies. Artemisinin resistance is primarily conferred by Kelch13 propeller mutations which are phenotypically characterised by generalised growth quiescence, altered haemoglobin trafficking and downstream enhanced activity of the parasite stress pathways through the ubiquitin proteasome system (UPS). Previous work on artemisinin resistance selection in a rodent model of malaria, which we and others have recently validated using reverse genetics, has also shown that mutations in deubiquitinating enzymes, DUBs (upstream UPS component) modulates susceptibility of malaria parasites to both artemisinin and chloroquine. The UPS or upstream protein trafficking pathways have, therefore, been proposed to be not just potential drug targets, but also possible intervention points to overcome artemisinin resistance. Here we report the activity of small molecule inhibitors targeting mammalian DUBs in malaria parasites. We show that generic DUB inhibitors can block intraerythrocytic development of malaria parasites in vitro and possess antiparasitic activity in vivo and can be used in combination with additive effect. We also show that inhibition of these upstream components of the UPS can potentiate the activity of artemisinin in vitro as well as in vivo to the extent that ART resistance can be overcome. Combinations of DUB inhibitors anticipated to target different DUB activities and downstream 20s proteasome inhibitors are even more effective at improving the potency of artemisinins than either inhibitors alone providing proof that targeting multiple UPS activities simultaneously could be an attractive approach to overcoming artemisinin resistance. These data further validate the parasite UPS as a target to both enhance artemisinin action and potentially overcome resistance. Lastly, we confirm that DUB inhibitors can be developed into in vivo antimalarial drugs with promise for activity against all of human malaria and could thus further exploit their current pursuit as anticancer agents in rapid drug repurposing programs.Graphical abstract

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Effect of phenyl vinyl sulphone cysteine protease inhibitor on Schistosoma mansoni: in vitro and in vivo experimental studies

Journal of Parasitic Diseases ◽

10.1007/s12639-017-0933-3 ◽

2017 ◽

Vol 41 (4) ◽

pp. 1049-1058

Author(s):

Manal Salah El-Din Mahmoud ◽

Ayman Nabil Ibrahim ◽

Abeer Fathy Badawy ◽

Nourhan Mohamed Abdelmoniem

Keyword(s):

Protease Inhibitor ◽

Schistosoma Mansoni ◽

Cysteine Protease ◽

Experimental Studies ◽

Cysteine Protease Inhibitor ◽

Phenyl Vinyl

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ABT-378, a Highly Potent Inhibitor of the Human Immunodeficiency Virus Protease

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.42.12.3218 ◽

1998 ◽

Vol 42 (12) ◽

pp. 3218-3224 ◽

Cited By ~ 304

Author(s):

Hing L. Sham ◽

Dale J. Kempf ◽

Akhteruzammen Molla ◽

Kennan C. Marsh ◽

Gondi N. Kumar ◽

...

Keyword(s):

Human Immunodeficiency Virus ◽

Protease Inhibitor ◽

Response To Therapy ◽

Hiv Protease ◽

Healthy Human ◽

Human Volunteers ◽

Immunodeficiency Virus ◽

Potent Protease Inhibitor

ABSTRACT The valine at position 82 (Val 82) in the active site of the human immunodeficiency virus (HIV) protease mutates in response to therapy with the protease inhibitor ritonavir. By using the X-ray crystal structure of the complex of HIV protease and ritonavir, the potent protease inhibitor ABT-378, which has a diminished interaction with Val 82, was designed. ABT-378 potently inhibited wild-type and mutant HIV protease (Ki = 1.3 to 3.6 pM), blocked the replication of laboratory and clinical strains of HIV type 1 (50% effective concentration [EC50], 0.006 to 0.017 μM), and maintained high potency against mutant HIV selected by ritonavir in vivo (EC50, ≤0.06 μM). The metabolism of ABT-378 was strongly inhibited by ritonavir in vitro. Consequently, following concomitant oral administration of ABT-378 and ritonavir, the concentrations of ABT-378 in rat, dog, and monkey plasma exceeded the in vitro antiviral EC50 in the presence of human serum by >50-fold after 8 h. In healthy human volunteers, coadministration of a single 400-mg dose of ABT-378 with 50 mg of ritonavir enhanced the area under the concentration curve of ABT-378 in plasma by 77-fold over that observed after dosing with ABT-378 alone, and mean concentrations of ABT-378 exceeded the EC50 for >24 h. These results demonstrate the potential utility of ABT-378 as a therapeutic intervention against AIDS.

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PROTEASE INHIBITOR GABEXATE MESILATE INHIBITS TNF RELEASE IN VITRO AND IN VIVO

Shock ◽

10.1097/00024382-199512001-00086 ◽

1995 ◽

Vol 4 (Supplement) ◽

pp. 22

Author(s):

Fukushima Ryoji ◽

Saito Hideaki ◽

Lin Ming-Tsan ◽

Inoue Tomomi ◽

Fakatsu Kazuhiko ◽

...

Keyword(s):

Protease Inhibitor ◽

Gabexate Mesilate ◽

Release In Vitro

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Mesenchymal stem cells express serine protease inhibitor to evade the host immune response

Blood ◽

10.1182/blood-2010-06-287979 ◽

2011 ◽

Vol 117 (4) ◽

pp. 1176-1183 ◽

Cited By ~ 30

Author(s):

Najib El Haddad ◽

Dean Heathcote ◽

Robert Moore ◽

Sunmi Yang ◽

Jamil Azzi ◽

...

Keyword(s):

Stem Cells ◽

Mesenchymal Stem Cells ◽

T Cell ◽

Protease Inhibitor ◽

Serine Protease ◽

Serine Protease Inhibitor ◽

Cytotoxic T Cell ◽

Wild Type

Abstract Clinical trials using mesenchymal stem cells (MSCs) have been initiated worldwide. An improved understanding of the mechanisms by which allogeneic MSCs evade host immune responses is paramount to regulating their survival after administration. This study has focused on the novel role of serine protease inhibitor (SPI) in the escape of MSCs from host immunosurveillance through the inhibition of granzyme B (GrB). Our data indicate bone marrow–derived murine MSCs express SPI6 constitutively. MSCs from mice deficient for SPI6 (SPI6−/−) exhibited a 4-fold higher death rate by primed allogeneic cytotoxic T cells than did wild-type MSCs. A GrB inhibitor rescued SPI6−/− MSCs from cytotoxic T-cell killing. Transduction of wild-type MSCs with MigR1-SPI6 also protected MSCs from cytotoxic T cell–mediated death in vitro. In addition, SPI6−/− MSCs displayed a shorter lifespan than wild-type MSCs when injected into an allogeneic host. We conclude that SPI6 protects MSCs from GrB-mediated killing and plays a pivotal role in their survival in vivo. Our data could serve as a basis for future SPI-based strategies to regulate the survival and function of MSCs after administration and to enhance the efficacy of MSC-based therapy for diseases.

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PIVL, a snake venom Kunitz-type serine protease inhibitor, inhibits in vitro and in vivo angiogenesis

Microvascular Research ◽

10.1016/j.mvr.2014.08.006 ◽

2014 ◽

Vol 95 ◽

pp. 149-156 ◽

Cited By ~ 14

Author(s):

Maram Morjen ◽

Stéphane Honoré ◽

Amine Bazaa ◽

Zaineb Abdelkafi-Koubaa ◽

Ameneallah Ellafi ◽

...

Keyword(s):

Protease Inhibitor ◽

Serine Protease ◽

Snake Venom ◽

Serine Protease Inhibitor ◽

Kunitz Type

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Oxidation of alpha 1-protease inhibitor: role of lipid peroxidation products

Journal of Applied Physiology ◽

10.1152/jappl.1989.66.5.2211 ◽

1989 ◽

Vol 66 (5) ◽

pp. 2211-2215 ◽

Cited By ~ 9

Author(s):

V. Mohsenin ◽

J. L. Gee

Keyword(s):

Lipid Peroxidation ◽

Fatty Acid ◽

Linoleic Acid ◽

Stearic Acid ◽

Protease Inhibitor ◽

Alpha Tocopherol ◽

Inhibitory Capacity ◽

Buffer Control

Previously we demonstrated that in vivo exposure of humans to NO2 resulted in significant inactivation of alpha 1-protease inhibitor (alpha 1-PI) in the bronchoalveolar lavage fluid. However, alpha 1-PI retains its elastase inhibitory activity in vitro when exposed to 10 times the concentration of NO2 used in vivo. We suggested exogenous oxidants such as O2 and NO2 exert their effect in vivo in part through lipid peroxidation. We investigated the mechanism of inactivation of alpha 1-PI in the presence or absence of lipids under oxidant atmosphere. alpha 1-PI in solutions containing phosphate buffer (control), 0.1 mM stearic acid (saturated fatty acid, 18:0), or 0.1 mM linoleic acid (polyunsaturated fatty acid, 18:2) was exposed to either N2 or NO2 (50 ppm for 4 h). Elastase inhibitory capacity of alpha 1-PI was significantly diminished in the presence of 0.1 mM linoleic acid and under NO2 atmosphere (75 +/- 8% of control, P less than 0.01), whereas there was no change in elastase inhibitory capacity of alpha 1-PI in the presence or absence (buffer only) of 0.1 mM stearic acid under a similar condition (109 +/- 11 and 94 +/- 6%, respectively). The inactivated alpha 1-PI as the result of peroxidized lipid could be reactivated by dithiothreitol and methionine sulfoxide peptide reductase, suggesting oxidation of methionine residue at the elastase inhibitory site. Furthermore the inhibitory effect of peroxidized lipid on alpha 1-PI could be prevented by glutathione and glutathione peroxidase and to some extent by alpha-tocopherol.

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