Defeating Leishmania resistance to Miltefosine (hexadecylphosphocholine) by peptide-mediated drug smuggling: A proof of mechanism for trypanosomatid chemotherapy

Centro de Investigaciones Biológicas, CSIC, Ramiro de Maeztu 9, 28040 Madrid, Spain.
Journal of Controlled Release (Impact Factor: 7.26). 05/2012; 161(3):835-42. DOI: 10.1016/j.jconrel.2012.05.023
Source: PubMed

ABSTRACT Miltefosine (hexadecylphosphocholine, HePC), the first orally active drug successful against leishmaniasis, is especially active on the visceral form of the disease. Resistance mechanisms are almost exclusively associated to dysfunction in HePC uptake systems. In order to evade the requirements of its cognate receptor/translocator, HePC-resistant Leishmania donovani parasites (R40 strain) were challenged with constructs consisting of an ω-thiol-functionalized HePC analogue conjugated to the cell-penetrating peptide (CPP) Tat(48-60), either through a disulfide or a thioether bond. The conjugates enter and kill both promastigote and intracellular amastigote forms of the R40 strain. Intracellular release of HePC by reduction of the disulfide-based conjugate was confirmed by means of double tagging at both the CPP (Quasar 670) and HePC (BODIPY) moieties. Scission of the conjugate, however, is not mandatory, as the metabolically more stable thioether conjugate retained substantial activity. The disulfide conjugate is highly active on the bloodstream form of Trypanosoma b. brucei, naturally resistant to HePC. Our results provide proof-of-mechanism for the use of CPP conjugates to avert drug resistance by faulty drug accumulation in parasites, as well as the possibility to extend chemotherapy into other parasites intrinsically devoid of membrane translocation systems.

Download full-text


Available from: David Andreu, Jul 03, 2015
1 Follower
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: A novel class of cell-penetrating, nucleolar-targeting peptides (NrTPs), was recently developed from the rattlesnake venom toxin crotamine. Based on the intrinsic fluorescence of tyrosine or tryptophan residues, the partition of NrTPs and crotamine to membranes with variable lipid compositions was studied. Partition coefficient values (in the 10(2)-10(5) range) followed essentially the compositional trend POPC:POPG≤POPG<POPC≤POPC:cholesterol. Leakage assays showed that NrTPs induce minimal lipid vesicle disruption. Fluorescence quenching of NrTPs, either by acrylamide or lipophilic probes, revealed that NrTPs are buried in the lipid bilayer only for negatively-charged membranes. Adoption of partial secondary structure by the NrTPs upon interaction with POPC and POPG vesicles was demonstrated by circular dichroism. Translocation studies were conducted using a novel methodology, based on the confocal microscopy imaging of giant multilamellar vesicles or giant multivesicular liposomes. With this new procedure, which can now be used to evaluate the membrane translocation ability of other molecules, it was demonstrated that NrTPs are able to cross lipid membranes even in the absence of a receptor or transmembrane gradient. Altogether, these results indicate that NrTPs interact with lipid bilayers and can penetrate cells via different entry mechanisms, reinforcing the applicability of this class of peptide as therapeutic tools for the delivery of molecular cargoes.
    Biochimica et Biophysica Acta 06/2012; 1818(11):2707-17. DOI:10.1016/j.bbamem.2012.06.014 · 4.66 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: Here we studied ability of two naphthoquinones to inhibit Leishmania growth (2,3-dichloro-5,8-dihydroxy-1,4-naphthoquinone (TR 001) and 2,3-dibromo-1,4-naphthoquinone (TR 002). TR 001 was more efficient than TR 002 in inducing killing of promastigotes and intracellular amastigotes. These values compare well to those obtained with the standard first-line antileishmanial agent sodium stibogluconate (SSG). TR 001 also induced significantly more nitric oxide (NO) production than TR 002 or SSG. Taken together, these data show that TR 001 and TR 002 could be promising new drugs for treatment of visceral leishmaniasis.
    Biological & Pharmaceutical Bulletin 01/2012; 35(10):1761-4. DOI:10.1248/bpb.b12-00419 · 1.78 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: Human visceral leishmaniasis (VL) continues to be a life-threatening neglected tropical disease, with close to 200 million people at risk of infection globally. Epidemics and resurgence of VL are associated with negligence by the policy makers, economic decline and population movements. Control of the disease is hampered by the lack of proficient vaccination, rapid diagnosis in a field setting and severe side effects of current drug therapies. The diagnosis of VL relied largely on invasive techniques of detecting parasites in splenic and bone marrow aspirates. rK39 and PCR, despite problems related to varying sensitivities and specificities and field adaptability, respectively, are considered the best options for VL diagnosis today. No single therapy of VL currently offers satisfactory efficacy along with safety. The field of VL research only recently shifted toward actively identifying new drugs for safe and affordable treatment. Oral miltefosine and safe AmBisome along with better use of amphotericin B have been rapidly implemented in the last decade. A combination therapy will substantially reduce the required dose and duration of drug administration and reduce the chance of the development of resistance. In addition, identification of asymptomatic cases, vector control and treatment of post-kala-azar dermal leishmaniasis would allow new perspectives in VL control and management.
    Expert Review of Anticancer Therapy 01/2013; 11(1):79-98. DOI:10.1586/eri.12.148 · 3.06 Impact Factor