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: 7.71). 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, Sep 28, 2015
1 Follower
54 Reads
  • Source
    • "Due to their translocating properties, they were named cell-penetrating peptides (CPPs) more than two decades ago [1] [2] [3] [4]. CPPs can be covalently conjugated or complexed with a variety of payloads, such as nucleic acids, proteins, nanoparticles or quantum dots [5] [6] [7], enabling their use in a broad range of applications, including transfection, siRNA technology, organelle imaging or delivery of low-permeability drugs [8] [9] [10]. The most studied CPPs in terms of cell translocation and effectiveness as biomedical agents are the HIV-1 transcriptional activator (Tat) [1], the antennapedia homeodomain from Drosophila (Antp) [11], and the VP22 protein from the Herpes simplex virus [12]. "
    [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 10/2012; 35(10):1761-4. DOI:10.1248/bpb.b12-00419 · 1.83 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Neglected diseases (NDs) are a group of diseases endemic in underdeveloped and developing countries and have been a global health problem, such as leishmaniasis, tuberculosis, cryptococcosis, leprosy among other. Drugs used in the clinic are toxic, they do not always result in a cure, and many parasites have shown resistance to them. In the last years, the incidence and prevalence rates of some Neglected Diseases have decreased in the world, opposite to the multi-drugresistance (MDT) levels observed. Drug resistance in leprosy and tuberculosis becomes even more important because they’re very limited alternative drugs to MDT. Molecular studies on the mechanism of action of these drugs have elucidated the genetic basis of drug resistance in Mycobacterium tuberculosis and Mycobacterium leprae, respectively. C. neoformans has been shown to present different susceptibility profiles to antifungal drugs according to in vitro studies, although little reports of resistant cases have been described. C. neoformans resistance can be seen to azoles, especially fluconazole, to amphotericin B (AmB) and to 5-flucytosine (5FC). This chapter will summarize the main mechanisms of resistance of some neglected diseases such as leishmaniasis, leprosy, cryptococcosis and other.
    Microbial pathogens and strategies for combating them: science, technology and education., 4 edited by A. Méndez-Vilas, 01/2013: chapter Mechanism of Resistance of Some Neglected Diseases: pages 480-489; Formatex Research Center., ISBN: 978-84-942134-1-0
Show more