RESUMEN
The parasite Leishmania donovani is one of the species causing visceral leishmaniasis in humans, a deadly infection claiming up to 40,000 lives each year. The current drugs for leishmaniasis treatment have severe drawbacks and there is an urgent need to find new anti-leishmanial compounds. However, the search for drug candidates is complicated by the intracellular lifestyle of Leishmania. Here, we investigate the use of human induced pluripotent stem cell (iPS)-derived macrophages (iMACs) as host cells for L. donovani. iMACs obtained through embryoid body differentiation were infected with L. donovani promastigotes, and high-content imaging techniques were used to optimize the iMACs seeding density and multiplicity of infection, allowing us to reach infection rates up to 70% five days after infection. IC50 values obtained for miltefosine and amphotericin B using the infected iMACs or mouse peritoneal macrophages as host cells were comparable and in agreement with the literature, showing the potential of iMACs as an infection model for drug screening.
Asunto(s)
Antiprotozoarios , Células Madre Pluripotentes Inducidas , Leishmania donovani , Leishmaniasis Visceral , Animales , Humanos , Ratones , Antiprotozoarios/farmacología , Antiprotozoarios/uso terapéutico , Leishmaniasis Visceral/parasitología , Macrófagos/parasitologíaRESUMEN
Positive modulation of the muscarinic M1-receptor has for a long time attracted scientists and drug developers for the potential treatment of Alzheimer's disease or Schizophrenia. The precognitive potential of M1 activation has however not been clinically demonstrated as a result of side effects associated both with agonists and positive allosteric modulators (PAM's) of the M1-receptor. To avoid excessive activation of the M1-receptor we have designed a new screening format and developed the first low-shift positive allosteric modulators for the M1 receptor. Low-shift PAM's offer the potential of "use-dependent" attenuation of transmitter-signaling while avoiding pseudo-agonistic behavior in vivo as a common limitation of the so far described high-shift PAM's. With these novel M1-PAM's, the M1 receptor is potentially the first GPCR for which both, high- and low shift PAM's have become available.
Asunto(s)
Receptor Muscarínico M1/metabolismo , Regulación Alostérica , Animales , Células CHO , Cricetinae , Cricetulus , Evaluación Preclínica de Medicamentos , Agonistas de Receptores de GABA-A/química , Agonistas de Receptores de GABA-A/metabolismo , Humanos , Agonistas Muscarínicos/química , Mutagénesis Sitio-Dirigida , Receptor Muscarínico M1/química , Receptor Muscarínico M1/genética , Receptores de GABA-A/química , Receptores de GABA-A/metabolismo , Relación Estructura-ActividadRESUMEN
Profiling of eight stereoisomeric T. cruzi growth inhibitors revealed vastly different in vitro properties such as solubility, lipophilicity, pKa, and cell permeability for two sets of four stereoisomers. Using computational chemistry and NMR spectroscopy, we identified the formation of an intramolecular NHâNR3 hydrogen bond in the set of stereoisomers displaying lower solubility, higher lipophilicity, and higher cell permeability. The intramolecular hydrogen bond resulted in a significant pKa difference that accounts for the other structure-property relationships. Application of this knowledge could be of particular value to maintain the delicate balance of size, solubility, and lipophilicity required for cell penetration and oral administration for chemical probes or therapeutics with properties at, or beyond, Lipinski's rule of 5.