The Leishmania nicotinamidase is essential for NAD+ production and parasite proliferation.

NAD+ is a central cofactor that plays important roles in cellular metabolism and energy production in all living cells. Genomics-based reconstruction of NAD+ metabolism revealed that Leishmania protozoan parasites are NAD+ auxotrophs. Consequently, these parasites require assimilating NAD+ precursors (nicotinamide, nicotinic acid, nicotinamide riboside) from their host environment to synthesize NAD+ by a salvage pathway. Nicotinamidase is a key enzyme of this salvage pathway that catalyses conversion of nicotinamide (NAm) to nicotinic acid (Na), and that is absent in higher eukaryotes. We present here the biochemical and functional characterizations of the Leishmania infantum nicotinamidase (LiPNC1). Generation of Lipnc1 null mutants leads to a decrease in NAD+ content, associated with a metabolic shutdown-like phenotype with an extensive lag phase of growth. Both phenotypes could be rescued by an add-back construct or by addition of exogenous Na. In addition, Lipnc1 null mutants were unable to establish a sustained infection in a murine experimental model. Altogether, these results illustrate that NAD+ homeostasis is a fundamental component of Leishmania biology and virulence, and that NAm constitutes its main NAD+ source in the mammalian host. The crystal structure of LiPNC1 we solved allows now the design of rational inhibitors against this new promising therapeutic target.

Synthesis of aminophenylhydroxamate and aminobenzylhydroxamate derivatives and in vitro screening for antiparasitic and histone deacetylase inhibitory activity.

A series of aminophenylhydroxamates and aminobenzylhydroxamates were synthesized and screened for their antiparasitic activity against Leishmania, Trypanosoma, and Toxoplasma. Their anti-histone deacetylase (HDAC) potency was determined. Moderate to no antileishmanial or antitrypanosomal activity was found (IC50 > 10 µM) that contrast with the highly efficient anti-Toxoplasma activity (IC50 < 1.0 µM) of these compounds. The antiparasitic activity of the synthetized compounds correlates well with their HDAC inhibitory activity. The best-performing compound (named 363) express a high anti-HDAC6 inhibitory activity (IC50 of 0.045 ±â€¯0.015 µM) a moderate cytotoxicity and a high anti-Toxoplasma activity in the range of known anti-Toxoplasma compounds (IC50 of 0.35-2.25 µM). The calculated selectivity index (10-300 using different human cell lines) of the compound 363 makes it a lead compound for the future development of anti-Toxoplasma molecules.

Strength and character of peptide/anion interactions.

The binding free energy of complex [Co(C(2)O(4))(3)](3-) to three peptides H-Lys-Gly-Lys-Gly-Lys-Gly-Lys-NH(2) (P-1), H-(Lys-Gly-Lys-Gly-Lys-Gly-Lys)(2)-NH(2) (P-2), H-(Lys-Gly-Lys-Gly-Lys-Gly-Lys)(3)-NH(2) (P-3) and to the monomers (amino acids) forming the peptides has been obtained using the kinetics of the electron-transfer reaction between [Ru(NH(3))(5)py](2+) and [Co(C(2)O(4))(3)](3-) as the probe. The polymerization of the monomers increases the negative free energy of binding and changes its character, noncooperative for the monomers and anticooperative for the peptides. This increase in the negative free energy represents a driving force for the polymerization process. The magnitude of the gain in negative free energy, as a consequence of the anticooperative character of the binding of the cobalt complex to the peptide, depends on the ratio of [complex]/[monomers].