An adamantamine derivative as a drug candidate for the treatment of visceral leishmaniasis.

BACKGROUND: This study aimed to investigate compounds acting on the host cell machinery to impair parasite installation with the possible advantage of limiting drug resistance. The strategy therefore consisted of selecting compounds that are poorly active on the axenic parasite, but very active on the intramacrophage form of Leishmania. OBJECTIVES: To identify a drug candidate from focused screening of adamantamine derivatives that can inhibit the development of Leishmania infantum in macrophages. METHODS: In vitro screening was performed on a library of 142 adamantamine derivatives with axenic and intramacrophage forms of L. infantum, as well as cytotoxicity assays, allowing selection of the most promising compound. Absorption, distribution, metabolism and excretion (ADME) experiments, including pharmacokinetics and microsomal stability, were performed and finally the physicochemical stability of the compound was investigated to assess its suitability for further drug development. RESULTS: VP343 was identified first in vitro, with a CC50 value of 63.7 µM and an IC50 value of 0.32 µM for L. infantum intramacrophage amastigotes and then in vivo, with a 59% reduction of the liver parasite burden after oral administration at 10 mg/kg/day for 5 days. In addition, the ADME data were compatible with moving this compound further through the antileishmanial drug candidate pipeline. CONCLUSIONS: VP343 has the properties of a good drug candidate and merits further investigations.

Structure-Activity Relationship Studies of Retro-1 Analogues against Shiga Toxin.

High-throughput screening has shown that Retro-1 inhibits ricin and Shiga toxins by diminishing their intracellular trafficking via the retrograde route, from early endosomes to the Golgi apparatus. To improve the activity of Retro-1, a structure-activity relationship (SAR) study was undertaken and yielded an analogue with a roughly 70-fold better half-maximal effective concentration (EC50) against Shiga toxin cytotoxicity measured in a cell protein synthesis assay.

DABMA: A Derivative of ABMA with Improved Broad-Spectrum Inhibitory Activity of Toxins and Viruses.

The small molecule ABMA has been previously shown to protect cells against multiple toxins and pathogens including virus, intracellular bacteria, and parasite. Its mechanism of action is directly associated with host endolysosomal pathway rather than targeting toxin or pathogen itself. However, the relationship of its broad-spectrum anti-infection activity and chemical structure is not yet resolved. Here, we synthesized a series of derivatives and compared their activities against diphtheria toxin (DT). Dimethyl-ABMA (DABMA), one of the most potent analogs with about 20-fold improvement in protection efficacy against DT, was identified with a similar mechanism of action to ABMA. Moreover, DABMA exhibited enhanced efficacy against Clostridium difficile toxin B (TcdB), Clostridium sordellii lethal toxin (TcsL), Pseudomonas Exotoxin A (PE) as well as Rabies and Ebola viruses. The results revealed a structure-activity relationship of ABMA, which is a starting point for its clinical development as broad-spectrum drug against existing and emerging infectious diseases.

ABMA, a small molecule that inhibits intracellular toxins and pathogens by interfering with late endosomal compartments.

Intracellular pathogenic microorganisms and toxins exploit host cell mechanisms to enter, exert their deleterious effects as well as hijack host nutrition for their development. A potential approach to treat multiple pathogen infections and that should not induce drug resistance is the use of small molecules that target host components. We identified the compound 1-adamantyl (5-bromo-2-methoxybenzyl) amine (ABMA) from a cell-based high throughput screening for its capacity to protect human cells and mice against ricin toxin without toxicity. This compound efficiently protects cells against various toxins and pathogens including viruses, intracellular bacteria and parasite. ABMA provokes Rab7-positive late endosomal compartment accumulation in mammalian cells without affecting other organelles (early endosomes, lysosomes, the Golgi apparatus, the endoplasmic reticulum or the nucleus). As the mechanism of action of ABMA is restricted to host-endosomal compartments, it reduces cell infection by pathogens that depend on this pathway to invade cells. ABMA may represent a novel class of broad-spectrum compounds with therapeutic potential against diverse severe infectious diseases.

Inhibitors of retrograde trafficking active against ricin and Shiga toxins also protect cells from several viruses, Leishmania and Chlamydiales.

Medical countermeasures to treat biothreat agent infections require broad-spectrum therapeutics that do not induce agent resistance. A cell-based high-throughput screen (HTS) against ricin toxin combined with hit optimization allowed selection of a family of compounds that meet these requirements. The hit compound Retro-2 and its derivatives have been demonstrated to be safe in vivo in mice even at high doses. Moreover, Retro-2 is an inhibitor of retrograde transport that affects syntaxin-5-dependent toxins and pathogens. As a consequence, it has a broad-spectrum activity that has been demonstrated both in vitro and in vivo against ricin, Shiga toxin-producing O104:H4 entero-hemorrhagic E. coli and Leishmania sp. and in vitro against Ebola, Marburg and poxviruses and Chlamydiales. An effect is anticipated on other toxins or pathogens that use retrograde trafficking and syntaxin-5. Since Retro-2 targets cell components of the host and not directly the pathogen, no selection of resistant pathogens is expected. These lead compounds need now to be developed as drugs for human use.

Retro-1 Analogues Differentially Affect Oligonucleotide Delivery and Toxin Trafficking.

Retro-1 is a small molecule that displays two important biological activities: First, it blocks the actions of certain toxins by altering their intracellular trafficking. Second, it enhances the activity of oligonucleotides by releasing them from entrapment in endosomes. This raises the question of whether the two actions involve the same cellular target. Herein we report the effects of several Retro-1 analogues on both toxins and oligonucleotides. We found analogues that affect toxins but not oligonucleotides and vice-versa, while Retro-1 is the only compound that affects both. This indicates that the molecular target(s) involved in the two processes are distinct.

Proteomic analysis of the Simkania-containing vacuole: the central role of retrograde transport.

Simkania negevensis is an obligate intracellular bacterial pathogen that grows in amoeba or human cells within a membrane-bound vacuole forming endoplasmic reticulum (ER) contact sites. The membrane of this Simkania-containing vacuole (SnCV) is a critical host-pathogen interface whose origin and molecular interactions with cellular organelles remain poorly defined. We performed proteomic analysis of purified ER-SnCV-membranes using label free LC-MS(2) to define the pathogen-containing organelle composition. Of the 1,178 proteins of human and 302 proteins of Simkania origin identified by this strategy, 51 host cell proteins were enriched or depleted by infection and 57 proteins were associated with host endosomal transport pathways. Chemical inhibitors that selectively interfere with trafficking at the early endosome-to-trans-Golgi network (TGN) interface (retrograde transport) affected SnCV formation, morphology and lipid transport. Our data demonstrate that Simkania exploits early endosome-to-TGN transport for nutrient acquisition and growth.

(S)-N-Methyldihydroquinazolinones are the Active Enantiomers of Retro-2 Derived Compounds against Toxins.

This study reports the synthesis, chromatographic separation, and pharmacological evaluation of the two enantiomers of a new compound, named Retro-2.1, active against toxins by inhibiting intracellular trafficking via the retrograde route. The absolute configuration of the bioactive enantiomer has been assigned from X-ray diffraction to the (S)-enantiomer. To date, (S)-Retro-2.1 is the most potent molecule to counteract the cytotoxic potential of ricin and Shiga toxin, with EC50 values of 23 and 54 nM, respectively.

N-methyldihydroquinazolinone derivatives of Retro-2 with enhanced efficacy against Shiga toxin.

The Retro-2 molecule protects cells against Shiga toxins by specifically blocking retrograde transport from early endosomes to the trans-Golgi network. A SAR study has been carried out to identify more potent compounds. Cyclization and modifications of Retro-2 led to a compound with roughly 100-fold improvement of the EC50 against Shiga toxin cytotoxicity measured in a cell protein synthesis assay. We also demonstrated that only one enantiomer of the dihydroquinazolinone reported herein is bioactive.

Nitrene chemistry in organic synthesis: still in its infancy?

The element nitrogen is essential to life. Considerable attention is thus paid to the development of synthetic methods for its introduction into molecules. Nitrenes, long regarded as highly reactive but poorly selective species, have recently emerged as useful tools for the formation of C-N bonds. Practical metal-catalyzed protocols are now available for the preparation of amines through either the aziridination of alkenes or the C-H amination of alkanes. Recent results highlighted in this Minireview suggest that synthetic nitrene chemistry is maturing with a wider scope not limited to these two reactions.

Rigid Analogues of Antimitotic Indolobenzazepinones: New Insights into Tubulin Binding via Molecular Modeling.

Two rigid analogues of 5-ethylindolobenzazepinone 4, a potent cytotoxic agent and inhibitor of tubulin polymerization, were prepared. The first was the indane derivative 5, in which the ethyl group is attached to the benzo moiety. The second was the pyrrolidine analogue 6, in which the ethyl chain was bound to the lactam nitrogen. While both compounds were considerably less active inhibitors of KB cell growth as compared to 4, inhibition of tubulin polymerization was only moderately reduced. Tubulin docking studies indicated that the aR and aS atropoisomers of 5 and 6 occupy different binding pockets at the colchicine binding site. Conversely, both aS-5 and aS-6 occupy the same binding pocket as aSS-4 but do not benefit from the favorable hydrophobic interactions provided by the C5 alkyl group of 4, thus possibly explaining their lower activities.