Visceral leishmaniasis (VL) is associated with treatment complications due to the continued growth of resistant parasites toward currently available pathogen-directed therapeutics. To limit the emergence and combat resistant parasites there is a need to develop new anti-leishmanial drugs and alternative treatment approaches, such as host-directed therapeutics (HDTs). Discovery of new anti-leishmanial drugs including HDTs requires suitable
in vitro
assay systems. Herein, we modified and evaluated a series of resazurin assays against different life-stages of the VL causing parasite,
Leishmania donovani
to identify novel HDTs. We further analyzed the synergy of combinatorial interactions between traditionally used pathogen-directed drugs and HDTs for clearance of intracellular
L. donovani
. The inhibitory concentration at 50% (IC
50
) of the five evaluated therapies [amphotericin B (AMB), miltefosine, paromomycin, DNER-4, and AR-12 (OSU-03012)] was determined against promastigotes, extracellular amastigotes, and intracellular amastigotes of
L. donovani
via a resazurin-based assay and compared to image-based microscopy. Using the resazurin-based assay, all evaluated therapies showed reproducible anti-leishmanial activity against the parasite's different life-stages. These results were consistent to the traditional image-based technique. The gold standard of therapy, AMB, showed the highest potency against intracellular
L. donovani
, and was further evaluated for combinatorial effects with the HDTs. Among the combinations analyzed, pathogen-directed AMB and host-directed AR-12 showed a synergistic reduction of intracellular
L. donovani
compared to individual treatments. The modified resazurin assay used in this study demonstrated a useful technique to measure new anti-leishmanial drugs against both intracellular and extracellular parasites. The synergistic interactions between pathogen-directed AMB and host-directed AR-12 showed a great promise to combat VL, with the potential to reduce the emergence of drug-resistant strains.
The human 20S proteasome inhibitor scytonemide A (1), a macrocyclic imine originally isolated from the cyanobacterium Scytonema hofmanni, was synthesized via a biomimetic solid-phase peptide synthesis (SPPS) approach employing the Weinreb AM resin. Utilizing this approach, cyclization of the protected heptapeptide via formation of the imine bond occurred spontaneously upon cleavage from the resin in the presence of a reducing agent and subsequent aqueous workup. The final deprotection step necessary to produce the natural product was accomplished under slightly basic conditions, facilitating cleavage of the silyl ether group while leaving the macrocycle intact. Purification of the synthetic scytonemide A was accomplished via normal-phase flash column chromatography, potentially facilitating larger scale preparation of the compound necessary for future mechanistic and SAR studies. The structure of the target compound was confirmed by NMR spectroscopy, which also shed light on differences in the spectroscopic data obtained for the synthetic and natural scytonemide A samples for some of the amide and alcohol signals in the H NMR spectrum.
Avian pathogenic
Escherichia coli
(APEC), a subgroup of ExPEC, is a zoonotic pathogen with public health importance. Quorum sensing is a mechanism that regulates virulence, biofilm formation, and pathogenesis in bacteria.
Optimization of degrader properties is often a challenge due to their beyond-rule-of-5 nature. Given the paucity of known E3 ligases and the often-limited choice of ligands with varied chemical structures for a given protein target, degrader linkers represent the best position within the chimeric molecules to modify their overall physicochemical properties. In this work, a series of AT7519-based CDK9 degraders was assembled using click chemistry, facilitating the tuning of aqueous solubility and lipophilicity while retaining their linker type and molecular weight. Using chromatographic logD and kinetic solubility experiments, we show that degraders with similar chemical constitution but varied position of the embedded triazole demonstrate different lipophilicity and aqueous solubility properties. Overall, this work highlights the impact of triazole placement on linker composition through application of click chemistry for degrader synthesis and its ability to be used to promote the achievement of favorable physicochemical properties.
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