Several trypanosomatid
cyclic nucleotide phosphodiesterases (PDEs)
possess a unique, parasite-specific cavity near the ligand-binding
region that is referred to as the P-pocket. One of these enzymes, Trypanosoma brucei PDE B1 (TbrPDEB1), is considered a drug
target for the treatment of African sleeping sickness. Here, we elucidate
the molecular determinants of inhibitor binding and reveal that the
P-pocket is amenable to directed design. By iterative cycles of design,
synthesis, and pharmacological evaluation and by elucidating the structures
of inhibitor-bound TbrPDEB1, hPDE4B, and hPDE4D complexes, we have
developed 4a,5,8,8a-tetrahydrophthalazinones as the first selective
TbrPDEB1 inhibitor series. Two of these, 8 (NPD-008)
and 9 (NPD-039), were potent (Ki = 100 nM) TbrPDEB1 inhibitors with antitrypanosomal effects
(IC50 = 5.5 and 6.7 μM, respectively). Treatment
of parasites with 8 caused an increase in intracellular
cyclic adenosine monophosphate (cAMP) levels and severe disruption
of T. brucei cellular organization, chemically validating
trypanosomal PDEs as therapeutic targets in trypanosomiasis.
Screening a library of small-molecule compounds using a cell line expressing human GABA transporter 3 (hGAT3) in a [(3)H]GABA uptake assay identified isatin derivatives as a new class of hGAT3 inhibitors. A subsequent structure-activity relationship (SAR) study led to the identification of hGAT3-selective inhibitors (i.e., compounds 20 and 34) that were superior to the reference hGAT3 inhibitor, (S)-SNAP-5114, in terms of potency (low micromolar IC50 values) and selectivity (>30-fold selective for hGAT3 over hGAT1/hGAT2/hBGT1). Further pharmacological characterization of compound 20 (5-(thiophen-2-yl)indoline-2,3-dione) revealed a noncompetitive mode of inhibition at hGAT3. This suggests that this compound class, which has no structural resemblance to GABA, has a binding site different from the substrate, GABA. This was supported by a molecular modeling study that suggested a unique binding site that matched the observed selectivity, inhibition kinetics, and SAR of the compound series. These compounds are the most potent GAT3 inhibitors reported to date that provide selectivity for GAT3 over other GABA transporter subtypes.
A methodology has been developed for an efficient and selective lipase-catalyzed aza-Michael reaction of various amines (primary and secondary) with a series of acrylates and alkylacrylates. Reaction parameters were tuned, and under the optimal conditions it was found that Pseudomonas stutzeri lipase and Chromobacterium viscosum lipase showed the highest selectivity for the aza-Michael addition to substituted alkyl acrylates. For the first time also, some CLEAs were examined that showed a comparable or higher selectivity and yield than the free enzymes and other formulations.
As
over 6 million people are infected with Chagas disease and only
limited therapeutic options are available, there is an urgent need
for novel drugs. The involvement of cyclic nucleotide phosphodiesterases
(PDE) in the lifecycle and biological fitness of a number of protozoan
parasites has been described and several of these enzymes are thought
to be viable drug targets. Within this context, a PDE-focused library
was screened for its ability to affect the viability of Trypanosoma cruzi parasites. 5-(3-(Benzyloxy)-4-methoxyphenyl)-2-isopropyl-4,4-dimethyl-2,4-dihydro-3H-pyrazol-3-one (4), previously reported as
a human PDE4 inhibitor, was identified as a hit. Upon optimization
on three positions of the phenylpyrazolone scaffold, 2-isopropyl-5-(4-methoxy-3-(pyridin-3-yl)phenyl)-4,4-dimethyl-2,4-dihydro-3H-pyrazol-3-one (34) proved to be the most
active compound against intracellular forms of T. cruzi (pIC50 = 6.4) with a 100-fold selectivity with respect
to toxicity toward human MRC-5 cells. Evaluation on different life
stages and clinically relevant T. cruzi strains revealed that the phenylpyrazolones are not active against
the bloodstream form of the Y strain but show submicromolar activity
against the intracellular form of the Y- and Tulahuen strains as well
as against the nitro-drug-resistant Colombiana strain. In vitro screening
of phenylpyrazolones against TcrPDEB1, TcrPDEC, and TcrCYP51 showed
that there was a poor correlation between enzyme inhibition and the
observed phenotypic effect. Among the most potent compounds, both
TcrCYP51 and non-TcrCYP51 inhibitors are identified, which were both
equally able to inhibit T. cruzi in
vitro.
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