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, 53 (14), 5281-9

Thiadiazole Carbamates: Potent Inhibitors of Lysosomal Acid Lipase and Potential Niemann-Pick Type C Disease Therapeutics

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Thiadiazole Carbamates: Potent Inhibitors of Lysosomal Acid Lipase and Potential Niemann-Pick Type C Disease Therapeutics

Anton I Rosenbaum et al. J Med Chem.

Abstract

Niemann-Pick type C (NPC) disease is a lysosomal storage disorder characterized at the cellular level by abnormal accumulation of cholesterol and other lipids in lysosomal storage organelles. Lysosomal acid lipase (LAL) has been recently identified as a potential therapeutic target for NPC. LAL can be specifically inhibited by a variety of 3,4-disubstituted thiadiazole carbamates. An efficient synthesis of the C(3) oxygenated/C(4) aminated analogues has been developed that furnishes the products in high yields and high degrees of purity. Common intermediates can also be used for the synthesis of the C(3) carbon substituted derivatives. Herein we tested various thiadiazole carbamates, amides, esters, and ketones for inhibition of LAL. In addition, we tested a diverse selection of commercially available non-thiadiazole carbamates. Our studies show that, among the compounds examined herein, only thiadiazole carbamates are effective inhibitors of LAL. We present a mechanism for LAL inhibition by these compounds whereby LAL transiently carbamoylates the enzyme similarly to previously described inhibition of acetylcholinesterase by rivastigmine and other carbamates as well as acylation of various lipases by orlistat.

Figures

Scheme 1
Scheme 1
a Reagents and conditions: (a) 2° amine (4 equiv), 22 °C or Δ; (b) KOH (4 equiv), DMSO:H2O, reflux; (c) KOtBu, aminocarbonyl chloride, THF, 22 °C.
Scheme 2
Scheme 2
Scheme 3
Scheme 3
a Reagents and conditions: (a) 1.0 equiv LiHMDS, THF, 22 °C, 3 min; (b) 1.3 equiv LiHMDS, tBuOAc, THF, 60 °C, 77%; (c) 2.3 equiv LiHMDS, 1.1 equiv tBuOAc, THF, 60 °C, 80%.
Scheme 4
Scheme 4
Kinetic outline of transient LAL modification by 3,4-disubstituted thiadiazole carbamates.
Figure 1
Figure 1
An example of LAL inhibitor previously observed to reduce lysosomal cholesterol levels in NPC-deficient cells.
Figure 2
Figure 2. Lineweaver-Burk analysis of LAL inhibition
Compound 13 was pre-incubated for 30 min at 37 °C with phLAL, the reaction was started by addition of 4MUO at various concentrations and monitored for 45 min. Compound concentration indicated refers to the concentration in the final reaction mixture. Linear regression fits to the data were computed in MATLAB. R2>0.95 for all fits. Data are from two independent experiments (6<n<8 for treated samples, where n is total number of wells per condition used for quantification).
Figure 3
Figure 3
Time-dependence of LAL inhibition by thiadiazole carbamates. phLAL was pre-incubated with the various compounds at 833 nM for the indicated times at 37 °C. The reaction was started by addition of 125 μM 4MUO and monitored for 30 min at 37 °C. The final compound concentration in the assay was 500 nM. Data represent averages ± SEM of 2 independent experiments normalized to control (DMSO) average value for each experiment (n=12, where n is total number of wells per condition used for quantification).
Figure 4
Figure 4
Compound efficacy in cells. Enzymatic assay for inhibition of LAL in cell lysates of cells treated with compound continuously for ~4 days (A) or treated for ~3 days and allowed to recover in growth media for ~1 additional day (B). Data represent averages ± SEM of 3 independent experiments normalized to control (DMSO) average value for each experiment (n=12, where n is total number of wells per condition used for quantification). Quantification of thiadiazole effects after ~3 days of treatment on cholesterol accumulation as measured by filipin LSO assay in NPC1 mutant cells (C) or neutral lipid accumulation in apparently normal human fibroblasts as measured by quantification of LipidTOX green staining (D). Data are presented as percentage of the average value of the DMSO controls (C) or normalized to the 10 μM 12 average value (D) in each experiment. Error bars, where visible, are SEM. (n=8 for treated samples, where n is total number of wells per condition used for quantification).

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