A high throughput screening approach to identify isocitrate lyase inhibitors from traditional Chinese medicine sources

Abstract

Isocitrate lyase (ICL) catalyses the first step of the glyoxylate bypass pathway, which reversibly cleaves isocitrate into succinate and glyoxylate. This pathway occurs in a wide range of pathogens and plays a key role in the pathogenesis of Mycobacterium tuberculosis (MTB) suggesting that it may represent a drug target for the treatment of tuberculosis. ICL was cloned, expressed, and purified, and a high-throughput screen (HTS) developed to screen active extracts derived from traditional Chinese medicines (TCMs) for inhibition of ICL. A colorimetric assay based on the formation of glyoxylatephenylhydrazone was used to measure ICL activity. The assay had signal to noise (S/N) of 12.74 and Z' factor of 0.72, indicating that the assay was suitable for HTS. Screening of a collection of 465 extracts derived from TCMs resulted in the identification of two extracts from Illicium verum Hook.f (Illiciaceae, XHD-1) and Zingiber officinale Rosc (Zingiberaceae, XHD-2), which inhibited ICL with IC 50 values of 47.7716.9 and 18.270.9 mg/ml, respectively. Drug Dev. Res. 67:818-823, 2006.

Major roles of isocitrate lyase and malate synthase in bacterial and fungal pathogenesis

“…1). Efforts to isolate natural ICL inhibitors from plants revealed that extracts of Illicium verum and Zingiber officinale inhibit the ICL of Mycobacterium tuberculosis (Bai et al, 2007).…”

In vitro and in vivo Activities of a New Lead Compound I2906 against <i>Mycobacterium tuberculosis</i>

“…This finding indicated that I2906 likely possesses cross-resistance with current anti-TB drugs, especially with second-line drugs. I2906 was firstly identified as an inhibitor of ICL according to a high throughput screening approach [6] . Although it showed week inhibitory effect on the enzyme, I2906 still displayed high activity against all susceptible clinical isolates and most tested clinical drug-resistant strains.…”

“…ICL plays an important role in the persistent infection of Mycobacterium tuberculosis in inflammatory macrophages, but it does not exist in vertebrates [5] , so ICL provides a unique and selective target for the mechanism of action of tuberculostatic drugs. In order to identify new inhibitors of this enzyme, the high throughput screening against our chemical library containing 5,000 compounds was used to discover new candidates [6] . One compound I2906, 1-ethyl-4-hydroxy-2-oxo-N -tridecanoyl-1,2-dihydroquinoline-3-carbohydrazide ( table 1 ), was selected as a hit with inhibitory effect of IC 50 of 134.3 g/ml.…”

5-Nitro-2,6-dioxohexahydro-4-pyrimidinecarboxamides: synthesis, in vitro antimycobacterial activity, cytotoxicity, and isocitrate lyase inhibition studies

“…Isocitrate lyase activity was determined at 37°C by measuring the formation of glyoxylate phenylhydrazone at 324 nm 12 . The reaction mixture contained 100 µL of 0.5 mM potassium phosphate buffer, 1.2 µL of 1 mM magnesium chloride, 24 µL of 100 mM 2-mercaptoethanol, 7 µL of 4 mM phenylhydrazine hydrochloride, 6 µL of 50 mM trisodium isocitric acid, and ICL enzyme (usually 3-6 µL).…”

“…Several small-molecule inhibitors have been described 16 as MTB ICL inhibitors; however, none has been developed as a drug for MTB. Isocitrate lyase activity was determined at 37°C by measuring the formation of glyoxylate phenylhydrazone in the presence of phenylhydrazine and isocitrate lyase at 324 nm, based on the method described 12 . The compounds were screened at a single concentration of 10 µM, and percentage inhibitions of the screened compounds along with that of the standard MTB ICL inhibitor 3-NPA (at 100 µM) for comparison are reported (Table 3).…”