Immobilised metal affinity chromatography for the capture of hydroxamate-containing siderophores and other Fe(III)-binding metabolites directly from bacterial culture supernatants.

Nickel(II)-based immobilized metal affinity chromatography (IMAC) has been used to capture from standard samples the hydroxamate-containing siderophores, acetohydroxamic acid (ahaH), suberodihydroxamic acid (shaH(2)) or desferrioxamine B (DFOB) in recoveries ranging between 35-90%. The capacity of a 1 mL Ni(II)-charged IMAC column towards DFOB capture at the pH optima of 8.9 is approximately 3000 nmol. This method has been used for the direct capture of DFOB (approximately 65% recovery) from the untreated supernatant of iron-deprived cultures of Streptomyces pilosus, the soil bacterium from which DFOB was first discovered. In addition to selecting for DFOB and a related siderophore, two other Fe(III)-responsive species have been identified from RP-HPLC analysis of the IMAC-processed eluant from the S. pilosus supernatant. Since the characterisation of siderophores from natural systems is hampered by the low yields obtained from traditional purification methods, this IMAC-based affinity method offers significant potential for improving yields of this key class of bioligands and other small molecule metabolites with affinities to IMAC-compatible metal ions.

Zn(II)-dependent histone deacetylase inhibitors: suberoylanilide hydroxamic acid and trichostatin A.

Suberoylanilide hydroxamic acid (SAHA, vorinostat, Zolinza) and trichostatin A (TSA) are inhibitors of the Zn(II)-dependent class I and class II histone deacetylases (HDACs), which are enzymes that operate in concert with histone acetyltransferases (HATs) to regulate the acetylation status of the epsilon-amino group of lysine residues of nucleosomal histones in chromatin. An increased level of histone acetylation resulting from the SAHA or TSA inhibition of Zn(II)-dependent HDACs relaxes the chromatin structure and upregulates transcription. The links made in the 1990s between the inhibition of HDAC activity and the suppression of tumor growth have brought the design of HDAC inhibitors (HDACi) to the forefront of oncology research. SAHA has anticancer activity against hematologic and solid tumors and has been approved by the FDA for the treatment of cutaneous T-cell lymphoma. The increased molecular-level understanding of class I and class IIa HDACs from X-ray crystallography highlights differences in the residues distal to the active site and in the cavity size, which has implications for HDACi substrate specificity and enzyme mechanism. Results from HDAC-focussed activity-based protein profiling experiments may lead to the design of molecules that are class-specific HDACi.