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.2.3.7 Carboxylic AcidsThe antiproliferative activity of sodium butyrate (Na+ salt of 20, Fig.8) towardseveral types of carcinogenic cells has long been known, but it was not until muchlater that its anticancer activity was linked to HDAC inhibition [75, 76].Pivanex 23is a prodrug, which is metabolized in vivo to release butyric acid [77].Other short-chain fatty acids such as valproic acid 21 and phenyl butyrate 22 have beeninvestigated in the same context [78, 79].These alkyl carboxylates show HDACinhibitory activity typically in the low millimolar range and are much weaker thanthe strong chelators described above.Interestingly methotrexate 24, a well-known dihydrofolate reductase inhibitorthat is clinically used for a number of indications such as leukemia and severepsoriasis, has recently been shown to interfere with HDAC activity [80].Structur-ally, it may be regarded as a butyric acid derivative.OOO OOOHOHO OOH20 21 22 23(butyric acid) (valproic acid, VPA) (phenyl butyrate, 4PBA) (pivanex, AN-9)O OHOOHNH2 NHN ON NFig.8 Structure of short fattyacid inhibitors andH2N N N24 (methotrexate)methotrexateThe Role of Histone Deacetylases in Neurodegenerative Diseases 152.3.8 Selectivity DeterminationDetermination of the selectivity of HDAC inhibitors toward isolated HDAC iso-forms has been challenging.As a result, many publications describe inhibitoryactivity using cell lysates.Understanding in this area has been dogged by difficul-ties in the purification of individual isoforms, leading at times to contradictoryresults.This problem has subsequently been overcome by the isolation of proteinfrom transfected Escherichia coli, which contain no endogenous HDACs [7].Little is known about the natural substrate specificities of the HDAC isoforms,indeed, as stated earlier, it may be that class IIa HDACs have no meaningful catalyticdeacetylation activity in vivo [7]; rather, they may act as Ne-acetyl lysine recognitiondomains [81].Various assays used to measure the inhibition of HDACs have beendeveloped.These range from high resolution mass spectrometry profiling of wholecell systems [1] to isolated enzyme assays using a synthetic acetyl lysine substrate[82].The low intrinsic catalytic activity of class IIa HDACs has been particularlyproblematic.To overcome this, gain-of-function mutants have been used [7].Theseresults have the caveat that they rely on modification of the active site and may notgive an accurate reflection of the true binding event.Indeed, for some inhibitors, amarked difference is observed between gain-of-function mutant and wild-typeHDAC4 [74].Recently, an activated substrate has been designed which is successfully turnedover by class IIa isoforms.This trifluoroacetate derivative is much more labile thanthe corresponding acetate and as such does not require the stabilizing tyrosineresidue found in class I, IIb, and IV HDACs for the enzymatic conversion [7].This substrate is efficiently deacetylated by the class IIa HDACs and intriguinglyseems to show selectivity over class I [81].This observed selectivity is likely to be aresult of the more sterically demanding CF3 group clashing with the more con-gested catalytic site of the class I isoforms.The tools to de-convolute HDAC isoform activity have now been developed.Recent results highlighting the difficulty in purification of individual isoforms frommammalian systems suggest that a cautious interpretation of historical data isrequired.In summary, the HDAC inhibitors known to date interact with the metal ion withinthe catalytic domain of the enzymes.However, besides recognition and deacetylationof substrates bearing Ne-acetylated lysine residues, HDACs clearly adopt other cellu-lar functions such as recruitment of other HDAC family members or transcriptionfactors via protein protein interactions.Molecules that selectively interfere with sucha non-catalytic function would serve as excellent tools to further understand anddissect the function of these enzymes [83].As compared to the currently investigatedHDAC inhibitors, such molecules would likely exhibit a different selectivity profile.In addition, the cellular localization of certain HDAC subtypes is governedby chaperone proteins (e.g., 14-3-3) which, depending on the phosphorylation state,transport the enzyme from the nucleus to the cytoplasm [ Pobierz całość w formacie PDF ]