Novel Sirtuin Inhibitory Warheads Derived From The N~?-acetyl-lysine Analog L-2-Amino-7-carboxamidoheptanoic Acid(L-ACAH)

Sirtuins are a family of enzymes able to catalyze the ?-nicotinamide adenine dinucleotide(?-NAD+)-dependent N?-acyl-lysine deacylation reaction, which serves as one way of reversing the post-translational lysine N?-acylation on proteins. Another family of protein N?-acyl-lysine deacylases is the Zn2+-containing amidohydrolases.The sirtuin-catalyzed protein N?-acyl-lysine deacylation reaction is not merely an amide hydrolysis reaction, instead, in this reaction, a sirtuin utilizes NAD+as the co-substrate to achieve the deacylation with the generation of three enzymatic products, i.e. nicotinamide, the deacylated protein species, and 2'-O-acyl-ADP-ribose(2'-O-AADPR). Yeast silent information regulator 2(Sir2) was the first sirtuin identified, and so far seven sirtuins(i.e.SIRT1-7) have been found in mammals including humans. Since the discovery in 2000 of the yeast Sir2, the sirtuin-catalyzed deacylation reaction has been increasingly demonstrated to play an important role in regulating crucial cellular processes such as transcription, DNA damage repair, and metabolism. This enzymatic reaction has also been regarded as a contemporary therapeutic target for human diseases such as cancer and metabolic diseases.Therefore, developing chemical modulators(inhibitors and activators) of the sirtuin-catalyzed deacylation reaction has been of interest to many researchers. While a decent number of sirtuin chemical modulators(mainly inhibitors) have been reported, identifying truly potent, selective, and cell permeable sirtuin chemical modulators still warrants further efforts. Among the currently known sirtuin inhibitors,the catalytic mechanism-based sirtuin inhibitors constitute a major type and multiple catalytic mechanism-based sirtuin inhibitory warheads have been identified since the first of such warheads, i.e. N?-thioacetyl-lysine, was reported in 2006. It should be noted that the inhibitory action of a catalytic mechanism-based sirtuin inhibitor was endowed by the catalytic mechanism-based sirtuin inhibitory warhead that it harbors.In order to address the concern over the potential metabolic toxicity associated with the use of the thioamide-based warhead N?-thioacetyl-lysine, researchers identified L-2-amino-7-carboxamido-heptanoic acid(L-ACAH, a close structural analog of N?-acetyl-lysine) as another catalytic mechanism-based SIRT1/2/3 inhibitory warhead.Considering that SIRT1/2/3 are also able to catalyze the removal of the lysine N?-acyl groups bulkier than acetyl, such as propionyl and myristoyl, in the current thesis work,we prepared the analogs of L-ACAH in whch its carboxamide NH2 was substitutedwith simple alkyl and aryl groups for a structure activity study. It should be noted that long-chain fatty acyl groups were not selectedbecause the human sirtuin family member SIRT6 is also a strong N?-long-chain fatty acyl deacylase(typically demyristoylase). However, SIRT6 has a relatively weaker deacylase activity on the N?-short-chain fatty acyl lysine. In light of this, and due to the catalytic mechanism-based nature of the SIRT1/2/3 inhibitory warhead L-ACAH and the chemical mechanism conservation among the sirtuin family members, the carboxamide NH2-dodecylated analog of L-ACAH was also prepared and evaluated in the current study to assess its inhibitory power against the SIRT6-catalyzed lysine deacylation reaction. In the human sirtuin family, SIRT5 is another weak lysine N?-deacetylase, yet it is a strong lysine N?-demalonylase/desuccinylase/ deglutarylase.The carboxamide NH2-carboxyalkylated analogs of L-ACAH were thus prepared and their inhibitory power against the SIRT5-catalyzed N?-acyl-lysine deacylation reaction was examined. Of note, these analogs are close structural mimics of N?-malonyl-,N?-succinyl-, and N?-glutaryl-lysine. Our findings in the current work include:(1) the carboxamide NH2-ethylated analog was a ~2.4–6.6-fold stronger SIRT1/2/3 inhibitory warhead than L-ACAH,(2) the carboxamide NH2-dodecylated analog of L-ACAH was a strong SIRT6 inhibitory warhead,(3) the carboxamide NH2-carboxymethylated analog of L-ACAH was a strong SIRT5 inhibitory warhead.