Weiping Zheng

Novel thiourea-based sirtuin inhibitory warheads

N(ε)-Thiocarbamoyl-lysine was recently demonstrated by our laboratory to be a potent catalytic mechanism-based SIRT1/2/3 inhibitory warhead, in the current study, among the prepared analogs of N(ε)-thiocarbamoyl-lysine with its terminal NH2 mono-substituted with alkyl and aryl groups, we found that N(ε)-methyl-thiocarbamoyl-lysine and N(ε)-carboxyethyl-thiocarbamoyl-lysine, respectively, also behaved as strong inhibitory warheads against SIRT1/2/3 and SIRT5, typical deacetylases and deacylase in the human sirtuin family, respectively. Moreover, N(ε)-methyl-thiocarbamoyl-lysine was found in the study to be a ∼ 2.5-18.4-fold stronger SIRT1/2/3 inhibitory warhead than its lead warhead N(ε)-thiocarbamoyl-lysine.

Sirtuins as emerging anti-parasitic targets

Silent information regulator 2 (Sir2) enzymes or sirtuins are a family of NAD(+)-dependent protein N(ε)-acetyl-lysine (AcK) deacetylases. Sirtuins are also evolutionarily conserved proteins that are present in all kingdoms of life ranging from bacteria to humans. Interestingly, it was recently found that the sirtuins found in various human parasites (especially the Plasmodium, Trypanosoma, and Leishmania species) were pro-survival for the parasites under various conditions. Therefore, these parasitic sirtuins have emerged as novel anti-parasitic therapeutic targets. This article reviews the currently available structural, biochemical, pharmacological, and medicinal chemistry studies on these enzymes, and discusses the perspectives of selectively targeting the parasitic sirtuins as a novel therapeutic strategy for the human parasitic diseases.

Sirtuin Inhibition: Strategies, Inhibitors, and Therapeutic Potential

The β-NAD+-dependent Nε-acyl-lysine deacylation reaction catalyzed by sirtuin family members has been increasingly demonstrated to be important in regulating multiple crucial cellular processes and has also been proposed to be a therapeutic target for multiple human diseases. Accordingly, its inhibitors have been actively pursued over the past few years. In addition, we have also seen the pharmacological assessment of sirtuin inhibitory compounds, although to a lesser extent. In this review, we first discuss how sirtuin inhibitors were discovered with the use of various approaches. We then follow with a discussion of pharmacological studies using sirtuin inhibitors. Our aim here is to set a stage for developing future superior sirtuin inhibitors and for an expanded effort in exploiting inhibitors to explore and/or validate the therapeutic potential stemming from the inhibition of the sirtuin-catalyzed deacylation reaction.

Simple N(ε)-thioacetyl-lysine-containing cyclic peptides exhibiting highly potent sirtuin inhibition.

Transforming a linear pentapeptidic pan-SIRT1/2/3 inhibitor harboring the catalytic mechanism-based sirtuin inhibitory warhead N(ε)-thioacetyl-lysine into its side chain-to-side chain cyclized derivatives was able to furnish highly potent SIRT1/2/3 inhibition (low nM). This finding attests to the feasibility of developing structurally simple yet highly potent catalytic mechanism-based cyclic peptidic sirtuin inhibitors.

Human SIRT3 tripeptidic inhibitors containing Nε-thioacetyl-lysine

Built upon our lead pan-SIRT1/2/3 tripeptidic inhibitors that contain the catalytic mechanism-based sirtuin inhibitory warhead N(ε)-thioacetyl-lysine, three of their analogs (i.e., 7, 9, and 19) were discovered in the current study to exhibit a significantly enhanced SIRT3 inhibitory selectivity while maintaining the SIRT3 inhibitory potency. These compounds represent novel lead compounds for developing more potent and selective SIRT3 inhibitors of the catalytic mechanism-based type.

A Selective Cyclic Peptidic Human SIRT5 Inhibitor

In the current study, we discovered that a side chain-to-side chain cyclic pentapeptide harboring a central Nε-carboxyethyl-thiocarbamoyl-lysine residue behaved as a strong and selective (versus human SIRT1/2/3/6) inhibitor against human SIRT5-catalyzed deacylation reaction. This compound was also found to be proteolytically much more stable than its linear counterpart. This compound could be a valuable lead for developing stronger, selective, metabolically stable, and cell permeable human SIRT5 inhibitors.

An enhanced functional interrogation/manipulation of intracellular signaling pathways with the peptide ‘stapling’ technology

Specific protein–protein interactions (PPIs) constitute a key underlying mechanism for the presence of a multitude of intracellular signaling pathways, which are essential for the survival of normal and cancer cells. Specific molecular blockers for a crucial PPI would therefore be invaluable tools for an enhanced functional interrogation of the signaling pathway harboring this particular PPI. On the other hand, if a particular PPI is essential for the survival of cancer cells but is absent in or dispensable for the survival of normal cells, its specific molecular blockers could potentially be developed into effective anticancer therapeutics. Due to the flat and extended PPI interface, it would be conceivably difficult for small molecules to achieve an effective blockade, a problem which could be potentially circumvented with peptides or proteins. However, the well-documented proteolytic instability and cellular impermeability of peptides and proteins in general would make their developing into effective intracellular PPI blockers quite a challenge. With the advent of the peptide ‘stapling’ technology which was demonstrated to be able to stabilize the α-helical conformation of a peptide via bridging two neighboring amino-acid side chains with a ‘molecular staple’, a linear parent peptide could be transformed into a stronger PPI blocker with enhanced proteolytic stability and cellular permeability. This review will furnish an account on the peptide ‘stapling’ technology and its exploitation in efforts to achieve an enhanced functional interrogation or manipulation of intracellular signaling pathways especially those that are cancer relevant.

Bivalent SIRT1 inhibitors

In the current study, bivalent compounds 1-17 constructed by covalently linking the ɛ-amino group of lysine in a tripeptidic scaffold to a functionality via a linker were prepared and examined for their inhibitory potencies against SIRT1, a prototypical member of the β-nicotinamide adenine dinucleotide (β-NAD+)-dependent sirtuin family of protein Nε-acyl-lysine deacylases. A few of them were found to be stronger SIRT1 inhibitors than the Nɛ-acetyl-lysine-containing monovalent counterparts 18 and 19. As exemplified with compounds 6 and 18, a bivalent SIRT1 inhibitor could exhibit a greater degree of inhibitory selectivity among SIRT1/2/3 than the corresponding monovalent counterpart. This study has laid a foundation for the future development of superior bivalent inhibitors against the (patho)physiologically and therapeutically important sirtuin family of deacylase enzymes.

Cyclic peptide-based potent and selective SIRT1/2 dual inhibitors harboring Nε-thioacetyl-lysine

In the current study, we discovered that several N-terminus-to-side chain cyclic tripeptides harboring the catalytic mechanism-based SIRT1/2/3 inhibitory warhead Nε-thioacetyl-lysine at their central positions exhibited a comparably strong inhibition (nM level) against the SIRT1/2-catalyzed Nε-acetyl-lysine deacetylation reactions. Their dual SIRT1/2 inhibitory action was also found to be stronger than that against SIRT3/5/6. Considering the previous demonstration that a SIRT1/2 dual inhibition could be instrumental in achieving an anti-cancer effect on those cancers retaining the wild-type tumor suppresser p53 protein, these compounds could be employed as leads for developing novel anti-cancer agents.

Chemical Probes in Sirtuin Research

Sirtuins refer to a family of intracellular enzymes that are the yeast silent information regulator 2 (sir2) protein homologs found in organisms from all the three kingdoms of life. This family of enzymes primarily catalyze the protein Nɛ-acyl-lysine deacylation reaction despite the report for a type of bacterial/fungal sirtuins to robustly catalyze a protein mono-ADP-ribosylation reaction, however, these two group transfer reactions employ the redox coenzyme β-nicotinamide adenine dinucleotide (β-NAD+) as the obligatory cosubstrate. Since 2000, in addition to histone proteins, more and more nonhistone proteins have also been identified as native substrates for the sirtuin-catalyzed deacylation, consistent with the ever-increased demonstration that this enzymatic reaction plays an important regulatory role in a variety of cellular processes, such as gene transcription and metabolism. This latter role is also consistent with the absolute dependence on β-NAD+ of the deacylation reaction catalyzed by sirtuin family members. The sirtuin-catalyzed deacylation has further been proposed as a contemporary therapeutic target for human diseases, such as cancer, neurodegenerative and metabolic diseases. In order to fully tap the therapeutic potential of the sirtuin-catalyzed deacylation, the past few years have witnessed a tremendous advancement in mechanistic elucidation, chemical modulator (inhibitor and activator) development, (chemical) biological and pharmacological exploration of the sirtuin-catalyzed deacylation reaction. During the journey of this knowledge advancement, the use of carefully designed chemical probes has played an elegant role. This chapter will delineate the development and application of these chemical probes in sirtuin research.