Cyclic tetrapeptide HDAC inhibitors as potential therapeutics for spinal muscular atrophy: Screening with iPSC-derived neuronal cells.

Spinal muscular atrophy (SMA) is an autosomal recessive neuromuscular disorder that is caused by inactivating mutations in the Survival of motor neuron 1 (SMN1) gene, resulting in decreased SMN protein expression. Humans possess a paralog gene, SMN2, which contains a splicing defect in exon 7 leading to diminished expression of full-length, fully functional SMN protein. Increasing SMN2 expression has been a focus of therapeutic development for SMA. Multiple studies have reported the efficacy of histone deacetylase inhibitors (HDACi) in this regard. However, clinical trials involving HDACi have been unsatisfactory, possibly because previous efforts to identify HDACi to treat SMA have employed non-neuronal cells as the screening platform. To address this issue, we generated an SMA-patient specific, induced pluripotent stem cell (iPSC) derived neuronal cell line that contains homogenous Tuj1+neurons. We screened a small library of cyclic tetrapeptide HDACi using this SMA neuronal platform and discovered compounds that elevate SMN2 expression by an impressive twofold or higher. These candidates are also capable of forming gems intranuclearly in SMA neurons, demonstrating biological activity. Our study identifies new potential HDACi therapeutics for SMA screened using a disease-relevant SMA neuronal cellular model.

Selective detection and inhibition of active caspase-3 in cells with optimized peptides.

Caspases are a family of cysteine-aspartyl proteases that are well recognized for their essential roles in apoptosis and inflammation. Recently, caspases have also been linked to the promotion of other biologically important phenomena, such as cellular differentiation and proliferation. Dysregulation of the multifaceted and indispensable activities of caspases has been globally linked to several diseases, including cancer and neurodegenerative disorders; however, the specific caspase members responsible for these diseases have yet to be assigned. Activity-based probes (ABPs) and peptide-based inhibitors are instrumental in the detection and control of protease activity and serve as alternative methods to genetic approaches. Such molecules aid in the interrogation of specific proteases within cellular and animal models as well as help elucidate aberrant proteolytic function correlated to disease phenotypes. No ABPs or inhibitors have been discovered that specifically target one of the eleven human caspases in a cellular context. Therefore, ascribing distinct contributions to an individual caspase activity within naturally occurring biological systems is not possible. Herein, we describe a peptide series optimized for the selective detection and inhibition of active caspase-3 in cells. These compounds exhibit low nanomolar potency against caspase-3 with >120-fold selectivity over caspase-7 which shares 77% active site identity. Our ability to individually target wild-type active caspase-3 for detection and cell permeable inhibition is a valuable proof-of-concept methodology that can be readily employed to probe the significance of caspase-3 in apoptosis, neurological disorders, cardiovascular diseases, and sepsis.

Small-molecule procaspase activators identified using fluorescence polarization.

Wake up, protein! Small molecules that directly activate proteins are rare and their discovery opens new avenues for the development of drugs and chemical tools to probe the functions and mechanisms of protein targets. To address the one-sided dichotomy between enzyme inhibition and activation, we describe a series of procaspase activators as chemical tools in the study of caspase biology.

Discovery of a highly selective caspase-3 substrate for imaging live cells.

Caspases are a family of cysteine proteases that are well-known for their roles in apoptosis and inflammation. Recent studies provide evidence that caspases are also integral to many additional cellular processes, such as differentiation and proliferation. Likewise, aberrant caspase activity has been implicated in the progression of several diseases, including neurodegenerative disorders, cancer, cardiovascular disease, and sepsis. These observations establish the importance of caspases to a diverse array of physiological functions and future endeavors will undoubtedly continue to elucidate additional processes that require caspase activity. Unfortunately, the existence of 11 functional human caspases, with overlapping substrate specificities, confounds the ability to confidently assign one or more isoforms to biological phenomena. Herein, we characterize a first-in-class FRET substrate that is selectively recognized by active caspase-3 over other initiator and executioner caspases. We further apply this substrate to specifically image caspase-3 activity in live cells undergoing apoptosis.

Selective inhibition of initiator versus executioner caspases using small peptides containing unnatural amino acids.

Caspases are fundamental to many essential biological processes, including apoptosis, differentiation, and inflammation. Unregulated caspase activity is also implicated in the development and progression of several diseases, such as cancer, neurodegenerative disorders, and sepsis. Unfortunately, it is difficult to determine exactly which caspase(s) of the 11 isoforms that humans express is responsible for specific biological functions. This lack of resolution is primarily due to highly homologous active sites and overlapping substrates. Currently available peptide-based inhibitors and probes are based on specificity garnered from peptide substrate libraries. For example, the canonical tetrapeptide LETD was discovered as the canonical sequence that is optimally recognized by caspase-8; however, LETD-based inhibitors and substrates promiscuously bind to other isoforms with equal affinity, including caspases-3, -6, and -9. In order to mitigate this problem, we report the identification of a new series of compounds that are >100-fold selective for inhibiting the initiator caspases-8 and -9 over the executioner caspases-3, -6, and -7.

Selective detection of caspase-3 versus caspase-7 using activity-based probes with key unnatural amino acids.

Caspases are required for essential biological functions, most notably apoptosis and pyroptosis, but also cytokine production, cell proliferation, and differentiation. One of the most well studied members of this cysteine protease family includes executioner caspase-3, which plays a central role in cell apoptosis and differentiation. Unfortunately, there exists a dearth of chemical tools to selectively monitor caspase-3 activity under complex cellular and in vivo conditions due to its close homology with executioner caspase-7. Commercially available activity-based probes and substrates rely on the canonical DEVD tetrapeptide sequence, which both caspases-3 and -7 recognize with similar affinity, and thus the individual contributions of caspase-3 and/or -7 toward important cellular processes are irresolvable. Here, we analyzed a variety of permutations of the DEVD peptide sequence in order to discover peptides with biased activity and recognition of caspase-3 versus caspases-6, -7, -8, and -9. Through this study, we identify fluorescent and biotinylated probes capable of selective detection of caspase-3 using key unnatural amino acids. Likewise, we determined the X-ray crystal structures of caspases-3, -7, and -8 in complex with our lead peptide inhibitor to elucidate the binding mechanism and active site interactions that promote the selective recognition of caspase-3 over other highly homologous caspase family members.

Discovery of HDAC Inhibitors That Lack an Active Site Zn(2+)-Binding Functional Group.

Natural and synthetic histone deacetylase (HDAC) inhibitors generally derive their strong binding affinity and high potency from a key functional group that binds to the Zn(2+) ion within the enzyme active site. However, this feature is also thought to carry the potential liability of undesirable off-target interactions with other metalloenzymes. As a step toward mitigating this issue, here, we describe the design, synthesis, and structure-activity characterizations of cyclic α3ß-tetrapeptide HDAC inhibitors that lack the presumed indispensable Zn(2+)-binding group. The lead compounds (e.g., 15 and 26) display good potency against class 1 HDACs and are active in tissue culture against various human cancer cell lines. Importantly, enzymological analysis of 26 indicates that the cyclic α3ß-tetrapeptide is a fast-on/off competitive inhibitor of HDACs 1-3 with K i values of 49, 33, and 37 nM, respectively. Our proof of principle study supports the idea that novel classes of HDAC inhibitors, which interact at the active-site opening, but not with the active site Zn(2+), can have potential in drug design.

Potential Agents for Treating Cystic Fibrosis: Cyclic Tetrapeptides that Restore Trafficking and Activity of ΔF508-CFTR.

Cystic fibrosis (CF) is a loss-of-function disease caused by mutations in the CF transmembrane conductance regulator (CFTR) protein, a chloride ion channel that localizes to the apical plasma membrane of epithelial cells. The most common form of the disease results from the deletion of phenylalanine-508 (ΔF508), leading to the accumulation of CFTR in the endoplasmic reticulum with a concomitant loss of chloride flux. We discovered that cyclic tetrapeptides, such as 11, 14, and 15, are able to correct the trafficking defect and restore cell surface activity of ΔF508-CFTR. Although this class of cyclic tetrapeptides is known to contain inhibitors of certain histone deacetylase (HDAC) isoforms, their HDAC inhibitory potencies did not directly correlate with their ability to rescue ΔF508-CFTR. In full HDAC profiling, 15 strongly inhibited HDACs 1, 2, 3, 10 and 11, but not HDACs 4-9. Although 15 had less potent IC(50) values than reference agent vorinostat (2) in HDAC profiling, it was markedly more potent than 2 in rescuing ΔF508-CFTR. We suggest that specific HDACs can have a differential influence on correcting ΔF508-CFTR, which may reflect both deacetylase and protein scaffolding actions.

Pd(II)-catalyzed o-C-H acetoxylation of phenylalanine and ephedrine derivatives with MeCOOO(t)Bu/Ac2O.

Pd(II)-catalyzed ortho C-H acetoxylation of triflate protected phenethyl- and phenpropylamines has been achieved with tert-butyl peroxyacetate as the stoichiometric oxidant and either DMF or CH(3)CN as the promoter. The reaction was found to tolerate a large variety of functional groups and could be combined with subsequent intramolecular amination to afford functionalized indoline derivatives.