Cysteine proteases as therapeutic targets: does selectivity matter? A systematic review of calpain and cathepsin inhibitors

AbstractCysteine proteases continue to provide validated targets for treatment of human diseases. In neurodegenerative disorders, multiple cysteine proteases provide targets for enzyme inhibitors, notably caspases, calpains, and cathepsins. The reactive, active-site cysteine provides specificity for many inhibitor designs over other families of proteases, such as aspartate and serine; however, a) inhibitor strategies often use covalent enzyme modification, and b) obtaining selectivity within families of cysteine proteases and their isozymes is problematic. This review provides a general update on strategies for cysteine protease inhibitor design and a focus on cathepsin B and calpain 1 as drug targets for neurodegenerative disorders; the latter focus providing an interesting query for the contemporary assumptions that irreversible, covalent protein modification and low selectivity are anathema to therapeutic safety and efficacy

Covalent Enzyme Inhibitors in Drug Design

Epoxysuccinates have shown promising activity as irreversible inhibitors of calpain and cathepsin in indications from cancer to Alzheimer’s disease (AD). Their poor pharmacokinetic properties have been addressed by use of simple epoxysuccinate ester prodrugs in preclinical and clinical trials. A prodrug strategy requires the prodrug to be converted efficiently to the drug molecule and for the prodrug itself not to engage off-target biomolecules. We observe that the epoxysuccinate esters are both more reactive and provide an alternative reaction pathway for covalent modification of biomolecules. Reactivity was studied towards GSH, glutathione-S-transferase (GST-P1), and in plasma. The parent epoxysuccinates are stable, whereas ester prodrugs undergo ready base-catalyzed reaction. The enhanced thiophilic reactivity of the neutral epoxide forces a dominant reaction pathway that does not yield the parent drug. This unappreciated reactivity of epoxysuccinyl prodrugs creates challenges in drug design and must be taken into account in interpretation of preclinical and clinical results, since these prodrugs will engage targets other than the cysteine proteases that the parent drug is designed selectively to inhibit. These findings make it obvious that epoxysuccinate esters are unsuitable as clinical candidates due to off-target reactivity. To obtain clinical leads maintaining the desirable pro-cognitive effects of the compounds but lacking the general chemical reactivity, several routes can be taken. Firstly, the electrophilic warhead can be modified in order to solve the problem of high off-target reactivity. A tight-binding reversible inhibitor would be superior in many respects to an irreversible inhibitor. Since a peptidomimetic recognition group is available that confers potent binding to the target enzymes, an alternative electrophile has the potential of solving the problems associated with the epoxysuccinate reactivity. A library of molecular probes was synthesized with the goal of maintaining potent inhibition of target cysteine proteases with attenuated reactivity towards general nucleophiles. An increased membrane permeability compared to the epoxysuccinates through the absence of negative charge was also desired. Several novel electrophilic warheads as well as groups known to react with active cysteines were explored with the intent to identify inhibitors of the target enzymes, calpain 1 and cathepsin B

Synthesis and structure-activity relationship of several aromatic ketone-based two-photon initiators

Several novel aromatic ketone-based two-photon initiators containing triple bonds and dialkylamino groups were synthesized and the structure-activity relationships were evaluated. Branched alkyl chains were used at the terminal donor groups to improve the solubility in the multifunctional monomers. Because of the long conjugation length and good coplanarity, the evaluated initiators showed large two-photon cross section values, while their fluorescence lifetimes and quantum yields strongly depend on the solvent polarity. All novel initiators exhibited high activity in terms of two-photon-induced microfabrication. This is especially true for fluorenone-based derivatives, which displayed much broader processing windows than well-known highly active initiators from the literature and commercially available initiators. While the new photoinitiators gave high reactivity in two-photon-induced photopolymerization at concentration as low as 0.1% wt, these compounds are surprisingly stable under one photon condition and nearly no photo initiation activity was found in classical photo DSC experiment

Pharmacological perturbation of the phase-separating protein SMNDC1

Abstract SMNDC1 is a Tudor domain protein that recognizes di-methylated arginines and controls gene expression as an essential splicing factor. Here, we study the specific contributions of the SMNDC1 Tudor domain to protein-protein interactions, subcellular localization, and molecular function. To perturb the protein function in cells, we develop small molecule inhibitors targeting the dimethylarginine binding pocket of the SMNDC1 Tudor domain. We find that SMNDC1 localizes to phase-separated membraneless organelles that partially overlap with nuclear speckles. This condensation behavior is driven by the unstructured C-terminal region of SMNDC1, depends on RNA interaction and can be recapitulated in vitro. Inhibitors of the protein’s Tudor domain drastically alter protein-protein interactions and subcellular localization, causing splicing changes for SMNDC1-dependent genes. These compounds will enable further pharmacological studies on the role of SMNDC1 in the regulation of nuclear condensates, gene regulation and cell identity

Design, Synthesis, and Optimization of Novel Epoxide Incorporating Peptidomimetics as Selective Calpain Inhibitors

Hyperactivation of the calcium-dependent cysteine protease calpain 1 (Cal1) is implicated as a primary or secondary pathological event in a wide range of illnesses and in neurodegenerative states, including Alzheimer’s disease (AD). E-64 is an epoxide-containing natural product identified as a potent nonselective, calpain inhibitor, with demonstrated efficacy in animal models of AD. By use of E-64 as a lead, three successive generations of calpain inhibitors were developed using computationally assisted design to increase selectivity for Cal1. First generation analogues were potent inhibitors, effecting covalent modification of recombinant Cal1 catalytic domain (Cal1_cat), demonstrated using LC–MS/MS. Refinement yielded second generation inhibitors with improved selectivity. Further library expansion and ligand refinement gave three Cal1 inhibitors, one of which was designed as an activity-based protein profiling probe. These were determined to be irreversible and selective inhibitors by kinetics studies comparing full length Cal1 with the general cysteine protease papain