Peptide Nitriles Research Articles

Cathepsin-Targeting SARS-CoV-2 Inhibitors: Design, Synthesis, and Biological Activity.

Cathepsins (Cats) are proteases that mediate the successful entry of SARS-CoV-2 into host cells. We designed and synthesized a tailored series of 21 peptidomimetics and evaluated their inhibitory activity against human cathepsins L, B, and S. Structural diversity was realized by combinations of different C-terminal warhead functions and N-terminal capping groups, while a central Leu-Phe fragment was maintained. Several compounds were identified as promising cathepsin L and S inhibitors with Ki values in the low nanomolar to subnanomolar range, for example, the peptide aldehydes 9a and 9b (9a, 2.67 nM, CatL; 0.455 nM, CatS; 9b, 1.76 nM, CatL; 0.512 nM, CatS). The compounds' inhibitory activity against the main protease of SARS-CoV-2 (Mpro) was additionally investigated. Based on the results at CatL, CatS, and Mpro, selected inhibitors were subjected to investigations of their antiviral activity in cell-based assays. In particular, the peptide nitrile 11e exhibited promising antiviral activity with an EC50 value of 38.4 nM in Calu-3 cells without showing cytotoxicity. High metabolic stability and favorable pharmacokinetic properties make 11e suitable for further preclinical development.

Dipeptide-Derived Alkynes as Potent and Selective Irreversible Inhibitors of Cysteine Cathepsins.

The potential of designing irreversible alkyne-based inhibitors of cysteine cathepsins by isoelectronic replacement in reversibly acting potent peptide nitriles was explored. The synthesis of the dipeptide alkynes was developed with special emphasis on stereochemically homogeneous products obtained in the Gilbert-Seyferth homologation for C≡C bond formation. Twenty-three dipeptide alkynes and 12 analogous nitriles were synthesized and investigated for their inhibition of cathepsins B, L, S, and K. Numerous combinations of residues at positions P1 and P2 as well as terminal acyl groups allowed for the derivation of extensive structure-activity relationships, which were rationalized by computational covalent docking for selected examples. The determined inactivation constants of the alkynes at the target enzymes span a range of >3 orders of magnitude (3-10 133 M-1 s-1). Notably, the selectivity profiles of alkynes do not necessarily reflect those of the nitriles. Inhibitory activity at the cellular level was demonstrated for selected compounds.

Selective Synthesis of Lysine Peptides and the Prebiotically Plausible Synthesis of Catalytically Active Diaminopropionic Acid Peptide Nitriles in Water.

Why life encodes specific proteinogenic amino acids remains an unsolved problem, but a non-enzymatic synthesis that recapitulates biology's universal strategy of stepwise N-to-C terminal peptide growth may hold the key to this selection. Lysine is an important proteinogenic amino acid that, despite its essential structural, catalytic, and functional roles in biochemistry, has widely been assumed to be a late addition to the genetic code. Here, we demonstrate that lysine thioacids undergo coupling with aminonitriles in neutral water to afford peptides in near-quantitative yield, whereas non-proteinogenic lysine homologues, ornithine, and diaminobutyric acid cannot form peptides due to rapid and quantitative cyclization that irreversibly blocks peptide synthesis. We demonstrate for the first time that ornithine lactamization provides an absolute differentiation of lysine and ornithine during (non-enzymatic) N-to-C-terminal peptide ligation. We additionally demonstrate that the shortest lysine homologue, diaminopropionic acid, undergoes effective peptide ligation. This prompted us to discover a high-yielding prebiotically plausible synthesis of the diaminopropionic acid residue, by peptide nitrile modification, through the addition of ammonia to a dehydroalanine nitrile. With this synthesis in hand, we then discovered that the low basicity of diaminopropionyl residues promotes effective, biomimetic, imine catalysis in neutral water. Our results suggest diaminopropionic acid, synthesized by peptide nitrile modification, can replace or augment lysine residues during early evolution but that lysine's electronically isolated sidechain amine likely provides an evolutionary advantage for coupling and coding as a preformed monomer in monomer-by-monomer peptide translation.

Peptide ligation by chemoselective aminonitrile coupling in water.

Amide bond formation is one of the most important reactions in both chemistry and biology1-4, but there is currently no chemical method of achieving α-peptide ligation in water that tolerates all of the 20 proteinogenic amino acids at the peptide ligation site. The universal genetic code establishes that the biological role of peptides predates life's last universal common ancestor and that peptides played an essential part in the origins of life5-9. The essential role of sulfur in the citric acid cycle, non-ribosomal peptide synthesis and polyketide biosynthesis point towards thioester-dependent peptide ligations preceding RNA-dependent protein synthesis during the evolution of life5,9-13. However, a robust mechanism for aminoacyl thioester formation has not been demonstrated13. Here we report a chemoselective, high-yielding α-aminonitrile ligation that exploits only prebiotically plausible molecules-hydrogen sulfide, thioacetate12,14 and ferricyanide12,14-17 or cyanoacetylene8,14-to yield α-peptides in water. The ligation is extremely selective for α-aminonitrile coupling and tolerates all of the 20 proteinogenic amino acid residues. Two essential features enable peptide ligation in water: the reactivity and pKaH of α-aminonitriles makes them compatible with ligation at neutral pH and N-acylation stabilizes the peptide product and activates the peptide precursor to (biomimetic) N-to-C peptide ligation. Our model unites prebiotic aminonitrile synthesis and biological α-peptides, suggesting that short N-acyl peptide nitriles were plausible substrates during early evolution.

ChemInform Abstract: Inhibition of Papain by Peptide Nitriles: Conversion of the Nitrile Group into Other Functionalities via the Papain Nitrile Thioimidate Ester Adduct.

ChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 100 leading journals. To access a ChemInform Abstract of an article which was published elsewhere, please select a “Full Text” option. The original article is trackable via the “References” option.

Development of Nitrile-Based Peptidic Inhibitors of Cysteine Cathepsins

It is now becoming clear that several papain-like cysteine cathepsins are involved in the pathophysiology of diseases such as osteoporosis, autoimmune disorders, and cancer. Therefore, the development of potent and selective cathepsin inhibitors is an attractive subject for medicinal chemists. New advances have been made for nitrile-based inhibitors, leading to the identification of the cathepsin K inhibitor odanacatib and other candidates with potential for therapeutic use. This review summarizes the development of peptidic and peptidomimetic compounds with an electrophilic nitrile 'warhead' as inhibitors of the cysteine cathepsins B, S, L, C, and K. Peptide nitriles have been shown to reversibly react with the active site cysteine under formation of a covalent thioimidate adduct. The structural optimization with respect to the positions P3, P2, P1, P1', and P2' resulted in the identification of potent and selective inhibitors of the corresponding cathepsins. The underlying structure-activity relationships are discussed herein.

Inhibition of the Activation of Multiple Serine Proteases with a Cathepsin C Inhibitor Requires Sustained Exposure to Prevent Pro-enzyme Processing

Cathepsin C is a cysteine protease required for the activation of several pro-inflammatory serine proteases and, as such, is of interest as a therapeutic target. In cathepsin C-deficient mice and humans, the N-terminal processing and activation of neutrophil elastase, cathepsin G, and proteinase-3 is abolished and is accompanied by a reduction of protein levels. Pharmacologically, the consequence of cathepsin C inhibition on the activation of these serine proteases has not been described, due to the lack of stable and non-toxic inhibitors and the absence of appropriate experimental cell systems. Using novel reversible peptide nitrile inhibitors of cathepsin C, and cell-based assays with U937 and EcoM-G cells, we determined the effects of pharmacological inhibition of cathepsin C on serine protease activity. We show that indirect and complete inhibition of neutrophil elastase, cathepsin G, and proteinase-3 is achievable in intact cells with selective and non-cytotoxic cathepsin C inhibitors, at concentrations approximately 10-fold higher than those required to inhibit purified cathepsin C. The concentration of inhibitor needed to block processing of these three serine proteases was similar, regardless of the cell system used. Importantly, cathepsin C inhibition must be sustained to maintain serine protease inhibition, because removal of the reversible inhibitors resulted in the activation of pro-enzymes in intact cells. These findings demonstrate that near complete inhibition of multiple serine proteases can be achieved with cathepsin C inhibitors and that cathepsin C inhibition represents a viable but challenging approach for the treatment of neutrophil-based inflammatory diseases.

Enzymatic hydrolysis of nitrides by an engineered nitrile hydratase (Papain Gln19Glu) in aqueous‐organic media

A mutant of the cysteine protease papain, displaying nitrile hydratase and amidase activities, was expressed in Pichia pastoris and used for the hydrolysis of peptide nitriles in aqueous-organic media. The rate of hydrolysis of these nitriles is lowered by increasing acetone concentration. This is caused by an increase of the Michaelis constant, and a variation of Vmax proportional to the amount of water in the mixture. The hydrolysis of the amide is less affected by the increase in co-solvent, which results in lower accumulation of this intermediate product. With the peptide nitrile tested, high nitrile concentrations could be used to promote the production of the amide and prevent its hydrolysis to the acid by diminishing the relative rate of amide hydrolysis. A number of non-peptidyl nitriles were also tested as potential substrates but activity was detected for only one compound with structural resemblance to peptide nitriles.

Synthesis of amidrazones using an engineered papain nitrile hydratase

To demonstrate the usefulness of an engineered papain nitrile hydratase as a biocatalyst, a peptide amidrazone was prepared by incubation of the nitrile MeOCO-Phe-Ala-nitrile with the Gln 19Glu papain mutant in the presence of salicylic hydrazide as a nucleophile. The amidrazone results from nucleophilic attack by salicylic hydrazide at the imino carbon of the thioimidate adduct formed between the enzyme and the peptide nitrile substrate. Compared to wild-type enzyme, the engineered nitrile hydratase causes a better than 4000-fold increase in the rate of amidrazone formation and yields a product of much higher purity. The advantages over other nitrile-hydrolyzing enzymes and current limitations of the papain nitrile hydratase are discussed.

ChemInform Abstract: Direct Dialkylation of Peptide Nitriles. Application to the Synthesis of 1‐Aminocyclopropane‐1‐carboxylic Acid (Acc)‐Containing Dipeptides.

AbstractChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 100 leading journals. To access a ChemInform Abstract of an article which was published elsewhere, please select a “Full Text” option. The original article is trackable via the “References” option.

Engineering nitrile hydratase activity into a cysteine protease by a single mutation.

A peptide nitrile hydratase activity has been engineered into the cysteine protease papain by a single carefully selected mutation at the active site of the enzyme. The papain variant Gln19Glu hydrolyzes the substrate MeOCO-PheAla-CN to the corresponding amide with a kcat/KM value of 1.15 x 10(3) M-1 s-1. The reaction leads to an accumulation of the corresponding amide, which is then further hydrolyzed to the acid by the natural amidase activity of the enzyme. The pH-dependency of the nitrile hydratase activity of Gln19Glu supports the involvement of the acid form of the Glu19 residue in the reaction. The wild type enzyme displays very weak nitrile hydratase activity, and the introduction of a glutamic acid residue in the oxyanion hole of papain causes the kcat at pH 5 to increase by a factor of at least 4 x 10(5). Peptide nitriles react with cysteine proteases to form thioimidates, and the role of the glutamic acid residue introduced at position 19 in the Gln19Glu enzyme is to participate in the acid-catalyzed hydrolysis of the thiomidate to the amide by the provision of a proton to form the more reactive protonated thioimidate. This dramatically decreases the energy barrier for the hydrolysis of the thioimidate, as shown by the impressive increase in kcat. The success of the rational approach undertaken is a consequence of the level of understanding of the basic catalytic properties of cysteine proteases of the papain family.

Peptide aldehydes and nitriles as transition state analog inhibitors of cysteine proteases.

Enzymes efficiently catalyze reactions by stabilizing inherently unstable transition states. For cysteine proteases, part of the stabilization is provided by a region of the enzyme termed the oxyanion hole. Site-directed mutagenesis has been used to investigate further the role of the oxyanion hole of papain in the binding of putative transition state analog inhibitors of cysteine proteases. The dissociation constants Ki(obs) for inhibition of wild-type and mutant enzymes (Gln19Ala, Gln19Glu, and Gln19His) by the aldehyde Ac-Phe-Gly-CHO and the nitrile MeOCO-Phe-Gly-CN have been determined in the pH range 3.5-9.0. For the peptide nitrile inhibitor, mutation of Gln19 was found to cause important increases in Ki(obs), and thioimidate adducts with the papain mutants Gln19Ala and Gln19Glu are less stable by 1.4-2.4 kcal/mol. However, for the peptide aldehyde inhibitor, the mutations resulted in a small but significant increase in stability of the tetrahedral hemithioacetal adduct (0.4-1.2 kcal/mol). In that respect, the hemithioacetal formed between papain and a peptide aldehyde cannot be considered a good model of the transition state for cysteine protease-catalyzed reactions. The influence of the mutations on the pH dependency of inhibition also indicates that with respect to oxyanion hole interaction, the inhibition of papain by peptide nitriles is a process closer to that of substrate hydrolysis than is the inhibition by the corresponding peptide aldehydes. The nature of the intermediates and transition states in hydrolysis reactions catalyzed by cysteine proteases, as well as the use of enzyme-inhibitor adducts as their models, is discussed.

Inhibition of papain by peptide nitriles: conversion of the nitrile group into other functionalities via the papain:nitrile thioimidate ester adduct

Peptide nitriles are reversible inhibitors of papain that form thioimidates with the cysteine thiol in the enzyme's active site. These thioimidates undergo reactions with thiols and amines to form acids and amidines, respectively. These reactions were also found to be stereospecific. Only a thioimidate derived from an L-amino acid nitrile will react with exogenous nucleophiles. These reactions were followed by 13C and 15N NMR techniques. Key words: papain, nitrile, l3C NMR, 15N NMR, thioimidate.

Inhibition of papain by peptide nitriles: Reactions of external nucleophiles with the thioimidate ester adduct

N-(N- Acetyl- l-phenylalanyl)glycinenitrile is a reversible inhibitor of papain which forms a thioimidate adduct with the cysteine thiol in the enzyme's active site. It is possible for this thioimidate to undergo reactions with external thiols, namely β-mercaptoethanol and N-acetylcysteamine. The reactions were followed by 13C- and 15N-NMR techniques which indicated that the initial nitrile was converted to N-(N- acetyl- l-phenylalanyl)glycinamide and then further hydrolysed to N-(N- acetyl- l-phenylalanyl)glycine . In essence papain is acting as a nitrile hydratase, and this activity, coupled with papain's well-characterized amidase activity, results in the synthesis of a carboxylic acid from a nitrile).

Reversible binding of peptide aldehydes to papain. Structure-activity relationships

The hydration of eleven peptide and hippuryl aldehydes has been measured as a function of temperature by means of NMR spectroscopy. In all cases the aldehydes were strongly hydrated (i.e., 90–95%) in aqueous solution. Dehydration of the hydrates was strongly endothermic, but this was partly offset by a positive entropy for dehydration. The binding of the aldehydes to papain was measured by fluorescence titration, and from these data dissociation constants for the hemithioacetal enzyme adducts were derived. Binding of N-Ac-l-PheNHCH2CHO (1) was particularly tight (Kd,corr = 0.00043 μM) whereas that of its d-enantiomer (2) was 300-fold weaker (Kd,corr = 0.129 μM). The binding constants of the eleven aldehydes correlated with those for the reversible covalent binding of the analogous nitriles according to the equation log Kd(CHO) = −2.687 + 1.016 log Kd,corr(CN) (r = 0.99), lending support to previous suggestions that both peptide aldehydes and peptide nitriles behave as transition-state- or reactive intermediate analogs for papain. This finding is particularly striking in view of the obvious differences in hybridization (sp2 vs. sp3) and geometry (trigonal vs. tetrahedral) at the reactive P1 carbon center in their covalent adduct forms (thioimidate ester vs. hemithioacetal, respectively). A model for the binding of substrates, their transition states and analogs thereof is proposed. A key feature of the model is an obligatory covalent (or developing covalent) interaction between Cys-25-SH and the carbonyl or equivalent carbon of P1, augmented by intermolecular P1NHOC(Asp-158), P2COHN(Gly-66) and P2NHOC(Gly-66) hydrogen bonds and a hydrophobic P2S2 interaction. The latter three interactions are optimum or nearly optimum when P2 is a hydrophobic l-amino acid with an N-acyl substituent. Data presented suggest that hippuryl derivatives are relatively non-specific substrates or inhibitors for papain and, consequently, are of diminished value as probes for binding and catalytic studies.

Reversible covalent binding of peptide nitriles to papain

The dissociation constants for reversible covalent binding of twelve peptide nitrile inhibitors to the active site of papain have been measured by means of flourescence titration. The binding constants generally the kinetic specificity constants ( k cat/ K m) for related papain substrates, supporting earlier suggestions that peptide nitriles behave as transition state analog inhibitors of papain. In ten cases the temperature dependence of binding was analyzed to determine the enthalpic and entropic contributions to the binding energy. A compensation plot of ΔH vs. TΔS resulted in two parallel lines, one for ‘specific’ nitriles (i.e., N- Ac- L -aa-NHCH 2CN ; aa = Phe, Leu, Met)_and the other for ‘non-specific’ nitriles (e.g., N- Ac- D-Phe-NHCH 2CN, PhCH 2CH 2CONHCH 2CN hippurylnitrile, etc.). For both specific and nonspecific nitriles representing an 1800-fold range of K d values (0.27 μM–490 μM), the solvent deuterium isotope effect on binding ( K d ( H 2O /K d ( D 2O ) = D K d ) was very close to 2.0. This isotope effect could be accounted for entirely by the simple protonic change which occurs upon the reversible addition of the active site sulfhydryl of papain to the nitrile group of the peptide derivative to form a covalent thioimidate linkage. In contrast, six closely related non-nitrile ligands containing identical peptide side chains but having C-terminal groups incapable of binding covalently to papain had unmeasureably high dissociation constants. Collectively, these results indicate that strong binding of peptide nitrile substrate analogs to papain requires a combination of (1) hydrophobic interaction (especially at the P2 position), (2) specific intermolecular hydrogen bonding and (3) covalent interaction of the nitrile with the active site sulfhydryl group.

ChemInform Abstract: Reversible Covalent Inhibition of Papain by a Peptide Nitrile.

Abstract13C NMR experiments indicate that papain (I) interacts with the nitrile (II) via reversible formation of an adduct such as (III).

Reversible covalent inhibition of papain by a peptide nitrile. Carbon-13 NMR evidence for a thioimidate ester adduct

Reversible covalent inhibition of papain by a peptide nitrile. Carbon-13 NMR evidence for a thioimidate ester adduct

The synthesis and chromatography of peptine nitriles.

Di- and tripeptide nitriles, glycylaminoacetonitrile (Gly-AAN), diglycylaminoacetonitrile (Gly-Gly-AAN), alany-alpha-aminopropionitrile (Ala-APN), and dialanyl-alpha-aminopropionitrile (Ala-Ala-APN) were synthesized first. These peptide nitriles and related peptides and peptide amides were analyzed by means of ion-exchange chromatography. The every two diastereomers of dialanine, dialanine amide, and Ala-APN were separated into two peaks by using a pH 3.25 buffer as an eluent. The four isomers of trialanine, trialanine amide, and Ala--Ala APN gave four, two, and one peak, respectively under the same conditions. The trimethylsilyl derivatives of alanyl peptides and related compounds were analyzed by means of gas chromatogrpahy combined with mass-spectrometry. The parent (M+ and/or M+-15) and other mass numbers observed in their mass-spectra supported the introduction of various numbers of trimethylsilyl groups.