The synthesis of peptidylfluoromethanes and their properties as inhibitors of serine proteinases and cysteine proteinases

A synthesis of peptidylfluoromethanes is described that utilizes the conversion of phthaloyl amino acids into their fluoromethane derivatives. These can be deblocked and elongated. The inactivation of chymotrypsin by Cbz-Phe-CH2F (benzyloxycarbonylphenylalanylfluoromethane) was found to be considerably slower than that of the analogous chloromethane. The fluoromethane analogue inactivates chymotrypsin with an overall rate constant that is 2% of that observed for the inactivation of the enzyme with the chloromethane. However, the result is the same. The reagent complexes in a substrate-like manner, with Ki = 1.4 × 10(-4) M, and alkylates the active-centre histidine residue. Cbz-Phe-Phe-CH2F and Cbz-Phe-Ala-CH2F were investigated as inactivators of the cysteine proteinase cathepsin B. The difference in reactivity between fluoromethyl ketones and chloromethyl ketones is less pronounced in the case of the cysteine proteinase than for the serine proteinase. Covalent bond formation takes place in this case also, as demonstrated by the use of a radiolabelled reagent.

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The synthesis of lysylfluoromethanes and their properties as inhibitors of trypsin, plasmin and cathepsin B

Biochemical Journal ◽

10.1042/bj2410871 ◽

1987 ◽

Vol 241 (3) ◽

pp. 871-875 ◽

Cited By ~ 36

Author(s):

H Angliker ◽

P Wikstrom ◽

P Rauber ◽

E Shaw

Keyword(s):

Proximity Effect ◽

Cathepsin B ◽

Chemical Reactivity ◽

Bond Formation ◽

Synthesis Method ◽

Covalent Bond ◽

Active Centre ◽

Fluoro Derivative ◽

Order Of Magnitude ◽

Covalent Bond Formation

The synthesis of two lysylfluoromethanes is described by an extension of the synthesis method of Rauber, Angliker, Walker & Shaw [(1986) Biochem. J. 239, 633-640]. Ala-Phe-Lys-CH2F was found to be an active-centre-directed inhibitor of plasmin and trypsin, as is the corresponding chloromethane. However, the rate of covalent-bond formation is about an order of magnitude lower at 25 degrees C for the fluoro derivative. It was, in addition, an extremely effective inactivator of cathepsin B at pH 5.4 and 6.4. The chemical reactivity of fluoromethanes was compared with that of chloromethanes as alkylators of GSH. At pH 7.4 and 37 degrees C, a fluoromethane has 1/500th the reactivity of a chloromethane. A comparison of the rates of reaction of the fluoromethane with cathepsin B and with GSH at pH 6.4 revealed an enhancement of 10(8)-fold for the alkylation of the enzyme, ascribable largely to a proximity effect.

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Inactivation of papain by antithrombin due to autolytic digestion: a model of serpin inactivation of cysteine proteinases

Biochemical Journal ◽

10.1042/bj3350701 ◽

1998 ◽

Vol 335 (3) ◽

pp. 701-709 ◽

Cited By ~ 12

Author(s):

Ingemar BJÖRK ◽

Kerstin NORDLING ◽

Elke RAUB-SEGALL ◽

Ulf HELLMAN ◽

Steven T. OLSON

Keyword(s):

Cysteine Proteinase ◽

Cathepsin L ◽

Serine Proteinase ◽

Peptide Bond ◽

Enzyme Inactivation ◽

Serine Proteinases ◽

Cysteine Proteinases ◽

Sds Page ◽

Reaction Proceeds ◽

Proteinase Inhibition

Cross-class inhibition of cysteine proteinases by serpins differs from serpin inhibition of serine proteinases primarily in that no stable serpin–cysteine proteinase complex can be demonstrated. This difference in reaction mechanism was elucidated by studies of the inactivation of the cysteine proteinases, papain and cathepsin L, by the serpin antithrombin. The two proteinases were inactivated with second-order rate constants of (1.6±0.1)×103 and (8.6±0.4)×102 M-1·s-1 respectively. An antithrombin to papain inactivation stoichiometry of ∼ 3 indicated extensive cleavage of the inhibitor concurrent with enzyme inactivation, a behaviour verified by SDS/PAGE. N-terminal sequence analyses showed cleavage predominantly at the P2–P1 bond, but also at the P2´–P3´ bond of antithrombin. The papain band in SDS/PAGE progressively disappeared on reaction of the enzyme with increasing amounts of antithrombin, but no band representing a stable antithrombin–papain complex appeared. SDS/PAGE with 125I-labelled papain showed that the disappearance of papain was caused by cleavage of the enzyme into small fragments. These results suggest a mechanism in which papain attacks a peptide bond in the reactive-bond loop of antithrombin adjacent to that involved in serine proteinase inhibition. The reaction proceeds, similarly to that between serpins and serine proteinases, to form an inactive acyl-intermediate complex, although with the substrate pathway dominating in the papain reaction. In this complex, papain is highly susceptible to proteolysis and is degraded by still active papain, which greatly decreases the lifetime of the complex and results in liberation of fragmented, inactive enzyme. This model may have relevance also for the inactivation of physiologically or pathologically important cysteine proteinases by serpins.

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L-trans-Epoxysuccinyl-leucylamido(4-guanidino)butane (E-64) and its analogues as inhibitors of cysteine proteinases including cathepsins B, H and L

Biochemical Journal ◽

10.1042/bj2010189 ◽

1982 ◽

Vol 201 (1) ◽

pp. 189-198 ◽

Cited By ~ 703

Author(s):

A J Barrett ◽

A A Kembhavi ◽

M A Brown ◽

H Kirschke ◽

C G Knight ◽

...

Keyword(s):

Active Site ◽

Cathepsin B ◽

Cysteine Proteinase ◽

Cathepsin L ◽

Competitive Inhibitor ◽

Serine Proteinases ◽

Cysteine Proteinases ◽

Leucocyte Elastase ◽

Structural Analogues ◽

Stoichiometric Reaction

1. L-trans-Epoxysuccinyl-leucylamido(4-guanidino)butane (E-64) at a concentration of 0.5 mM had no effect on the serine proteinases plasma kallikrein and leucocyte elastase or the metalloproteinases thermolysin and clostridial collagenase. In contrast, 10 muM-E-64 rapidly inactivated the cysteine proteinases cathepsins B, H and L and papain (t0.5 = 0.1-17.3s). The streptococcal cysteine proteinase reacted much more slowly, and there was no irreversible inactivation of clostripain. The cysteine-dependent exopeptidase dipeptidyl peptidase I was very slowly inactivated by E-64. 2. the active-site-directed nature of the interaction of cathepsin B and papain with E-64 was established by protection of the enzyme in the presence of the reversible competitive inhibitor leupeptin and by the stereospecificity for inhibition by the L as opposed to the D compound. 3. It was shown that the rapid stoichiometric reaction of the cysteine proteinases related to papain can be used to determine the operational molarity of solutions of the enzymes and thus to calibrate rate assays. 4. The apparent second-order rate constants for the inactivation of human cathepsins B and H and rat cathepsin L by a series of structural analogues of E-64 are reported, and compared with those for some other active-site-directed inhibitors of cysteine proteinases. 5. L-trans-Epoxysuccinyl-leucylamido(3-methyl)butane (Ep-475) was found to inhibit cathepsins B and L more rapidly than E-64. 6. Fumaryl-leucylamido(3-methyl)butane (Dc-11) was 100-fold less reactive than the corresponding epoxide, but was nevertheless about as effective as iodoacetate.

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Chemical Investigations Into the Biosynthesis of the Gymnastatin and Dankastatin Alkaloids

Chemical Science ◽

10.1039/d1sc02613e ◽

2021 ◽

Author(s):

Bingqi Tong ◽

Bridget Belcher ◽

Daniel Nomura ◽

Thomas Maimone

Keyword(s):

Natural Products ◽

Protein Function ◽

Bond Formation ◽

Covalent Bond ◽

Fungal Strain ◽

Fertile Ground ◽

Covalent Bond Formation

Electrophilic natural products have provided fertile ground for understanding how nature inhibits protein function using covalent bond formation. The fungal strain Gymnascella dankaliensis has provided an especially interesting collection of...

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Material Adhesion through Direct Covalent Bond Formation Assisted by Noncovalent Interactions

ACS Applied Polymer Materials ◽

10.1021/acsapm.1c00223 ◽

2021 ◽

Author(s):

Motofumi Osaki ◽

Tomoko Sekine ◽

Hiroyasu Yamaguchi ◽

Yoshinori Takashima ◽

Akira Harada

Keyword(s):

Noncovalent Interactions ◽

Bond Formation ◽

Covalent Bond ◽

Covalent Bond Formation

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The cysteine proteinases of Schistosoma mansoni cercariae

Parasitology ◽

10.1017/s003118209600830x ◽

1997 ◽

Vol 114 (2) ◽

pp. 105-112 ◽

Cited By ~ 42

Author(s):

J. P. DALTON ◽

K. A. CLOUGH ◽

M. K. JONES ◽

P. J. BRINDLEY

Keyword(s):

Schistosoma Mansoni ◽

Cathepsin B ◽

Cathepsin L ◽

Serine Proteinase ◽

Skin Penetration ◽

Scanning Electron Micrographs ◽

Cysteine Proteinases ◽

Similar Function ◽

Substrate Preferences ◽

Serine Proteinase Activity

Based on substrate preferences, cercariae of Schistosoma mansoni were seen to express both cathepsin L and cathepsin B cysteine proteinases, although the former activity was many -fold greater. Two cathepsin L activities identified in cercarial extracts by zymography co-migrated with activities in extracts of 3 h and 24 h schisotosomula and in extracts of adult worms. Since these enzymes have been implicated in haemoglob in digestion by adult worms, they may perform a similar function in schistosomula. Immunolocalization using scanning electron micrographs showed that cathepsin L and cathepsin B proteinases were present in the cercarial post-acetabular glands. In addition, cercarial serine proteinase activities considered to facilitate skin penetration efficiently cleaved the substrates Z-Gly-Pro-Arg-NHMec and Z-Gly-Pro-Lys-NHMec. Cercariae release most of this serine proteinase activity when induced to secrete the contents of their acetabular glands. In contrast, newly transformed 3 h and 24 h schistosomula did not express this activity.

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Mechanism of Covalent Binding of Ibrutinib to Bruton’s Tyrosine Kinase revealed by QM/MM Calculations

10.26434/chemrxiv.13149893 ◽

2020 ◽

Author(s):

Angus Voice ◽

Gary Tresadern ◽

Rebecca Twidale ◽

Herman Van Vlijmen ◽

Adrian Mulholland

Keyword(s):

Tyrosine Kinase ◽

Covalent Binding ◽

Bond Formation ◽

Covalent Bond ◽

Covalent Modification ◽

Mechanistic Study ◽

Bruton’S Tyrosine Kinase ◽

Bruton's Tyrosine Kinase ◽

Covalent Inhibitors ◽

Covalent Bond Formation

Ibrutinib is the first covalent inhibitor of Bruton’s tyrosine kinase (BTK) to be used in the treatment of B-cell cancers. Understanding the mechanism of covalent inhibition is crucial for the design of safer and more selective covalent inhibitors that target BTK. There are questions surrounding the precise mechanism of covalent bond formation in BTK as there is no appropriate active site residue that can act as a base to deprotonate the cysteine thiol prior to covalent bond formation. To address this, we have investigated several mechanistic pathways of covalent modification of C481 in BTK by ibrutinib using QM/MM reaction simulations. The lowest energy pathway we identified involves a direct proton transfer from C481 to the acrylamide warhead in ibrutinib, followed by covalent bond formation to form an enol intermediate. There is a subsequent rate-limiting keto-enol tautomerisation step (DG‡=10.5 kcal mol-1) to reach the inactivated BTK/ibrutinib complex. Our results represent the first mechanistic study of BTK inactivation by ibrutinib to consider multiple mechanistic pathways. These findings should aid in the design of covalent drugs that target BTK and related proteins.

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Supramolecular Assembly-Enabled Homochiral Polymerization of Short (dA)n Oligonucleotides

Chemical Communications ◽

10.1039/d1cc05420a ◽

2021 ◽

Author(s):

Sreejith Mangalath ◽

Suneesh C Karunakaran ◽

Gary Newnam ◽

Gary Schuster ◽

Nicholas Hud

Keyword(s):

Supramolecular Chemistry ◽

Bond Formation ◽

Supramolecular Assembly ◽

Covalent Bond ◽

Complex Mixtures ◽

Covalent Bond Formation

A goal of supramolecular chemistry is to create covalent polymers of precise composition and stereochemistry from complex mixtures by the reversible assembly of specific monomers prior to covalent bond formation....

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