Switching the Switch: Ligand Induced Disulfide Formation in HDAC8

Jänsch, Niklas; Sugiarto, Wisely Oki; Muth, Marius; Desczyk, Charlotte; Ballweg, Matthias; Kirschhöfer, Frank; Brenner‐Weiß, Gerald; Meyer‐Almes, Franz‐Josef

doi:10.1002/chem.202001712

Cited by 8 publications

(18 citation statements)

References 27 publications

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“…C244, which is unique to HDAC8, is one of the most buried cysteines with the highest calculated pK a value. 31 It is thus surprising that this cysteine with theoretically low reactivity showed ligandability with BDHI compound 8. Interestingly, a screening of maleimide analogues appended with the 3,5-bis(trifluoromethyl)phenyl group (similar to 8) has shown to label C244 and C275 of HDAC8 and inhibit the enzyme activity.…”

Section: ■ Results and Discussionmentioning

confidence: 99%

Covalent Inhibition by a Natural Product-Inspired Latent Electrophile

et al. 2023

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Strategies to target specific protein cysteines are critical to covalent probe and drug discovery. 3-Bromo-4,5-dihydroisoxazole (BDHI) is a natural product-inspired, synthetically accessible electrophilic moiety that has previously been shown to react with nucleophilic cysteines in the active site of purified enzymes. Here, we define the global cysteine reactivity and selectivity of a set of BDHI-functionalized chemical fragments using competitive chemoproteomic profiling methods. Our study demonstrates that BDHIs capably engage reactive cysteine residues in the human proteome and the selectivity landscape of cysteines liganded by BDHI is distinct from that of haloacetamide electrophiles. Given its tempered reactivity, BDHIs showed restricted, selective engagement with proteins driven by interactions between a tunable binding element and the complementary protein sites. We validate that BDHI forms covalent conjugates with glutathione S-transferase Pi (GSTP1) and peptidyl-prolyl cis–trans isomerase NIMA-interacting 1 (PIN1), emerging anticancer targets. BDHI electrophile was further exploited in Bruton’s tyrosine kinase (BTK) inhibitor design using a single-step late-stage installation of the warhead onto acrylamide-containing compounds. Together, this study expands the spectrum of optimizable chemical tools for covalent ligand discovery and highlights the utility of 3-bromo-4,5-dihydroisoxazole as a cysteine-reactive electrophile.

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Section: ■ Results and Discussionmentioning

confidence: 99%

Covalent Inhibition by a Natural Product-Inspired Latent Electrophile

et al. 2023

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“…C244, which is unique to HDAC8, is one of the most buried cysteines with the highest calculated p K a value. 31 It is thus surprising that this cysteine with theoretically low reactivity showed ligandability with BDHI compound 8 . Interestingly, a screening of maleimide analogs appended with the 3,5-bis(tri-fluoromethyl)phenyl group (similar to 8) has shown to label C244 and C275 of HDAC8 and inhibit the enzyme activity.…”

Section: Resultsmentioning

confidence: 99%

Covalent inhibition by a natural product-inspired latent electrophile

Byun

Ritchie

Holewinski

et al. 2023

Preprint

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Strategies to target specific protein cysteines are critical to covalent probe and drug discovery. 3-bromo-4,5-dihydroxazole is a natural product-inspired, synthetically accessible electrophilic moiety that has previously been shown to react with nucleophilic cysteines in the active site of purified enzymes. Here we define the global cysteine reactivity and selectivity of a set of 3-bromo-4,5-dihydroxazole-functionalized chemical fragments using competitive chemoproteomic profiling methods. Our study demonstrates that 3-bromo-4,5-dihydroxazoles capably engage reactive cysteine residues in the human proteome and the selectivity landscape of cysteines liganded by 3-bromo-4,5-dihydroxazoles is distinct from that of haloacetamide electrophiles. Given its tempered reactivity, 3-bromo-4,5-dihydroxazoles showed restricted, selective engagement with proteins driven by interactions between a tunable binding element and the complementary protein sites. We further validate that 3-bromo-4,5-dihydroxazoles form covalent conjugates with glutathione S-transferase Pi (GSTP1) and peptidyl-prolyl cis-trans isomerase NIMA-interacting 1 (PIN1), emerging anti-cancer targets. Together, this study expands the spectrum of optimizable chemical tools for covalent ligand discovery and highlights the utility of 3-bromo-4,5-dihydroxazole as a cysteine-reactive electrophile.

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“…This might be explained by the fast modification of cysteines C153 and C275, which are involved in redox-regulation of HDAC8. [10] The cysteine redox switch partner of C153 in HDAC8, C102, as well as C275 are not present in HDAC4. In comparison, 3(a-h) showed significantly lower inhibitory activity against HDAC8.…”

Section: Resultsmentioning

confidence: 99%

“…HDAC8 consists of 10 cysteines showing special redox regulative features. [10] We quantified the degree of labeling for seven solvent accessible cysteine residues of HDAC8 by tryptic digestion and high resolution HPLC-MS/MS. With this approach we investigated whether 5h has a beneficial labeling behavior by means of reactivity compared with Nbenzyl maleimide.…”

Section: Chembiochemmentioning

confidence: 99%

3‐Chloro‐5‐Substituted‐1,2,4‐Thiadiazoles (TDZs) as Selective and Efficient Protein Thiol Modifiers

et al. 2022

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The study of cysteine modifications has gained much attention in recent years. This includes detailed investigations in the field of redox biology with focus on numerous redox derivatives like nitrosothiols, sulfenic acids, sulfinic acids and sulfonic acids resulting from increasing oxidation, S-lipidation, and perthiols. For these studies selective and rapid blocking of free protein thiols is required to prevent disulfide rearrangement. In our attempt to find new inhibitors of human histone deacetylase 8 (HDAC8) we discovered 5-sulfonyl and 5-sulfinyl substituted 1,2,4-thiadiazoles (TDZ), which surprisingly show an outstanding reactivity against thiols in aqueous solution. Encouraged by these observations we investigated the mechanism of action in detail and show that these compounds react more specifically and faster than commonly used N-ethyl maleimide, making them superior alternatives for efficient blocking of free thiols in proteins. We show that 5-sulfonyl-TDZ can be readily applied in commonly used biotin switch assays. Using the example of human HDAC8, we demonstrate that cysteine modification by a 5-sulfonyl-TDZ is easily measurable using quantitative HPLC/ESI-QTOF-MS/MS, and allows for the simultaneous measurement of the modification kinetics of seven solvent-accessible cysteines in HDAC8.

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Switching the Switch: Ligand Induced Disulfide Formation in HDAC8

Cited by 8 publications

References 27 publications

Covalent Inhibition by a Natural Product-Inspired Latent Electrophile

Covalent Inhibition by a Natural Product-Inspired Latent Electrophile

Covalent inhibition by a natural product-inspired latent electrophile

3‐Chloro‐5‐Substituted‐1,2,4‐Thiadiazoles (TDZs) as Selective and Efficient Protein Thiol Modifiers

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