Relative Rates of Michael Reactions of 2‘-(Phenethyl)thiol with Vinyl Sulfones, Vinyl Sulfonate Esters, and Vinyl Sulfonamides Relevant to Vinyl Sulfonyl Cysteine Protease Inhibitors

Reddick, Jason J.; Cheng, Junjie; Roush, William

doi:10.1021/ol034555l

Cited by 110 publications

(72 citation statements)

References 24 publications

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“…It is interesting to note that other benzyl-substituted pyrazolopyrimidines bearing similar electrophiles such as an acrylamide (7) and an α-chloroacetamide (12) were poor inhibitors. In the former case, this fact may be because the double bond of the vinylsulfonamide (9) is expected to have greater rotational freedom than the planar acrylamide (7) as a result of the poor overlap between the sp 3 ∕d orbitals of nitrogen and sulfur, respectively (37). In the instance of the α-chloroacetamide, it is possible that the electrophile/nucleophile pair is unable to assume an orientation compatible with S N 2 attack.…”

Section: Discussionmentioning

confidence: 99%

Chemical genetic strategy for targeting protein kinases based on covalent complementarity

Garske

Peters

Cortesi

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

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The conserved nature of the ATP-binding site of the >500 human kinases renders the development of specific inhibitors a challenging task. A widely used chemical genetic strategy to overcome the specificity challenge exploits a large-to-small mutation of the gatekeeper residue (a conserved hydrophobic amino acid) and the use of a bulky inhibitor to achieve specificity via shape complementarity. However, in a number of cases, introduction of a glycine or alanine gatekeeper results in diminished kinase activity and ATP affinity. A new chemical genetic approach based on covalent complementarity between an engineered gatekeeper cysteine and an electrophilic inhibitor was developed to address these challenges. This strategy was evaluated with Src, a proto-oncogenic tyrosine kinase known to lose some enzymatic activity using the shape complementarity chemical genetic strategy. We found that Src with a cysteine gatekeeper recapitulates wild type activity and can be irreversibly inhibited both in vitro and in cells. A cocrystal structure of T338C c-Src with a vinylsulfonamide-derivatized pyrazolopyrimidine inhibitor was solved to elucidate the inhibitor binding mode. A panel of electrophilic inhibitors was analyzed against 307 kinases and MOK (MAPK/MAK/MRK overlapping kinase), one of only two human kinases known to have an endogenous cysteine gatekeeper. This analysis revealed remarkably few offtargets, making these compounds the most selective chemical genetic inhibitors reported to date. Protein engineering studies demonstrated that it is possible to increase inhibitor potency through secondary-site mutations. These results suggest that chemical genetic strategies based on covalent complementarity should be widely applicable to the study of protein kinases. chemical genetics | irreversible inhibitor | SgK494

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Section: Discussionmentioning

confidence: 99%

Chemical genetic strategy for targeting protein kinases based on covalent complementarity

Garske

Peters

Cortesi

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

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“…Roush and co-workers recently ranked the relative activities of Michael acceptors: enone > vinylsulfone > vinylsulfonate > enoate > vinylsulfonamide. 5 The vinylsulfonamide was chosen initially since this is the least reactive member in the series to minimize nonspecific thiol addition. Additionally, molecular modeling showed that the vinylsulfonamide adopted the required folded conformation of the bound acyladenylate ( Figure S11, Supporting Information).…”

mentioning

confidence: 99%

A Mechanism-Based Aryl Carrier Protein/Thiolation Domain Affinity Probe

et al. 2007

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The design, synthesis and biochemical characterization of a mechanism-based aryl carrier protein (ArCP) affinity probe that selectively modifies the terminal thiol of the aryl carrier protein phosphopantethein (Ppant) prosthetic group is described. Labeling of the aryl carrier protein was shown to require the cognate adenylating enzyme to channel the affinity probe onto the Ppant cofactor. The selective labeling was established by observation of the phosphopantetheinyl ejection ion via MS/MS and the probe was also found to stabilize an interaction between an aryl carrier protein and adenylating enzyme by an electrophoretic mobility shift assay.Carrier proteins (CPs) play a central role in the biosynthesis of polyketide synthase (PKS), nonribosomal peptide synthetase (NRPS), and fatty acid synthase (FAS) derived natural products. CPs, also referred to as thiolation domains, are responsible for transporting the substrate and chain intermediates to the catalytic centers of the PKS, NRPS, and FAS assembly lines. The biosynthetic chain intermediates are tethered as thioesters on the terminal thiol of a phosphopantetheine (Ppant) prosthetic group that is covalentiy attached to an invariant serine residue of the CP. 2 CPs are either freestanding or embedded in these multifunctional proteins and exist as three variants: an acyl carrier protein (ACP) found in PKSs and FASs, a peptidyl carrier protein (PCP) found in NRPS systems, and an aryl carrier protein (ArCP) commonly found in siderophore NRPS synthetases. 2 An understanding of how CPs recognize their upstream and downstream partner proteins is essential to "deciphering the logic" for assembly of these natural products. 1 CPs also constitute a potential target for the development of a new class of antibiotics since they are involved in the synthesis of several bacterial virulence factors and essential primary metabolites. For example, the natural antibiotic platensimycin acts by disrupting interactions between a CP and a ketosynthase (KS) domain in the bacterial type n FAS. 2 The groups of Burkart, Walsh and Johnsson demonstrated the utility of CPs as low molecular weight protein fusion tags that can be easily modified by exploiting the promiscuity of phosphopantetheinyl transferases (PPTase) to incorporate fluorescent and affinity tags onto the conserved serine residue of the carrier domain. 3 Mechanism-based affinity probes that specifically modify the terminal thiol of the Ppant prosthetic group of CPs could serve as new reagents for the site specific labeling of proteins, lead to the development of novel antibacterial agents, and provide powerful chemical probes to study the interactions of CPs with their partner enzyme domains. In this latter regard, Burkart and co-workers recently detailed a pantetheine analogue, which cross-links KS and ACP domains. 4 We report herein our complementary efforts at the design and biochemical characterization of a mechanism-based CP affinity probe that selectively modifies ArCPs. NIH-PA Author ManuscriptNIH-PA Author Man...

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“…[118] The vinyl sulfonamide was found to be the least reactive, with the rate of thiol addition to phenyl vinyl sulfonate esters proceeding approximately 3000 times faster. This suggested the vinyl sulfonamide as a potential group for use in KTGT, as it is less likely to react non-selectively than other Michael acceptors.…”

Section: Vinyl Sulfonamidementioning

confidence: 98%

Kinetic Template‐Guided Tethering of Fragments

2012

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This thesis is composed of two separate projects: Kinetic Template-Guided Tethering of Fragments and Design and Synthesis of a Chemical Probe to Dissect the Cellular Signalling Cascade leading to Cyclin D1 Degradation after DNA Damage. Kinetic Template-Guided Tethering of FragmentsThe development of a novel methodology for the site-directed discovery of small molecule, protein-binding ligands is described. The protein of interest, with a cysteine thiol (either native or engineered) adjacent to the desired binding pocket, is incubated with mixtures of low molecular weight compounds (fragments) modified with either an acrylamide or a vinyl sulfonamide capture group. Any ligand within the mixture that binds within the pocket brings the capture group into close proximity with the cysteine thiol, promoting a conjugate addition reaction at an increased rate over the background reaction. The capture reaction is designed to be slow, such that during the time course of an experiment, no adduct formation is observed unless the reaction is templated by the protein. By this method, binding ligands are rapidly identified by mass spectrometry analysis of the crude reaction mixtures. The methodology has been termed 'kinetic template-guided tethering'. Design and Synthesis of a Chemical Probe to Dissect the Cellular Signalling Cascade leading to Cyclin D1Degradation after DNA Damage An inhibitor described within the literature was found to attenuate the reduction of cyclin D1 after DNA damage to cells. In order to implement a two-step chemical proteomics strategy to find the molecular target of this inhibitor, a synthesis of the compound with an alkyne appendage was required. The alkyne acts as a functional handle for attachment of a reporter molecule in cells via the Huisgen cycloaddition ('Click') reaction. The design and synthesis of this inhibitor with the alkyne appendage is described.

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Relative Rates of Michael Reactions of 2‘-(Phenethyl)thiol with Vinyl Sulfones, Vinyl Sulfonate Esters, and Vinyl Sulfonamides Relevant to Vinyl Sulfonyl Cysteine Protease Inhibitors

Cited by 110 publications

References 24 publications

Chemical genetic strategy for targeting protein kinases based on covalent complementarity

Chemical genetic strategy for targeting protein kinases based on covalent complementarity

A Mechanism-Based Aryl Carrier Protein/Thiolation Domain Affinity Probe

Kinetic Template‐Guided Tethering of Fragments

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