Allele-specific inhibition of divergent protein tyrosine phosphatases with a single small molecule

Zhang, Xinyu; Chen, Vincent; Rosen, Mari S.; Blair, Elizabeth R.; Lone, Anna Mari; Bishop, Anthony C.

doi:10.1016/j.bmc.2008.07.053

Cited by 13 publications

(35 citation statements)

References 35 publications

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“…The plasmids encoding His 6 -tagged catalytic domains of Shp2 (pJGO0001), 24 Shp1 (pACB149), 24 PTP1B (pOBD0002), 21 PTPH-1 (pERB047), 26 HePTP (pBAD-HePTP), 16 and FAP-1 (pAML001) 16 have been previously described. All site-directed mutations were introduced using the Quikchange mutagenesis kit (Stratagene) according to the manufacturer’s instructions.…”

Section: Methodsmentioning

confidence: 99%

“…19–20 Targeting engineered cysteine-enriched WPD loops with biarsenicals has proven to be a general approach for PTP inhibition. 16, 21–22 However, the WPD loop constitutes part of the PTP active site (in its closed state) and plays a key role in the catalytic mechanism of PTPs, presenting the possibility that mutations on the loop itself could alter the inherent kinetic activity or substrate specificity of the enzyme. Moreover, the presence of catalytically essential residues on the WPD loop significantly constrains the options for its engineering in a manner that is functionally silent ( e.g.…”

Section: Introductionmentioning

confidence: 99%

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Rational design of allosteric-inhibition sites in classical protein tyrosine phosphatases

Chio

Bishop

2015

Bioorganic & Medicinal Chemistry

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Protein tyrosine phosphatases (PTPs), which catalyze the dephosphorylation of phosphotyrosine in protein substrates, are critical regulators of metazoan cell signaling and have emerged as potential drug targets for a range of human diseases. Strategies for chemically targeting the function of individual PTPs selectively could serve to elucidate the signaling roles of these enzymes and would potentially expedite validation of the therapeutic promise of PTP inhibitors. Here we report a novel strategy for the design of non-natural allosteric-inhibition sites in PTPs; these sites, which can be introduced into target PTPs through protein engineering, serve to sensitize target PTPs to potent and selective inhibition by a biarsenical small molecule. Building on the recent discovery of a naturally occurring cryptic allosteric site in wild-type Src-homology-2 domain containing PTP (Shp2) that can be targeted by biarsenical compounds, we hypothesized that Shp2’s unusual sensitivity to biarsenicals could be strengthened through rational design and that the Shp2-specific site could serve as a blueprint for the introduction of non-natural inhibitor sensitivity in other PTPs. Indeed, we show here that the strategic introduction of a cysteine residue at a position removed from the Shp2 active site can serve to increase the potency and selectivity of the interaction between Shp2’s allosteric site and the biarsenical inhibitor. Moreover, we find that “Shp2-like” allosteric sites can be installed de novo in PTP enzymes that do not possess naturally occurring sensitivity to biarsenical compounds. Using primary-sequence alignments to guide our enzyme engineering, we have successfully introduced allosteric-inhibition sites in four classical PTPs—PTP1B, PTPH-1, FAP-1, and HePTP—from four different PTP subfamilies, suggesting that our sensitization approach can likely be applied widely across the classical PTP family to generate biarsenical-responsive PTPs.

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Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Rational design of allosteric-inhibition sites in classical protein tyrosine phosphatases

Chio

Bishop

2015

Bioorganic & Medicinal Chemistry

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“…The PTP set included four mutants of TCPTP and one of HePTP (Figure 1), all of which have been previously described. 5,6,7 We chose TCPTP because the previously published optimization of localization of biarsenical-binding cysteines was performed on this enzyme; HePTP was also used to confirm that the results can be applied in other sensitized enzymes as well. The selected mutants have cysteine-enriched biarsenical binding motifs with either two (P181C/E187C TCPTP), three (P181C+2C TCPTP), or four cysteines (4C TCPTP, 4C@211 HePTP) within or adjacent to their respective WPD loops.…”

Section: Resultsmentioning

confidence: 99%

“…5–7 The appropriate plasmids were used to transform BL21(DE3)-codonPLUS-RIL E. coli . Single colonies were picked and used to inoculate 1000 ml LB cultures, which were grown to mid-log phase at 37°C, and induced with either 0.2 mM IPTG (TCPTP) or 0.04% arabinose (HePTP) for 16 h at 26°C.…”

Section: Methodsmentioning

confidence: 99%

Probing the target-specific inhibition of sensitized protein tyrosine phosphatases with biarsenical probes

et al. 2015

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Selective control of enzyme activity is critical for elucidating the roles of specific proteins in signaling pathways. One potential means for developing truly target-specific inhibitors involves the use of protein engineering to sensitize a target enzyme to inhibition by a small molecule that does not inhibit homologous wild-type enzymes. Previously, it has been shown that protein tyrosine phosphatases (PTPs) can be sensitized to inhibition by a biarsenical probe, FlAsH-EDT2, which inhibits PTP activity by specifically binding to cysteine residues that have been introduced into catalytically important regions. In the present study, we developed an array of biarsenical probes, some newly synthesized and some previously reported, to investigate for the first time the structure-activity relationships for PTP inhibition by biarsenicals. Our data show that biarsenical probes which contain substitutions at the 2′ and 7′ positions are more effective than FlAsH-EDT2 at inhibiting sensitized PTPs. The increased potency of 2′,7′-substituted probes was observed when PTPs were assayed with both para-nitrophenylphosphate and phosphopeptide PTP substrates and at multiple probe concentrations. The data further indicate that the enhanced inhibitory properties are the result of increased binding affinity between the 2′,7′-substituted biarsenical probes and sensitized PTPs. In addition we provide previously unknown physicochemical and stability data for various biarsenical probes.

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“…The plasmids encoding His 6 -tagged catalytic domains of SHP-1 (SHP-1cat) and FAP-1 (FAP-1cat) have been previously described [22,23]. The plasmid encoding His 6 -tagged full-length SHP-1 (SHP-1fl) was ordered from VectorBuilder.…”

Section: Methodsmentioning

confidence: 99%

A missense methionine mutation augments catalytic activity but reduces thermal stability in two protein tyrosine phosphatases

Bishop

2016

Biochemical and Biophysical Research Communications

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Recent data sets that catalog the missense mutations in thousands of human genomes have revealed a vast and largely unexplored world of non-canonical protein sequences that are expressed in humans. The functional consequences of most human missense mutations, however, are unknown, and the accuracy with which their effects can be predicted by computational algorithms remains unclear. Among humans of European descent, the most common missense mutation in the catalytic domain of SH2-containing protein tyrosine phosphatase 1 (SHP-1) converts the enzyme’s canonical valine 451 to methionine (V451M). The V451M mutation lies in a conserved motif adjacent to the protein tyrosine phosphatase (PTP) consensus sequence and is predicted to compromise catalytic function. In this study it is shown that, counter to prediction, V451M SHP-1 possesses increased catalytic activity as compared to the wild-type enzyme. Additionally, a PTP-wide search of missense-mutation data revealed a variant of one other PTP, Fas-associated PTP (FAP-1), that contains a methionine mutation at the position corresponding to 451 of SHP-1 (T2406M FAP-1). It is shown here that the T2406M mutation increases FAP-1’s PTP activity, to a degree that is comparable to the activation deriving from the V451M mutation in SHP-1. Although the two non-canonical methionine residues confer increased activity at moderate temperatures, both V451M SHP-1 and T2406M FAP-1 are less thermally stable than their canonical counterparts, as demonstrated by the mutants’ strongly reduced activities at high temperatures. These results highlight the challenges in predicting the functional consequences of missense mutations, which can differ under varying conditions, and suggest that, with regard to position 451/2406, canonical PTP domains have “chosen” stability over optimized activity during the course of evolution.

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Allele-specific inhibition of divergent protein tyrosine phosphatases with a single small molecule

Cited by 13 publications

References 35 publications

Rational design of allosteric-inhibition sites in classical protein tyrosine phosphatases

Rational design of allosteric-inhibition sites in classical protein tyrosine phosphatases

Probing the target-specific inhibition of sensitized protein tyrosine phosphatases with biarsenical probes

A missense methionine mutation augments catalytic activity but reduces thermal stability in two protein tyrosine phosphatases

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