Turning a protein kinase on or off from a single allosteric site via disulfide trapping

Sadowsky, Jack; Burlingame, Mark A.; Wolan, Dennis W.; McClendon, Christopher L.; Jacobson, Matthew P.; Wells, James A.

doi:10.1073/pnas.1102376108

Cited by 147 publications

(137 citation statements)

References 31 publications

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“…To test whether a given pocket communicates with the active site, we measured the activity of proteins with and without TNB (one half of DTNB) covalently bound within the pocket. A measurable change in activity would demonstrate that there is communication, although it should not be used as a quantitative measure of potential inhibition because other molecules that bind the same site could be more potent inhibitors or even enhance the protein's activity (39). Using this approach, we find that the specific activity of L286C is reduced from 361 ± 29 to 97 ± 6 nmol product/μg/min.…”

Section: Resultsmentioning

confidence: 83%

Discovery of multiple hidden allosteric sites by combining Markov state models and experiments

Bowman

Bolin

Hart

et al. 2015

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

196

232

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The discovery of drug-like molecules that bind pockets in proteins that are not present in crystallographic structures yet exert allosteric control over activity has generated great interest in designing pharmaceuticals that exploit allosteric effects. However, there have only been a small number of successes, so the therapeutic potential of these pockets-called hidden allosteric sites-remains unclear. One challenge for assessing their utility is that rational drug design approaches require foreknowledge of the target site, but most hidden allosteric sites are only discovered when a small molecule is found to stabilize them. We present a means of decoupling the identification of hidden allosteric sites from the discovery of drugs that bind them by drawing on new developments in Markov state modeling that provide unprecedented access to microsecond-to millisecond-timescale fluctuations of a protein's structure. Visualizing these fluctuations allows us to identify potential hidden allosteric sites, which we then test via thiol labeling experiments. Application of these methods reveals multiple hidden allosteric sites in an important antibiotic target-TEM-1 β-lactamase. This result supports the hypothesis that there are many as yet undiscovered hidden allosteric sites and suggests our methodology can identify such sites, providing a starting point for future drug design efforts. More generally, our results demonstrate the power of using Markov state models to guide experiments.thiol labeling | antibiotic resistance | molecular dynamics

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Section: Resultsmentioning

confidence: 83%

Discovery of multiple hidden allosteric sites by combining Markov state models and experiments

Bowman

Bolin

Hart

et al. 2015

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

196

232

View full text Add to dashboard Cite

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“…Not only do covalent ligand-protein complexes offer the advantage of an isolable, homogeneous sample for study, but also in this case we were able to access a continuum of cooperativity modes, significantly expanding the breadth of the analysis. Given the ease with which such covalent small molecules can be discovered via tethering, we anticipate the broader use of such ligands in mechanistic studies of conformationally dynamic protein complexes that comprise critical cellular functions (44)(45)(46).…”

Section: Discussionmentioning

confidence: 99%

Dissecting allosteric effects of activator–coactivator complexes using a covalent small molecule ligand

Wang

Lodge

Fierke³

et al. 2014

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

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Allosteric binding events play a critical role in the formation and stability of transcriptional activator-coactivator complexes, perhaps in part due to the often intrinsically disordered nature of one or more of the constituent partners. The kinase-inducible domain interacting (KIX) domain of the master coactivator CREB binding protein/ p300 is a conformationally dynamic domain that complexes with transcriptional activators at two discrete binding sites in allosteric communication. The complexation of KIX with the transcriptional activation domain of mixed-lineage leukemia protein leads to an enhancement of binding by the activation domain of CREB (phosphorylated kinase-inducible domain of CREB) to the second site. A transient kinetic analysis of the ternary complex formation aided by small molecule ligands that induce positive or negative cooperative binding reveals that positive cooperativity is largely governed by stabilization of the bound complex as indicated by a decrease in k off . Thus, this suggests the increased binding affinity for the second ligand is not due to an allosteric creation of a more favorable binding interface by the first ligand. This is consistent with data from us and from others indicating that the on rates of conformationally dynamic proteins approach the limits of diffusion. In contrast, negative cooperativity is manifested by alterations in both k on and k off , suggesting stabilization of the binary complex.IDP | protein-protein interaction P rotein-protein interactions (PPIs) underscore all cellular processes, and the mechanistic dissection of PPI networks is thus a high priority (1-4). A particular challenge is defining the mechanism of PPI formation between intrinsically disordered proteins (IDPs) where allosteric changes play a substantive role (5-7). Allosteric communication between binding sites is vital for the proper function of feedback and regulatory circuits in the cell (8, 9). For example, the conformationally dynamic kinase-inducible domain interacting (KIX) domain of the master transcriptional coactivator CREB binding protein (CBP) undergoes a structural shift and stabilization upon binding to a cognate ligand such as the intrinsically disordered transcriptional activation domain (TAD) of mixed-lineage leukemia protein (MLL) (Fig. 1A) (10). The interaction of a second ligand, phosphorylated kinase-inducible domain (pKID) of CREB, is enhanced (up to twofold) by the presence of MLL and several elegant studies have documented allosteric communication between the two binding sites (11-15). However, the relatively modest affinities (micromolar) of the native complexes and the aggregation propensities and the promiscuous binding profiles of the transcriptional activation domains have hampered kinetic dissection of this and other complexes (16,17).The coactivator CBP exists across metazoans (18)(19)(20) and is a transcription hub that interacts with numerous transcriptional activators, using several discrete domains (21,22). The KIX domain within CBP is a 90-residue motif ...

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“…Targeting the allosteric sites on protein kinases may also lead to the identification of activators rather than inhibitors, which could be useful for therapeutic intervention or as pharmacological tools. In particular, compounds targeting the PIF pocket (the hydrophobic motif present in the N-terminal lobe of AGC kinases) of either PDK1 or PKCz can either act as activators (Hindie et al, 2009) or substrate-selective inhibitors (Lopez-Garcia et al, 2011;Sadowsky et al, 2011;Busschots et al, 2012). Similarly, the myr-pocket binders of ABL can be converted into activators if they are designed not to bend helix I of the ABL kinase domain Yang et al, 2011).…”

Section: Downloaded Frommentioning

confidence: 99%

25 Years of Small Molecular Weight Kinase Inhibitors: Potentials and Limitations

2014

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Deregulation of protein and lipid kinase activities leads to a variety of pathologies, ranging from cancer inflammatory diseases, diabetes, infectious diseases, and cardiovascular disorders. Protein kinases and lipid kinases represent, therefore, an important target for the pharmaceutical industry. In fact, approximately one-third of all protein targets under investigation in the pharmaceutical industry are protein or lipid kinases. To date, 30 kinase inhibitors have been approved, which, with few exceptions, are mainly for oncological indications and directed against only a handful of protein and lipid kinases, leaving 70% of the kinome untapped. Despite these successes in kinase drug discovery, the development of kinase inhibitors with outstanding selectivity, identification and validation of driver kinase(s) in diseases, and the emerging problem of resistance to the inhibition of key target kinases remain major challenges. This minireview provides an insight into protein and lipid kinase drug discovery with respect to achievements, binding modes of inhibitors, and novel avenues for the generation of second-generation kinase inhibitors to treat cancers.

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Turning a protein kinase on or off from a single allosteric site via disulfide trapping

Cited by 147 publications

References 31 publications

Discovery of multiple hidden allosteric sites by combining Markov state models and experiments

Discovery of multiple hidden allosteric sites by combining Markov state models and experiments

Dissecting allosteric effects of activator–coactivator complexes using a covalent small molecule ligand

25 Years of Small Molecular Weight Kinase Inhibitors: Potentials and Limitations

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