Thermal Denaturation:  A Method to Rank Slow Binding, High-Affinity P38α MAP Kinase Inhibitors

Kroe, Rachel R.; Regan, John; Proto, A.; Peet, Gregory W.; Roy, Tapon; Landro, Laura D.; Fuschetto, Natalie G.; Pargellis, Christopher A.; Ingraham, Richard H.

doi:10.1021/jm030120s

Cited by 50 publications

(60 citation statements)

References 17 publications

(19 reference statements)

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“…An exact IC 50 (possibly subnanomolar) for the highly potent compounds could not be determined because an enzyme concentration of 4 nM was necessary to give a robust signal in the assay. The correlation between enzyme inhibitory activity and the thermal melt results was very tight (R 2 ϭ 0.821, P Ͻ 0.0001) and supports the use of the thermal method before an enzyme assay has been developed and optimized (17).…”

Section: Discussionmentioning

confidence: 60%

Selective Inhibitors of Methionyl-tRNA Synthetase Have Potent Activity against Trypanosoma brucei Infection in Mice

Shibata

Gillespie

Kelley

et al. 2011

Antimicrob Agents Chemother

125

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Human African trypanosomiasis continues to be an important public health threat in extensive regions of sub-Saharan Africa. Treatment options for infected patients are unsatisfactory due to toxicity, difficult administration regimes, and poor efficacy of available drugs. The aminoacyl-tRNA synthetases were selected as attractive drug targets due to their essential roles in protein synthesis and cell survival. Comparative sequence analysis disclosed differences between the trypanosome and mammalian methionyl-tRNA synthetases (MetRSs) that suggested opportunities for selective inhibition using drug-like molecules. Experiments using RNA interference on the single MetRS of Trypanosoma brucei demonstrated that this gene product was essential for normal cell growth. Small molecules (diaryl diamines) similar to those shown to have potent activity on prokaryotic MetRS enzymes were synthesized and observed to have inhibitory activity on the T. brucei MetRS (50% inhibitory concentration, <50 nM) and on bloodstream forms of T. brucei cultures (50% effective concentration, as low as 4 nM). Twenty-one compounds had a close correlation between enzyme binding/inhibition and T. brucei growth inhibition, indicating that they were likely to be acting on the intended target. The compounds had minimal effects on mammalian cell growth at 20 M, demonstrating a wide therapeutic index. The most potent compound was tested in the murine model of trypanosomiasis and demonstrated profound parasite suppression and delayed mortality. A homology model of the T. brucei MetRS based on other MetRS structures was used to model binding of the lead diaryl diamine compounds. Future studies will focus on improving the pharmacological properties of the MetRS inhibitors.

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Section: Discussionmentioning

confidence: 60%

Selective Inhibitors of Methionyl-tRNA Synthetase Have Potent Activity against Trypanosoma brucei Infection in Mice

Shibata

Gillespie

Kelley

et al. 2011

Antimicrob Agents Chemother

125

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“…This simple and cost-effective screening method allowed us to target both active and inactive kinases and has been shown to provide data that are consistent with orthogonal methods such as isothermal titration calorimetry and enzymatic assays (13,(15)(16)(17). A comparison of T m shift data with IC 50 values of two targets has been included in SI Fig.…”

Section: Resultsmentioning

confidence: 85%

A systematic interaction map of validated kinase inhibitors with Ser/Thr kinases

Fedorov

Marsden

Pogačić

et al. 2007

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

336

323

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Protein kinases play a pivotal role in cell signaling, and dysregulation of many kinases has been linked to disease development. A large number of kinase inhibitors are therefore currently under investigation in clinical trials, and so far seven inhibitors have been approved as anti-cancer drugs. In addition, kinase inhibitors are widely used as specific probes to study cell signaling, but systematic studies describing selectivity of these reagents across a panel of diverse kinases are largely lacking. Here we evaluated the specificity of 156 validated kinase inhibitors, including inhibitors used in clinical trials, against 60 human Ser/Thr kinases using a thermal stability shift assay. Our analysis revealed many unexpected cross-reactivities for inhibitors thought to be specific for certain targets. We also found that certain combinations of activesite residues in the ATP-binding site correlated with the detected ligand promiscuity and that some kinases are highly sensitive to inhibition using diverse chemotypes, suggesting them as preferred intervention points. Our results uncovered also inhibitor crossreactivities that may lead to alternate clinical applications. For example, LY333531, a PKC␤ inhibitor currently in phase III clinical trials, efficiently inhibited PIM1 kinase in our screen, a suggested target for treatment of leukemia. We determined the binding mode of this inhibitor by x-ray crystallography and in addition showed that LY333531 induced cell death and significantly suppressed growth of leukemic cells from acute myeloid leukemia patients.acute myeloid leukemia ͉ inhibitor selectivity ͉ LY333Ј531 ͉ PIM1 ͉ screening data

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“…Several p38 inhibitors are structurally and biophysically well-characterized (Table 1). [13][14][15] p38 inhibitors 1 and 4 bind to the DFG-in conformation [16] that is also observed for apo p38. [17] In contrast, p38 inhibitors 2 and 3 of the diarylurea class were found to bind to the DFG-out conformation.…”

mentioning

confidence: 71%

NMR Characterization of Kinase p38 Dynamics in Free and Ligand‐Bound Forms

et al. 2006

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In its apo state kinase p38 effects slow motions that can be detected in the NMR spectrum. One of the affected parts is the pharmacologically interesting DFG motif. Diarylurea inhibitors that bind to the DFG‐out conformation lock this motif in a defined state, whereas DFG‐in inhibitors that bind to the adjacent hinge region leave the flexibility of the DFG motif unaffected (see crystal structure of the complex of p38 with the inhibitor SB203580).

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Thermal Denaturation: A Method to Rank Slow Binding, High-Affinity P38α MAP Kinase Inhibitors

Cited by 50 publications

References 17 publications

Selective Inhibitors of Methionyl-tRNA Synthetase Have Potent Activity against Trypanosoma brucei Infection in Mice

Selective Inhibitors of Methionyl-tRNA Synthetase Have Potent Activity against Trypanosoma brucei Infection in Mice

A systematic interaction map of validated kinase inhibitors with Ser/Thr kinases

NMR Characterization of Kinase p38 Dynamics in Free and Ligand‐Bound Forms

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