In recent years an increased number of covalent protein kinase inhibitors has been approved for cancer therapy and many more are undertaking clinical trials. Covalent binding is usually obtained by introducing in these drugs an electrophilic warhead able to bind specific nucleophilic sites in the protein target. Covalent inhibition of oncogenic protein kinases allows to obtain a stronger and prolonged therapeutic effect compared to reversible inhibition; however, the choice of the dose to be administered to patients and the evaluation of the selectivity among the kinase family are mandatory to reach pharmacological results reducing possible side effects. From the DMPK perspective for covalent inhibitors, in vitro and in vivo data extrapolation, to obtain human pharmacokinetic projection, can be challenging and numerous efforts have to be undertaken in developing methods to accurately and quantitively determine inhibitor target engagement in preclinical and more importantly in clinical studies. Chemoproteomic approaches, taking advantage of the use of chemical probes, provide powerful tools to analyze binding characteristics between small molecules and proteins and validate the mode of action of these drug candidates. Click chemistry is a cycloaddition reaction between an azide and alkyne group to generate a 1,4-disubstituted 1,2,3-triazole ring. In this way, it is possible to functionalize the inhibitor under development with an alkyne moiety that could be labelled with a fluorescent-azide for target engagement detection and quantitation in native environments and a desthiobiotin-azide molecule for identification of the inhibitor off-target binders and proteome-wide selectivity. The functionalization of the inhibitor with a small group such as the alkyne group (the molecule obtained is called probe) allows the incubation of the compound directly in situ, with minimal alteration of inhibitor features (e.g. potency and permeability). Thus, the aim of this project is the optimization and application of click chemistry reaction in order to study covalent mechanism of action of ibrutinib, a covalent inhibitor of the tyrosine protein kinase BTK, approved by FDA in 2013 for the treatment of B cell malignancies. We used ibrutinib as case study, but the developed protocols can be applied to the study of other covalent inhibitors. Target engagement conditions were initially optimized on the recombinant BTK protein using an ibrutinib derivative, bearing an alkyne group, and then transferred to cell extracts. Competition experiments were set up on extracts and then an in cell target engagement experiment was conducted, treating cells with 5 μM ibrutinib. The significant decreasing of the signal intensity of fluorescent probe labeled BTK, after pre-incubation of cells with ibrutinib, suggested a full target occupancy of BTK. Nevertheless, with the aim to precisely calculate this target occupancy, a chemoproteomic workflow coupled to quantitative mass spectrometry analysis has been optimized and set. In this case, click chemistry reaction was performed coupling to ibrutinib probe an azide functionalized with a desthiobiotin moiety in order to capture desthiobiotinylated peptides with a streptavidin resin, taking advantage of high affinity between streptavidin and desthiobiotin. The developed protocols have been validated in cells treated with ibrutinib and, in addition to BTK, two additional protein kinases JAK3 and BLK have been identified modified on cysteine 909 and 319, respectively. These cysteines are located in the ATP-binding site of the two kinases in a position corresponding to the cysteine 481 in BTK. Interaction with ibrutinib for JAK3 and BLK was confirmed by additional analyses on recombinant protein and cell lysates. Total proteome analysis, both in label free quantitation and TMT mode, has been undertaken to additionally characterize ibrutinib treated cells. The work allowed to better understand the preclinical profile of an oncological target therapy drug in term of potency and selectivity in living cells. The process is transferable to other covalent drugs and applied not only in preclinical models but also in clinical trials, helping in the definition of the optimal dose for patients to obtain the best efficacy, limiting side effects.

QUANTITATIVE PROTEOMIC APPROACHES TO STUDY DRUG MECHANISM OF ACTION / S. Castelli ; tutor: G. Aldini, B. Valsasina ; coordinatore: G. Aldini. - : . Dipartimento di Scienze Farmaceutiche, 2022. ((35. ciclo, Anno Accademico 2022.

QUANTITATIVE PROTEOMIC APPROACHES TO STUDY DRUG MECHANISM OF ACTION

S. Castelli
2023

Abstract

In recent years an increased number of covalent protein kinase inhibitors has been approved for cancer therapy and many more are undertaking clinical trials. Covalent binding is usually obtained by introducing in these drugs an electrophilic warhead able to bind specific nucleophilic sites in the protein target. Covalent inhibition of oncogenic protein kinases allows to obtain a stronger and prolonged therapeutic effect compared to reversible inhibition; however, the choice of the dose to be administered to patients and the evaluation of the selectivity among the kinase family are mandatory to reach pharmacological results reducing possible side effects. From the DMPK perspective for covalent inhibitors, in vitro and in vivo data extrapolation, to obtain human pharmacokinetic projection, can be challenging and numerous efforts have to be undertaken in developing methods to accurately and quantitively determine inhibitor target engagement in preclinical and more importantly in clinical studies. Chemoproteomic approaches, taking advantage of the use of chemical probes, provide powerful tools to analyze binding characteristics between small molecules and proteins and validate the mode of action of these drug candidates. Click chemistry is a cycloaddition reaction between an azide and alkyne group to generate a 1,4-disubstituted 1,2,3-triazole ring. In this way, it is possible to functionalize the inhibitor under development with an alkyne moiety that could be labelled with a fluorescent-azide for target engagement detection and quantitation in native environments and a desthiobiotin-azide molecule for identification of the inhibitor off-target binders and proteome-wide selectivity. The functionalization of the inhibitor with a small group such as the alkyne group (the molecule obtained is called probe) allows the incubation of the compound directly in situ, with minimal alteration of inhibitor features (e.g. potency and permeability). Thus, the aim of this project is the optimization and application of click chemistry reaction in order to study covalent mechanism of action of ibrutinib, a covalent inhibitor of the tyrosine protein kinase BTK, approved by FDA in 2013 for the treatment of B cell malignancies. We used ibrutinib as case study, but the developed protocols can be applied to the study of other covalent inhibitors. Target engagement conditions were initially optimized on the recombinant BTK protein using an ibrutinib derivative, bearing an alkyne group, and then transferred to cell extracts. Competition experiments were set up on extracts and then an in cell target engagement experiment was conducted, treating cells with 5 μM ibrutinib. The significant decreasing of the signal intensity of fluorescent probe labeled BTK, after pre-incubation of cells with ibrutinib, suggested a full target occupancy of BTK. Nevertheless, with the aim to precisely calculate this target occupancy, a chemoproteomic workflow coupled to quantitative mass spectrometry analysis has been optimized and set. In this case, click chemistry reaction was performed coupling to ibrutinib probe an azide functionalized with a desthiobiotin moiety in order to capture desthiobiotinylated peptides with a streptavidin resin, taking advantage of high affinity between streptavidin and desthiobiotin. The developed protocols have been validated in cells treated with ibrutinib and, in addition to BTK, two additional protein kinases JAK3 and BLK have been identified modified on cysteine 909 and 319, respectively. These cysteines are located in the ATP-binding site of the two kinases in a position corresponding to the cysteine 481 in BTK. Interaction with ibrutinib for JAK3 and BLK was confirmed by additional analyses on recombinant protein and cell lysates. Total proteome analysis, both in label free quantitation and TMT mode, has been undertaken to additionally characterize ibrutinib treated cells. The work allowed to better understand the preclinical profile of an oncological target therapy drug in term of potency and selectivity in living cells. The process is transferable to other covalent drugs and applied not only in preclinical models but also in clinical trials, helping in the definition of the optimal dose for patients to obtain the best efficacy, limiting side effects.
ALDINI, GIANCARLO
ALDINI, GIANCARLO
covalent inhibitors; target engagement; chemical proteomic; click chemistry
Settore CHIM/08 - Chimica Farmaceutica
QUANTITATIVE PROTEOMIC APPROACHES TO STUDY DRUG MECHANISM OF ACTION / S. Castelli ; tutor: G. Aldini, B. Valsasina ; coordinatore: G. Aldini. - : . Dipartimento di Scienze Farmaceutiche, 2022. ((35. ciclo, Anno Accademico 2022.
Doctoral Thesis
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/2434/950653
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