Quantitating drug-target engagement in single cells in vitro and in vivo

Abstract

Quantitation of drug target engagement in single cells has proven difficult, often leaving unanswered questions in the drug development process. Here we show that intracellular target engagement of unlabeled new therapeutics can be quantitated using polarized microscopy combined with competitive binding of matched fluorescent companion imaging probes. We quantitate the dynamics of target engagement of covalent BTK inhibitors as well as reversible PARP inhibitors in populations of single cells as two examples using a single companion imaging probe for each target. We then determine average in vivo tumor concentrations and show marked population heterogeneity following systemic delivery, revealing single cells with low target occupancy at high average target engagement in vivo.

Molecular Imaging of PARP

“…Uptake specificity was shown by correlation of PARP1 protein expression and PARPi-FL retention, as well as by the ability to block PARPi-FL uptake by saturating PARP with olaparib (13). One set of studies (7,8) presented PARPi-FL as a tool to measure drugtarget interaction in real time at subcellular resolution in vitro and in vivo using multiphoton fluorescence anisotropy microscopy. The anisotropy value of an excited fluorophore is linked to its mobility and hence provides insight into its binding state.…”

“…The earlier study (7) showed anisotropy measurements of PARPi-FL in vivo using a window chamber model. Later (8), an indirect anisotropy approach was used to measure pharmacodynamic parameters of olaparib, allowing the identification of single cells that featured a low occupancy despite a high average target engagement.…”

Localizing Antifungal Drugs to the Correct Organelle Can Markedly Enhance their Efficacy

“…To date,d rug delivery research has focused primarily on enhancing drug absorption and on trafficking it to specific sites of infection within organs and tissues. [5][6][7][8][9] Life-threatening fungal infections are associated with patients suffering from ac ompromised immune system;t hat is,p atients with HIV/AIDS,c ancer patients undergoing chemotherapy,o rp atients treated with immunosuppressants to prevent the rejection of organ transplants. [3,4] Directing the drug to localize to the relevant organelle within the pathogen by altering its chemical structure and properties has the potential to increase its pharmacological activity.A ccordingly,i mproving the ability of as mall molecule to localize to the specific organelle or domain that harbors its target can significantly improve its efficacyd ue to an increase in the local concentration of the inhibitor.…”

Translational drug discovery and development with the use of tissue‐relevant biomarkers: Towards more physiological relevance and better prediction of clinical efficacy

“…imaging, [105] cellular thermal shift assays (CETSA) [106] and competitive binding with fluorescently labelled companion imaging probes (CIP) accompanied by fluorescence polarization microscopy [107] are further approaches to determine target occupancy in vivo.…”