Proteolysis targeting chimeras (PROTACs) are heterobifunctional small molecules that simultaneously bind to a target protein and an E3 ligase, thereby leading to ubiquitination and subsequent degradation of the target. They present an exciting opportunity to modulate proteins in a manner independent of enzymatic or signaling activity. As such, they have recently emerged as an attractive mechanism to explore previously "undruggable" targets. Despite this interest, fundamental questions remain regarding the parameters most critical for achieving potency and selectivity. Here we employ a series of biochemical and cellular techniques to investigate requirements for efficient knockdown of Bruton's tyrosine kinase (BTK), a nonreceptor tyrosine kinase essential for B cell maturation. Members of an 11-compound PROTAC library were investigated for their ability to form binary and ternary complexes with BTK and cereblon (CRBN, an E3 ligase component). Results were extended to measure effects on BTK-CRBN cooperative interactions as well as in vitro and in vivo BTK degradation. Our data show that alleviation of steric clashes between BTK and CRBN by modulating PROTAC linker length within this chemical series allows potent BTK degradation in the absence of thermodynamic cooperativity.

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ABSTRACT:With efforts to reduce cytochrome P450-mediated clearance (CL) during the early stages of drug discovery, transporter-mediated CL mechanisms are becoming more prevalent. However, the prediction of plasma concentration-time profiles for such compounds using physiologically based pharmacokinetic (PBPK) modeling is far less established in comparison with that for compounds with passively mediated pharmacokinetics (PK). In this study, we have assessed the predictability of human PK for seven organic aniontransporting polypeptide (OATP) substrates (pravastatin, cerivastatin, bosentan, fluvastatin, rosuvastatin, valsartan, and repaglinide) for which clinical intravenous data were available. In vitro data generated from the sandwich culture human hepatocyte system were simultaneously fit to estimate parameters describing both uptake and biliary efflux. Use of scaled active uptake, passive distribution, and biliary efflux parameters as inputs into a PBPK model resulted in the overprediction of exposure for all seven drugs investigated, with the exception of pravastatin. Therefore, fitting of in vivo data for each individual drug in the dataset was performed to establish empirical scaling factors to accurately capture their plasma concentration-time profiles. Overall, active uptake and biliary efflux were under-and overpredicted, leading to average empirical scaling factors of 58 and 0.061, respectively; passive diffusion required no scaling factor. This study illustrates the mechanistic and model-driven application of in vitro uptake and efflux data for human PK prediction for OATP substrates. A particular advantage is the ability to capture the multiphasic plasma concentration-time profiles for such compounds using only preclinical data. A prediction strategy for novel OATP substrates is discussed.

Early prediction of clearance mechanisms allows for the rapid progression of drug discovery and development programs, and facilitates risk assessment of the pharmacokinetic variability associated with drug interactions and pharmacogenomics. Here we propose a scientific framework--Extended Clearance Classification System (ECCS)--which can be used to predict the predominant clearance mechanism (rate-determining process) based on physicochemical properties and passive membrane permeability. Compounds are classified as: Class 1A--metabolism as primary systemic clearance mechanism (high permeability acids/zwitterions with molecular weight (MW) ≤400 Da), Class 1B--transporter-mediated hepatic uptake as primary systemic clearance mechanism (high permeability acids/zwitterions with MW >400 Da), Class 2--metabolism as primary clearance mechanism (high permeability bases/neutrals), Class 3A--renal clearance (low permeability acids/zwitterions with MW ≤400 Da), Class 3B--transporter mediated hepatic uptake or renal clearance (low permeability acids/zwitterions with MW >400 Da), and Class 4--renal clearance (low permeability bases/neutrals). The performance of the ECCS framework was validated using 307 compounds with single clearance mechanism contributing to ≥70% of systemic clearance. The apparent permeability across clonal cell line of Madin - Darby canine kidney cells, selected for low endogenous efflux transporter expression, with a cut-off of 5 × 10(-6) cm/s was used for permeability classification, and the ionization (at pH7) was assigned based on calculated pKa. The proposed scheme correctly predicted the rate-determining clearance mechanism to be either metabolism, hepatic uptake or renal for ~92% of total compounds. We discuss the general characteristics of each ECCS class, as well as compare and contrast the framework with the biopharmaceutics classification system (BCS) and the biopharmaceutics drug disposition classification system (BDDCS). Collectively, the ECCS framework is valuable in early prediction of clearance mechanism and can aid in choosing the right preclinical tool kit and strategy for optimizing drug exposure and evaluating clinical risk of pharmacokinetic variability caused by drug interactions and pharmacogenomics.

Oral bioavailability (F) is a product of fraction absorbed (Fa), fraction escaping gut-wall elimination (Fg), and fraction escaping hepatic elimination (Fh). In this study, using a database comprised of Fa, Fg, Fh, and F values for 309 drugs in humans, an analysis of the interrelation of physicochemical properties and the individual parameters was carried out in order to define the physicochemical space for optimum human oral bioavailability. Trend analysis clearly indicated molecular weight (MW), ionization state, lipophilicity, polar descriptors, and free rotatable bonds (RB) influence bioavailability. These trends were due to a combination of effects of the properties on Fa and first-pass elimination (Fg and Fh). Higher MW significantly impacted Fa, while Fg and Fh decreased with increasing lipophilicity. Parabolic trends were observed for bioavailability with polar descriptors. Interestingly, RB has a negative effect on all three parameters, leading to its pronounced effect on bioavailability. In conclusion, physicochemical properties influence bioavailability with typically opposing effects on Fa and first-pass elimination. This analysis may provide a rational judgment on the physicochemical space to optimize oral bioavailability.