Experimental and Computational Methods to Assess Central Nervous System Penetration of Small Molecules

Abstract

In CNS drug discovery, the estimation of brain exposure to lead compounds is critical for their optimization. Compounds need to cross the blood-brain barrier (BBB) to reach the pharmacological targets in the CNS. The BBB is a complex system involving passive and active mechanisms of transport and efflux transporters such as P-glycoproteins (P-gp) and breast cancer resistance protein (BCRP), which play an essential role in CNS penetration of small molecules. Several in vivo, in vitro, and in silico methods are available to estimate human brain penetration. Preclinical species are used as in vivo models to understand unbound brain exposure by deriving the Kp,uu parameter and the brain/plasma ratio of exposure corrected with the plasma and brain free fraction. The MDCK-mdr1 (Madin Darby canine kidney cells transfected with the MDR1 gene encoding for the human P-gp) assay is the commonly used in vitro assay to estimate compound permeability and human efflux. The in silico methods to predict brain exposure, such as CNS MPO, CNS BBB scores, and various machine learning models, help save costs and speed up compound discovery and optimization at all stages. These methods enable the screening of virtual compounds, building of a CNS penetrable compounds library, and optimization of lead molecules for CNS penetration. Therefore, it is crucial to understand the reliability and ability of these methods to predict CNS penetration. We review the in silico, in vitro, and in vivo data and their correlation with each other, as well as assess published experimental and computational approaches to predict the BBB penetrability of compounds.

Keywords: CNS drug discovery; P-glycoproteins (P-gp); active transport; blood–brain barrier (BBB); breast cancer resistance protein (BCRP); efflux transporters; in silico models; influx transporters; passive diffusion.

Figure 1

(a) The endothelial tight junctions of the BBB (shown in brown) severely restrict paracellular transport, whereas specialized transporters, e.g., P-gp (green diamond) and BCRP (blue oval) (efflux transporters) and OCT1 (orange triangle) and OCT2 (yellow hexagon) (influx transporters) regulate the transcellular transport of metabolic nutrients and other essential molecules across BBB. Enclosed in the basal lamina, pericyte cells partially surround these BBB endothelial cells. The complex tight cellular network of BBB is further maintained by astrocytes’ end-feet. Astrocytes maintain the cellular link between neurons and microglial cells. The transport across BBB involves concentration gradient-driven passive diffusion and active transport employing various efflux and influx transporters in the endothelial cell membrane. (b) Schematic of plasma and brain compartments presenting different modes of transport across BBB, i.e., passive diffusion and active transport using efflux (e.g., P-gp, BCRP) and influx (e.g., OCT1, OCT2) transporters. Kp,uu represents the unbound brain to unbound plasma drug concentration ratio, where Cu,b and Cu,p represent the unbound drug concentration in the brain and plasma, respectively. Different brain compartments, i.e., Blood, BBB, CSF, BCSFB, ISF, and ICF, correspond to blood, blood–brain barrier, cerebrospinal fluid, blood–cerebrospinal fluid barrier, interstitial fluid, and intracellular fluid, respectively.

Figure 2

Relative distribution of CNS class of compounds: CNS+ (green) and CNS− (red). The MPO, MPO_V2, pMPO, and BBB scores range from 0 to 6 (a score within a range of [4, 6] means better CNS penetration). Original pMPO scores range between 0 and 1. To be consistent with MPO scores, we scaled the pMPO scores from 0 to 6.

Figure 3

Relative distribution of CNS class of compounds: CNS+ (green) and CNS− (red). The MPO, MPO_V2, pMPO, and BBB scores range from 0 to 6 (a score in the range of [4, 6] means better CNS penetration). Original pMPO scores range between 0 and 1. To be consistent with MPO scores, we scaled the pMPO scores from 0 to 6. Percentage of CNS drugs and non-CNS drugs correctly identified (for CNS: MPO, MPO_V2, pMPO, BBB Score (4, 6]; for non-CNS: MPO, MPO_V2, BBB Scores [0,4)) in their respective CNS and non-CNS database is plotted on 100% stacked bar graph for MPO, MPO_V2, pMPO, and BBB Scores.

Figure 4

The schematic diagram of the proposed mechanism of P-gp (MDR1) is represented. Transmembrane (TBDs) and nucleotide (NBDs) binding domains of P-gp are presented in green and red, respectively. The P-gp substrates are shown by black triangles, which cross the BBB membrane by passive diffusion or active transport. The inward-facing, ADP-bound state structure (i) changes conformation, the NBDs dimerize, and the TMDs re-orientate to extracellular space to adopt an outward-facing (ATP-bound) state (ii). The extracellular segment’s transmembrane helices in the outward-facing conformation of P-gp reorient to release the substrate. Upon ATP hydrolysis, the transporter is reoriented to the inward-facing structure, and two phosphate molecules are released.

Figure 5

A plot of the CNS class of compounds CNS+ (green) and CNS− (red) against their measured efflux ratios. CNS+ compounds with good brain exposure have a higher probability of having lower efflux.

Figure 6

The bias of available drugs with MDCK (orange) and Kp,uu data (black). The grey and white dots represent CNS+ and CNS- compounds, respectively.

Figure 7

In silico methods (CNS MPO, MPO_V2, pMPO, and BBB Score) segregate low vs. high efflux compounds, but there is much room for improvement.

Figure 8

100% stacked bar graphs for low-to-high MPO, MPO_V2, pMPO, and BBB Scores for rat Kp,uu dataset. Compounds with a higher score tend to show higher unbound brain exposure.

Figure 9

The MDR1-MDCK in vitro assay predicts good in vivo Kp,uu when ER < 3. However, compounds with medium efflux (3,10] also show moderate in vivo brain exposure.

Figure 10

The Kp,uu of preclinical species is an important parameter for predicting human brain exposure. Most of the CNS drugs show rat Kp,uu over 0.3.