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Commentary Open Access
Volume 6 | Issue 1 | DOI: https://doi.org/10.33696/Pharmacol.6.054

Validating the Use of Rational Modification of Compounds to Reduce P-gp Efflux

  • 1Institute for Neurodegenerative Diseases, Weill Institute for Neurosciences, University of California, San Francisco, California 94158, United States
  • 2Department of Neurology, Weill Institute for Neurosciences, University of California, San Francisco, California 94158, United States
+ Affiliations - Affiliations

*Corresponding Author

Jay Conrad, Jay.Conrad@ucsf.edu; Roy J Vaz, Roy.Vaz@ucsf.edu

Received Date: June 21, 2024

Accepted Date: June 28, 2024

Abstract

In both the Central nervous system (CNS) as well as Oncology small molecule drug discovery programs, efflux due to P-glycoprotein (P-gp) could be a deterrent during the discovery phase to obtain in vitro or in vivo pharmacological readouts. Several different strategies have been utilized in the past in order to overcome efflux by P-gp, many of which have been described in a recent article [5] from our labs. We describe the use of Induced-fit docking (IFD) of matched pairs (pairs of molecules modified by a single group) in order to demonstrate that a change in the IFD score, destabilizing the complex, will afford a molecule with less P-gp efflux potential. In a particular discovery program, several iterations might need to be implemented after examining the IFD lowest scored pose to obtain a molecule in that chemical series without P-gp efflux. Examples of such an effort are currently underway and will be published shortly. This summary describes therefore what we consider, a validation of a unifying approach to obtaining molecules with low to no p-gp efflux potential.

Keywords

P-gp, MDR1, Blood-brain barrier, Kp, CNS penetration, P-glycoprotein, Drug interaction, CADD

Introduction

Successful neurodegenerative disease and central nervous system drugs require exposure in the target tissue. Often limiting drug levels and a major challenge in developing drugs for CNS indications are the efflux transporters expressed at the blood brain barrier (Figure 1). One such efflux transporter is permeability glycoprotein or P-glycoprotein, or the ATP-binding cassette subfamily B member 1 (ABCB1) [1]. Avoiding efflux by P-gp is also important for oncology drug discovery where the transporter limits drug exposure in cancer cells. Development of P-gp inhibitors as adjuvants for oncology drugs is an active area of research [2].

Figure 1. A cartoon representation of the blood brain barrier depicting various transporters present, including P-gp. Created with BioRender.com.

Structure based approaches to modelling efflux transporters has been made difficult by the challenge in obtaining atomic resolution structures of membrane bound proteins. Advances in Cryogenic electron microscopy (Cryo-EM) made in the last decade or so have made obtainment of transporter proteins a new possibility. Furthermore, structures of P-Glycoprotein (P-gp) have been available for several years, they are bound with inhibitor rather than substrate. When P-gp operates as an efflux pump the transporter cycles through various conformations with the aid of ATP. The first published structures of human P-gp with the substrate taxol (pdb code 6qex) [3] as well as the drug free structure in a similar conformation (pdb code 7a65) defined the substrate-binding, entrance tunnel and the vestibule regions [4].

The set of cryo-EM structures enabled the article recently published [5] describing a predictive model for P-gp efflux activity. The initial hypothesis that substrates interact more strongly with the transporter when docked into the active site was then evaluated. Test molecules were docked in the substrate binding pocket and an induced-fit docking (IFD) algorithm utilized and the overall binding energy assessed. Using matched pairs of compounds with single point changes that are reported to reduce P-gp efflux matched the computed decrease in binding energy. This correlation demonstrated that the computational methodology could be applied to the structure-based design of small molecules with lower P-gp efflux activity.

Five Chemical Structural Modifcations

The molecular changes that affect P-gp efflux described in the CNS and oncology drug discovery literature can be categorized into groups [6,7]. Five examples covering the types of changes will be covered in this summary. The compound names in this commentary are the same used in Conrad et al. [5].

Increasing molecular volume

The first example involves increasing the Molecular weight or molecular volume as shown in Table 1. This is exemplified by the molecular pair NAP and NAQ. Synthesized as mu-opioid receptor antagonists a 4-pyridyl amide in NAP is replaced with an isoquinoline in NAQ [8]. Both compounds have similar in vitro potency towards the opioid receptor. However, only NAQ with an efflux ratio of 1.3, displays in vivo efficacy whereas NAP, with an efflux ratio of >10, does not display any pharmacology. This is attributed to significantly lower brain exposure levels for NAP. The IFD pose of NAP is better accommodated by 7 kcal/mol with hydrogen bonds and cation-pi interactions which, due to the increase in size of the second aromatic ring, NAQ cannot form.

Table 1. Structure of NAP and NAQ. The highlighted portions show the changes in the pair accounting for the difference in P-gp efflux.

Changing conformation

The second type of change, conformation, is shown with the matched pair opioid receptor-like 1 (ORL1) antagonists 1a and 1b [9]. In this pair of molecules shown in Table 2, a small change from a thio ether in compound 1a to a ketone group in compound 1b, a small change in molecular weight, was found to not greatly impact the activity towards ORL1 antagonism. However, the P-gp efflux was reduced drastically. There is a large conformation change for 1a and 1b on binding P-pg where compound 1a was better accommodated in the IFD pose in P-gp by 26 kcal/mol.

Table 2. The highlighted single group change in compounds 1a and 1b which changes the conformation leading to a change in P-gp efflux.

Increase pKb

The third type of change is an increase of pKb or decrease of pKa (pKa + pKb = 14) exemplified by the molecules shown in Table 3 which were synthesized as Kinesin Spindle Protein Inhibitors 2c-2f [10]. In these compounds fluorine was iteratively incorporated, and the efflux measured by an MDR assay [11,12]. Many discovery compounds often contain a basic nitrogen which can also enhance efflux. Therefore, a common tactic to decrease amine pKa is the replacement of a hydrogen by a fluorine near the basic heteroatom. This can however have several effects. As well as decreasing the hydrogen bond accepting ability of the heteroatom, fluorine has a different size and impact on conformation than hydrogen. In this example the pKa of the N-H will also be lowered by increased fluorine incorporation. In Table 3, the pKa decreased progressively from 10.7 to 5.2. On docking these compounds into P-gp, it is clear, that the larger size of fluorine has an impact on the binding pose. Although this is a more complicated case the experimental MDR assay values match the IFD score.

Table 3. A series of molecules where the C, beta to the basic N is increasingly substituted with Fluorine (F), decreasing the pKa. The effects of the increasing Fluorine substitution are to make the basic N less basic. Also progressing from 2c through 2f, the lowest energy pose for 2c cannot be achieved by 2d through 2f due to the added steric bulk of each added F.

Removal of a hydrogen bond donor

Removal of a hydrogen bond (H-bond) donor can also decrease P-gp efflux and is exemplified by the matched pair in Table 4. This pair were synthesized as Cannabinoid-2 agonists for chronic pain [13] with both molecules showing similar in vitro CB2 activity. Here the molecular weight of the pair of molecules is identical. The removal of the H-bond donor leads to a decrease in interactions between molecule 5b with P-gp as the NH to P-gp amide C=O hydrogen bond present in 5a is lost. A lowering in IFD score for 5b correlates with a large change in P-gp efflux ratio.

Table 4. A pair of compounds where removal of an H-bond donor in 5a leads to a decrease in P-gp Efflux.

Removal of a hydrogen bond acceptor

The last example to reduce P-gp efflux is the removal of a H-bond acceptor in dual serotonin and noradrenaline reuptake inhibitors shown in examples 7a and 7b, (Table 5) [14]. The decrease in molecular weight by deleting acceptors can also improve ligand efficiency and in the example shown in Table 5, the molecular weight is reduced by 28 amu. The compounds dock into different pockets with compound 7a forming stronger interactions even though the tetrahydropyranyl O in compound 7a is not involved in an H-bond directly.

Table 5. A pair of compounds where removal of an H-bond acceptor in 7b leads to a decrease in P-gp Efflux.

Conclusions

Even though there are several different design strategies to reduce P-gp efflux, the change in the IFD score is unifying. Decrease in IFD score correlates with reduced P-gp efflux and can be employed in modifying a chemical structure from being a strong P-gp substrate to a non-substrate. With this computational tool in hand small molecule drug candidates can be tailored to maintain desired target activity or toxicological properties and minimize P-gp efflux.

Structures of other efflux transporters such as BCRP [15,16] and MRP4 [17,18] as well as in vitro data availability for these and other transporters [19] will enable the use of rational design of molecules to overcome efflux from those transporters. These efforts hopefully will lead to compounds with effective pharmacological CNS and oncology compounds with less resistance.

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