How Big Is Too Big for Cell Permeability? - Journal ... - ACS Publications

Feb 24, 2017 - ... M. W.; Vacca , J. P.; Olsen , D. B.; Liverton , N. J.; Carroll , S. S. MK-5172, a selective inhibitor of hepatitis C virus NS3/4a p...
3 downloads 13 Views 614KB Size
Viewpoint pubs.acs.org/jmc

How Big Is Too Big for Cell Permeability? Par̈ Matsson† and Jan Kihlberg*,‡ †

Department of Pharmacy, BMC, Uppsala University, Box 580, SE-751 23 Uppsala, Sweden Department of Chemistry, BMC, Uppsala University, Box 576, SE-751 23 Uppsala, Sweden



ABSTRACT: Understanding how to design cell permeable ligands for intracellular targets that have difficult binding sites, such as protein−protein interactions, would open vast opportunities for drug discovery. Interestingly, libraries of cyclic peptides displayed a steep drop-off in membrane permeability at molecular weights above 1000 Da and it appears likely that this cutoff constitutes an upper size limit also for more druglike compounds. However, chemical space from 500 to 1000 Da remains virtually unexplored and represents a vast opportunity for those prepared to venture into new territories of drug discovery.

T

(Figure 1a), i.e., at a MW of approximately 1000 Da. This permeability trend was also observed for a set of cyclic peptide natural products that displayed conformational preferences but had MWs in the same range as the investigated peptide libraries. As pointed out by the authors, this size-dependent permeability is not compatible with the traditional solubility− diffusion based theories for cell permeability. Instead other theories, for instance, approximating membrane diffusion with diffusion through polymer networks, may be required to provide mechanistic insight into the unexpected size dependence. The study of Pye et al. also points to the importance of keeping lipophilicity within a narrow window in order to combine cell permeability and aqueous solubility at high MW. This window is reduced as MW increases, putting an upper limit on the size of cell-permeable compounds. A further consequence of this narrow window is that exposed polar or charged groups can be expected to be prohibitive for permeability in this chemical space. It is important to note that the sharp drop-off in permeability (which becomes very pronounced at a MW > 1000 Da) agrees very well with previous investigations that have highlighted that almost no orally administered drugs and clinical candidates exist at higher MWs than this (Figure 1b).4 It thus appears that the results reported by Pye et al. are not limited to the hydrophobic cyclic peptides studied by the authors but are applicable across different classes of cell permeable and orally administered drugs. As recently suggested,5,6 it also appears to be essential for orally administered drugs with MW > 700 Da to possess a certain flexibility in the molecular structure; this allows them to adapt to their environment and thereby combine aqueous solubility, cell permeability, and efficient target binding.5,6 In the absence of such “chameleonic” behavior, these important drug properties would be mutually exclusive in this chemical space. A few orally available and cellpermeable outliers do exist at even greater molecular sizes. Of these, cyclosporin A has been studied most extensively and appears as the master among molecular chameleons due to its reversible formation of four intramolecular hydrogen bonds and shielding of polarity by lipophilic side chains.5,6 In fact, as

he human proteome is estimated to have 100 000 to 1 000 000 binary protein−protein interactions (PPIs), which may constitute one of the most important sources of novel targets for drug discovery. However, harnessing this potential has been challenging, since PPIs and several other nonclassical drug targets typically have large, featureless, and sometimes flexible interfaces that are difficult to drug with small molecules.1,2 In addition to the difficulties posed by the target binding site, the majority of PPIs and nonclassical targets are located inside cells, requiring drugs to cross cell membranes to elicit their effect. Thus, even though biologics are often ideal for difficult-to-drug targets, they are ruled out by their lack of cell permeability, as well as by their low bioavailability on oral administration. In order to unlock the potential of PPIs and other intracellular targets for drug discovery, it is therefore of utmost importance to understand how far chemical space that contains cell permeable, druglike molecules extends. In this issue of the Journal of Medicinal Chemistry, Cameron Pye et al. report that carefully designed cyclic peptides display a sharp reduction in passive cell permeability with increasing size.3 When put in the context of other recent studies, it appears likely that this cutoff is applicable also to nonpeptidic and more druglike compounds beyond traditional small molecule space. Cameron Pye et al. evaluated the impact of molecular size and lipophilicity on membrane permeability using libraries of cyclic octa-, nona- and decapeptides in the 800−1200 Da size range. Backbone amide bonds were completely N-methylated to eliminate the possibility of intramolecular hydrogen bonding, which could otherwise have influenced conformation preferences and thereby membrane permeability. As a consequence of the synthetic procedure, all peptides contained one Tyr and one Pro residue, while the remaining residues were restricted to amino acids with natural or non-natural aliphatic side chains, thereby reducing the influence of polar and charged groups. The parallel artificial membrane permeability assay (PAMPA) and a MDCK cell clone that expresses only low levels of Pglycoprotein were used for determination of membrane permeability to eliminate or reduce the influence of transporter mediated efflux. Interestingly, a sharp and unexpected reduction in passive permeability was observed, beginning at molecular sizes just over 800 Å3 and severely limiting permeability above 1000 Å3 © 2017 American Chemical Society

Received: February 12, 2017 Published: February 24, 2017 1662

DOI: 10.1021/acs.jmedchem.7b00237 J. Med. Chem. 2017, 60, 1662−1664

Journal of Medicinal Chemistry

Viewpoint

Figure 1. Membrane permeability of macrocyclic peptides, large drugs, and clinical candidates. (a) Using carefully designed libraries of cyclic peptides, Pye et al. demonstrate a sharp decrease in membrane diffusion at high molecular volume that is not accounted for by the traditional solubility-diffusion model.3 (b) A recent review of high-MW drugs and clinical candidates revealed that orally bioavailable compounds are extremely rare at MW > 1000 Da (red and gray symbols represent oral and nonoral compounds, respectively; shaded surfaces indicate extended regions of chemical space in which cell permeability is possible).4

clear. Analyses of cell permeability in traditional small-molecule drug space indicate that transporter-mediated efflux increases with greater molecular size.7 In line with this, cellular efflux that increased with molecular size was observed for macrocyclic compounds in the 400−800 Da size range.8 Thus, efflux mediated by ATP-binding cassette (ABC) transporters such as P-glycoprotein (MDR1/ABCB1), breast cancer resistance protein (BCRP/ABCG2), and members of the multidrug resistance-associated protein family (MRP/ABCC) can impose additional limitations on the cell permeability of high-MW molecules, below the drop-off described by Pye et al. However, transporter-mediated efflux has not been systematically explored in the size ranges discussed by Pye et al., with some notorious exceptions like cyclosporin A. Correspondingly, uptake transporters in the solute carrier (SLC) superfamily can have pronounced impact on cellular drug permeability, but this has not been systematically studied for larger molecules. Available literature suggests that only some of the known drug-transporting SLCs may accept substrates near the size ranges discussed by Pye et al.5 Examples include the transport of larger HCV NS3/4A protease inhibitors by members of the organic anion transporting polypeptide (OATP/SLCO) family.9 However, the SLC family is notably understudied,10 with the majority of human SLCs referred to in less than 15 reports in the scientific literature. Clearly, much work is needed in defining the substrate specificities of these transporters and their potential impact on drug disposition. Endocytosis and transcytosis mechanisms have mostly been studied for nanoparticulate drug delivery systems and biologics but could also play a role for intermediate-size drug molecules. For instance, a family of cell-penetrating peptides that for the first time provide for highly efficient delivery of drugs to the cytosol was recently described.11 Again, fundamental research is needed, but given that most transcellular permeability coefficients reported for nanodelivery systems12 are lower than those observed by Pye et al. at MW of ∼1000 Da, it appears that simple transmembrane diffusion may still be the more effective pathway for compounds in the size-range studied by Pye, et al, which are still, by comparison, rather small. In conclusion, intracellular targets such as protein−protein interactions constitute valuable new opportunities for drug

demonstrated by Pye et al., cyclosporin A outperforms the similarly sized model cyclic peptides when it comes to permeability (Figure 1a). Incorporation of chameleonic behavior in the design could thus be essential for discovery of “oversized” drugs that extend cell-permeable drug space beyond the current borders at approximately 1000 Å3 or 1000 Da (Figure 2).3,4 Even more importantly, it may also be an

Figure 2. Size boundaries for membrane-permeable molecules. Membrane permeability is typically limited when polar surface area (PSA) exceeds 140 Å2.13 Compounds that can alternately expose or shield polar functionality depending on the environment (“chameleonic molecules”) represent an important opportunity to venture beyond the borders of traditional drug space. The concept is exemplified by cyclosporin A, which can adopt both membranepermeable (low PSA) and water-soluble (high PSA) conformations. This extends the boundaries of cell-permeable chemical space and also appears to be essential for orally administered drugs with MW > 700 Da. TPSA: topological polar surface area.

effective means of improving the pharmacokinetic properties of compounds in the 700−1000 Da MW range, which is a challenging region of chemical space but one that still presents significant opportunities for discovery of orally administered drugs. The use of artificial membranes and efflux-reduced MDCK cells allowed Pye et al. to study the size dependency of simple transmembrane diffusion, in isolation from other complicating transport mechanisms. This includes transporter-mediated efflux and uptake and particle trafficking pathways like endoand transcytosis. The impact of such facilitated mechanisms in the cellular transport of larger druglike molecules is not yet 1663

DOI: 10.1021/acs.jmedchem.7b00237 J. Med. Chem. 2017, 60, 1662−1664

Journal of Medicinal Chemistry

Viewpoint

Edwards, A. M.; Superti-Furga, G. A call for systematic research on solute carriers. Cell 2015, 162, 478−487. (11) Qian, Z.; Martyna, A.; Hard, R. L.; Wang, J.; Appiah-Kubi, G.; Coss, C.; Phelps, M. A.; Rossman, J. S.; Pei, D. Discovery and mechanism of highly efficient cyclic cell-penetrating peptides. Biochemistry 2016, 55, 2601−2612. (12) Lundquist, P.; Artursson, P. Oral absorption of peptides and nanoparticles across the human intestine: Opportunities, limitations and studies in human tissues. Adv. Drug Delivery Rev. 2016, 106, 256− 276. (13) Palm, K.; Stenberg, P.; Luthman, K.; Artursson, P. Polar molecular surface properties predict the intestinal absorption of drugs in humans. Pharm. Res. 1997, 14, 568−571.

discovery but are often difficult to modulate using small molecules and inaccessible for biologics that lack cell permeability. It is therefore important to understand to what extent such “difficult” intracellular targets can be modulated by ligands outside traditional small molecule drug space and how such ligands should be designed to successfully reach their targets. A few recent studies have begun to provide a first level of understanding of how such nonconventional drugs interact with their targets,1,2 what the outer limits of cell permeability are, and what structural features are important for their design.4−6 Pye et al. have now taken another important step to clarify the scope and limitations of drug discovery beyond the rule of 5, but we still lack even a basic understanding of how to design drugs in this space. Thus, we can only second the conclusion of Pye et al. that mining of this chemical space for therapeutically valuable compounds will be a challenge for the coming decades.



AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Phone: +46 (0)18 4713801. ORCID

Pär Matsson: 0000-0002-9094-2581 Jan Kihlberg: 0000-0002-4205-6040



REFERENCES

(1) Scott, D. E.; Bayly, A. R.; Abell, C.; Skidmore, J. Small molecules, big targets: drug discovery faces the protein−protein interaction challenge. Nat. Rev. Drug Discovery 2016, 15, 533−550. (2) Doak, B. C.; Zheng, J.; Dobritzsch, D.; Kihlberg, J. How beyond rule of 5 drugs and clinical candidates bind to their targets. J. Med. Chem. 2016, 59, 2312−2327. (3) Pye, C. R.; Hewitt, W. M.; Schwochert, J.; Haddad, T. D.; Townsend, C. E.; Etienne, L.; Lao, Y.; Limberakis, C.; Furukawa, A.; Mathiowetz, A. M.; Price, D. A.; Liras, S.; Lokey, R. S. Nonclassical size dependence of permeation defines bounds for passive absorption of large drug molecules. J. Med. Chem. 2017, DOI: 10.1021/acs.jmedchem.6b01483. (4) Doak, B. C.; Over, B.; Giordanetto, F.; Kihlberg, J. Oral druggable space beyond the rule of 5: Insights from drugs and clinical candidates. Chem. Biol. 2014, 21, 1115−1142. (5) Matsson, P.; Doak, B. C.; Over, B.; Kihlberg, J. Cell permeability beyond the rule of 5. Adv. Drug Delivery Rev. 2016, 101, 42−61. (6) Whitty, A.; Zhong, M.; Viarengo, L.; Beglov, D.; Hall, D. R.; Vajda, S. Quantifying the chameleonic properties of macrocycles and other high-molecular-weight drugs. Drug Discovery Today 2016, 21, 712−717. (7) Desai, P. V.; Sawada, G. A.; Watson, I. A.; Raub, T. J. Integration of in silico and in vitro tools for scaffold optimization during drug discovery: predicting P-glycoprotein efflux. Mol. Pharmaceutics 2013, 10, 1249−1261. (8) Over, B.; Matsson, P.; Tyrchan, C.; Artursson, P.; Doak, B. C.; Foley, M. A.; Hilgendorf, C.; Johnston, S. E.; Lee, M. D., IV; Lewis, R. J.; McCarren, P.; Muncipinto, G.; Norinder, U.; Perry, M. W. D.; Duvall, J. R.; Kihlberg, J. Structural and conformational determinants of macrocycle cell permeability. Nat. Chem. Biol. 2016, 12, 1065−1074. (9) Summa, V.; Ludmerer, S. W.; McCauley, J. A.; Fandozzi, C.; Burlein, C.; Claudio, G.; Coleman, P. J.; DiMuzio, J. M.; Ferrara, M.; Di Filippo, M.; Gates, A. T.; Graham, D. J.; Harper, S.; Hazuda, D. J.; McHale, C.; Monteagudo, E.; Pucci, V.; Rowley, M.; Rudd, M. T.; Soriano, A.; Stahlhut, M. W.; Vacca, J. P.; Olsen, D. B.; Liverton, N. J.; Carroll, S. S. MK-5172, a selective inhibitor of hepatitis C virus NS3/ 4a protease with broad activity across genotypes and resistant variants. Antimicrob. Agents Chemother. 2012, 56, 4161−4167. (10) Cesar-Razquin, A.; Snijder, B.; Frappier-Brinton, T.; Isserlin, R.; Gyimesi, G.; Bai, X.; Reithmeier, R. A.; Hepworth, D.; Hediger, M. A.; 1664

DOI: 10.1021/acs.jmedchem.7b00237 J. Med. Chem. 2017, 60, 1662−1664