Effect of Chirality on Common in Vitro Experiments: An Enantiomeric

Jul 23, 2015 - This analysis elucidates the impact of small molecule architecture on common in vitro ADME assays. In vitro parameters considered in th...
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Effect of Chirality on Common in Vitro Experiments: An Enantiomeric Pair Analysis Jeffrey T. Bagdanoff,*,† Yongjin Xu,*,‡ Gavin Dollinger,‡ and Eric Martin‡ †

Global Discovery Chemistry/Oncology and Exploratory Chemistry, Novartis Institutes for BioMedical Research, 250 Massachusetts Avenue, Cambridge, Massachusetts 02139, United States ‡ Global Discovery Chemistry/Oncology and Exploratory Chemistry, Novartis Institutes for BioMedical Research, 4560 Horton Street, Building 4, Emeryville, California 94608, United States S Supporting Information *

ABSTRACT: This analysis elucidates the impact of small molecule architecture on common in vitro ADME assays. In vitro parameters considered in this analysis included Caco-2 permeability/efflux, CYP3A4 inhibition, hERG inhibition, and rat microsomal extraction ratio (ER). The statistical significance and practical meaningfulness of chirality were determined by comparison of the distribution of enantiomers with the experimental variation distribution observed from duplicate measurements. Statistical tools were applied to characterize the role of molecular architecture on the outcome of a given in vitro assay. We found that CYP3A4 inhibition, hERG inhibition, Caco-2 permeability, and efflux are unlikely to be modulated by chirality. However, rat microsomal ER provides a statistically significant, and quantitatively meaningf ul, chance of being influenced by chirality.

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INTRODUCTION The relationship between physicochemical descriptors of small molecules and in vitro/in vivo performance has been extensively evaluated and reviewed. An early report by Pidgeon1 examined the role of molecular weight in membrane permeability, preceding Lipinski’s omnipresent “rule of five”.2,3 Similar evaluations compared physiochemical properties of compounds from discovery phase through commercialization.4−7 Many other principal structural features have been correlated with clinical success, such as sp3 atom count8 and aromatic ring count.9 Publication of such retrospective analyses has been prolific. While the rapid proliferation of drug discovery metrics and guidelines has offered an armament of tools for triaging chemical space in search of drugs, it has been observed that the variety of, sometimes conflicting, metrics may be counterproductive to the medicinal chemist.10 Of particular concern, it has been observed that retrospective analyses are not informed by the base-rate frequency of non-drug-like properties11 presenting the opportunity for a logical fallacy known as base-rate fallacy12 to operate. When operative, seemingly intuitive, yet inappropriate conclusions can result.13 It has also been observed that many retrospective analyses are prone to “correlation inflation”, introduced by systematic errors that arise from binning and averaging noisy data sets.14 This data treatment may exaggerate the magnitude of a trend. Given the active literature discussion regarding appropriate role of physicochemical property based retrospective analyses, we were inspired to consider physicochemical independent determinants of in vitro ADME performance in a more © XXXX American Chemical Society

objective, statistically based manner. Specifically, we sought to ascertain impact of small molecule architecture on common in vitro ADME assays. In recognition of the potential for enantiomeric pairs to interact differently with the chiral environments presented by Caco-2 permeability, CYP inhibition, hERG, and microsomal stability matrices, we questioned whether differential responses between enantiomers would be observed in any of these assays. To this end, the Novartis database was queried for enantiomeric pairs where each member contained a valid Caco-2 permeability, CYP inhibition, hERG, or microsomal ER result. In the process, 26 691 valid pairs were identified. For each data set, a distribution of the differences between the experimental responses of enantiomeric pairs was determined. The statistical significance of each distribution was evaluated by comparison to the distribution of duplicate data points. Duplicate data are routinely obtained for individual compounds in the course of quality control protocols. Comparison of the duplicate data distribution with the enantiomeric pair shift distribution revealed the impact of chirality on each in vitro parameter. This comparison allowed for the determination of pvalues and D-values via the Kolmogorov−Smirnov (K−S) test, a simple, widely used, and easily understood nonparametric test of the equality between two distributions.15 The statistical foundation made possible the determination of whether, and by what magnitude, observed differences between enantiomers exceeded experimental error. Received: February 19, 2015

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DOI: 10.1021/acs.jmedchem.5b00552 J. Med. Chem. XXXX, XXX, XXX−XXX

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METHODS Compound structures and the corresponding data were retrieved using an in-house data query tool. The data set included 131 563 compounds with a measured solubility, Caco2 permeability and efflux, CYP inhibition, human ER, hERG binding value, or a biochemical activity for a single arbitrarily selected in-house target. Identification of enantiomer and isotope pairs was performed using code written in Python using RDKit16 by the following steps: (1) Compound structures were indexed by the InChI key from the canonical SMILES string with chiral information removed. (2) Compounds were labeled for the presence of a chiral feature. (3) Possible pairs were generated by enumerating all possible compound combinations with the same index from step 1. Identical pairs and the pairs without chiral features on both compounds in a pair were filtered. (4) Enantiomeric pairs were identified by inverting all chiral specifications on one compound in the pair and then comparing the resulting canonical smiles with the other compound. While our internal compound registration protocols require explicit designation of stereochemical descriptors, absolute enantiomeric purity for each compound in our collection may vary. While the enantiomeric purity of materials generated from chiral separation of a racemic mixture is generally >95%, the enantiomeric purity of commercial starting materials employed in synthesis is not. As a consequence, actual differences in the measurement of enantiomeric pairs may be underestimated. To determine whether the difference between in vitro values was significant beyond experimental variation, a variation decomposition method17 was applied. To compare the distribution of the end point response against the experimental variation, the K−S test was chosen. The K−S test is nonparametric and insensitive to the number of data points beyond what is required to calculate the cumulative distribution function (CDF). The CDF is built on a computational basis and can distinguish between statistical significance (p-value, the likelihood that an effect exists) and the predictive value of the difference (D-value). The D-value from the K−S test indicates the maximum magnitude of the difference, and the p-value indicates the likelihood that the two distributions are coincident. Three logical conclusions follow from the applied K−S test: (1) Low p-value (0.1) indicate two statistically significant and meaningfully distinct distributions are present, respectively. (2) Low p-value ( 0.5) change in pIC50 upon chiral inversion, and the chance of observing this by experimental variability is only 5%. The pie chart cutoff was chosen because 95% of replicate variations are below 0.5 in ΔpIC50. Caco-2 [A → B] Permeability. Since Caco-2 [A → B] permeability reflects the net involvement of multiple possible modes of membrane transportation,25,26 including paracellular transport,27,28 transcellular transport,29 active carrier mediated uptake,30 transcytosis mechanisms,31 and efflux pumps,32 it was unclear whether any chiral bias in the Caco-2 [A → B] membrane transfer of enantiomeric pairs would be detected. The participation of multiple parallel transport mechanisms in this complex cell-based system might obscure any single chirally biased process. In this analysis, 308 valid enantiomeric pairs across 126 scaffolds (level 3 scaffold tree; Shannon’s diversity index = 3.86; Pielou’s evenness index = 0.80) were examined against 1985 replicate measurement pairs. This data set was refined to exclude low (20 times) than the majority. These compounds created a sampling bias problem. The over-representation distorted the CDF curve. To combat the sampling problem, 104 compounds with more than four replicate measurements were removed. Following the adjustment, the obtained D-value (0.02) and p-value (0.1) indicated that no meaningful or statistically significant difference is observed between the distributions in the solubility data for replicates and enantiomers. Among all parameters studied, over-representation was unique to the solubility measurement; therefore, a similar treatment was neither required nor appropriate for other assays. Isocitrate Dehydrogenase (IDH) as a Model Biochemical Target. Because biochemical target proteins are composed of chiral, nonracemic amino acids and adopt asymmetric tertiary structures, it is generally understood that they present a selective chiral environment for their small molecule guests. The potency of IDH inhibition within certain chemical series is reported to be affected by chirality.20−22 Wild type IDH is a NADP+/NADH and metal dependent oxidoreductase that catalyzes the oxidative decarboxylation of endogenous isocitrate to α-ketoglutarate. Mutations in IDH1/2 yield a neomorphic activity, catalytic reduction of α-ketoglutate to 2-hydroxyglutarate (2-HG), and are associated with multiple cancers types.23 For example, leukemia patients with IDH1/2 mutations present elevated 2-HG levels in serum.24 As a positive control for the predictive value of the statistical method, the effect of chirality in the context of mutant IDH was evaluated. This analysis included 63 enantiomeric pairs across 16 scaffolds (level 3 scaffold tree; Shannon’s diversity index = 2.08; Pielou’s evenness index = 0.87) and 32 724 replicate measurement pairs (Figure 2). Despite the small number of enantiomeric pairs, the analysis was sufficiently powered to generate meaningful statistics; the p-value (3 × 10−10) and Dvalue (0.44) confirm a large difference between the distributions. Consequently, we confirm that chiral inversion of a fixed stereocenter in ligands has a statistically significant effect on the biochemical activity against the target, and the

Figure 3. Enantiomers do not demonstrate variable Caco-2 [A → B] permeability. Data summary: CDF curves (top) comparing Δlog(Caco-2 Papp A−B) of enantiomeric pairs (green) with replicate measurements (black); pie chart of the experimental variation with cutoff of 0.71 (bottom left); pie chart of enantiomeric pair measurement difference with cutoff of 0.71 (bottom right). The pie chart cutoff was chosen at around 95% of replicate variations.

(0.068) demonstrate these two distributions differ no more than expected by experimental variability, and there is no meaningful difference between the Caco-2 [A → B] permeability of enantiomeric pairs. In consideration of the prominent role of PGP efflux pumps in Caco-2 [B → A] measurements, and the possible role of chiral recognition in this process, we also applied the statistical method to the Caco-2 [B → A] assay. From the analysis (304 valid enantiomeric pairs with 1843 duplicate measurements), we obtain a low p-value (0.04) and a low D-value (0.09). Both of these measurements are just below our somewhat arbitrary

Figure 2. Enantiomers exhibit variable biochemical activity vs IDH. Data summary: difference distribution for enantiomeric pairs (top left); variation distribution for replicate measurements (low left); CDF curves (right) comparing ΔpIC50 of enantiomeric pairs (blue) with replicate measurements (black). C

DOI: 10.1021/acs.jmedchem.5b00552 J. Med. Chem. XXXX, XXX, XXX−XXX

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thresholds. Consequently, while we again conclude that the distributions are equivalent, no discrimination was observed between enantiomers in the Caco-2 [A → B] measurement. However, there is a slight chance (4%) that further sampling from this same population might indicate a marginally meaningful difference. hERG Inhibition. In contrast to the complex mixture of transcellular transport mechanisms operating in a Caco-2 measurement, in vitro measurement of hERG blockade reflects a discrete ligand−protein interaction. The hERG pharmacophore has been broadly characterized by two lipophilic moieties flanking a central basic nitrogen.33 Although a high-resolution crystal structure of the hERG channel is currently unavailable at the time of this report, homology models, low-resolution Xray,34 and electron microscopy35 studies have elucidated key structural features. The hERG channel is a homotetramer where each subunit contains six α-helical transmembrane segments. Aromatic residues situated near the mouth of the channel the pore helix are implicated as the primary interaction points for hERG blockers via π-stacking and hydrophobic interactions.36 The tetrameric architecture of the hERG ion channel renders the gross architecture C-4 symmetric, which is chiral like a propeller. Considering the chiral hERG channel environment, we evaluated the effect of ligand chirality on hERG ion channel blockade. Measurements were available for 464 valid enantiomeric pairs across 127 scaffolds (level 3 scaffold tree; Shannon’s diversity index = 3.80; Pielou’s evenness index = 0.78) and 2111 replicate measurement pairs (Figure 4). The low p-value (0.0087) indicates that the distributions of hERG measurement differences between enantiomeric pairs are statistically significant. However, the low D-value (0.085) suggests that the difference is small. On the basis of these data, we conclude that hERG ion channel blockade is not

meaningfully influenced by inversion of chirality. With 464 enantiomeric pairs and 2111 replicate measurement pairs, the distributions are very accurately characterized, so the small systematic difference between enantiomers can be detected but will rarely matter. Consequently, the interaction of small molecules with the hERG channel is primarily influenced by physicochemical properties. Rat Microsomal ER. Over 90% of drug oxidations in man are mediated by P450 enzymes, and the major catalysts of human drug metabolism are mediated by the CYP3A4, CYP2D6, and CYP2C subfamily enzymes. The architecture and catalytic mechanism of cytochrome P450 enzymes have been rigorously characterized.37 Principal structural features include a hexacoordinate Fe(III) cored porphryin flanked by the protein backbone. The P450 catalytic cycle has been thoroughly studied.38−40 The gross features include (1) substrate binding to the Fe(III) core of enzyme’s active site, (2) combination of oxygen and NADPH to provide a Fe(III) peroxy complex, (3) heterolytic O−O bond cleavage to form a reactive oxyferryl Fe(IV) complex, (4) oxygen atom transfer to the substrate, and (5) product dissociation concomitant with regeneration of the Fe(III) porphyrin complex. If CYP450 enzymes were a rigid framework with a welldefined catalytic mode, chiral antipodes of a xenobiotic would generate diasteromeric transition states upon interaction with the chiral CYP450 environment, possibly resulting in different observed rates of metabolism. However, this scenario is complicated by the heterogeneity of multiple structurally distinct CYP450 isoforms in a microsomal stability matrix,41 the presence of multiple substrate-binding pockets and effector binding regions, the contribution of multiple catalytic modes and conformations,42 the potential contribution of allosteric effects,43 and potential variability in source microsomes over years of data collection. A mitigating factor in this complex scenario is the recognition that CYP3A4 is the most abundant CYP450 isoform44 and a majority of mammalian xenobiotic metabolism is mediated by CYP3A4.45 Furthermore, the rat microsomal ER assay employed in this analysis comprises the subcellular S-9 fraction reconstituted with NADPH cofactor. Consequently, the assay matrix is simplified by exclusion of phase-2 metabolizing enzymes, aldehyde oxidase enzymes, etc. In this analysis (Figure 5), 1112 valid enantiomeric pairs across 243 scaffolds (level 3 scaffold tree; Shannon’s diversity index = 4.45; Pielou’s evenness index = 0.81) were examined against 148 406 replicate measurement pairs. A significant shift was observed in the distributions between enantiomeric pairs versus the background distributions in the replicate data. The low p-value (2.2 × 10−16) and high D-value (0.48) indicates that these distributions are distinct and the magnitude of the difference is large, respectively. As shown in the pie chart, there is a 41% of chance that one would observe >8% change in ER by inverting chirality, and the chance of observing such change by experimental variation is only 5.5%. From this analysis, it is concluded that chiral recognition is a statistically significant and meaningf ul event in an in vitro microsomal ER assay. Since enantiomers can have meaningfully different ER values, diastereomers might also meaningfully differ on the basis of molecular shape alone; they may differ by more than is predicted by log D values. The case of a high ER molecule containing multiple stereocenters may be a valid exercise to synthesize diastereomers. This is particularly true when crystallography demonstrates that a region of a small molecule is solvent exposed. Unfortunately, our internal data

Figure 4. Enantiomers do not demonstrate variable hERG inhibition. Data summary: CDF curves (top) comparing Δlog(hERG dofetilide binding human recombinant IC50) of enantiomeric pairs (green) with replicate measurements (black); pie chart of the experimental variation with cutoff of 0.9 (bottom left); pie chart of enantiomeric pair measurement difference with cutoff of 0.9 (bottom right). The pie chart cutoff was chosen at around 95% of replicate variations. D

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set was not large enough to generate meaningful statistics regarding the impact of diastereomeric inversion on ER. To illustrate the effect, several pairs of enantiomers were collected from the Novartis database, along with their associated rat microsomal ER values. As shown in Table 1, the structurally diverse enantiomer pairs represented by structures 1−7 demonstrate dramatically different rat microsomal ER values. In the examples shown, one member of the enantiomeric pair is characterized by an extremely high rat microsomal ER, while the other is characterized by an extremely low rat microsomal ER value. The effect is seen across a variety of chiral subunits. CYP3A4 Inhibition. Like other CYPs, CYP3A4 is subject to reversible and mechanism-based (irreversible) inhibition by xenobiotics.46 While reversible inhibition is characterized by classic binding kinetics, mechanism-based inactivation involves the formation of reactive metabolic intermediates that covalently modify the CYP.47 This process is characterized by NADPH-, time-, and concentration-dependent enzyme inactivation via chemical modification of the prosthetic heme and/or the CYP protein. On the basis of the analysis of structural features of CYP enzymes presented in the previous section, and the differential metabolism observed for enantiomeric pairs, we were interested in whether chirality would play a role in CYP3A4 inhibition. In this analysis (Figure 6), 249 valid enantiomeric pairs across 82 scaffolds (level 3 scaffold tree; Shannon’s diversity index = 3.61; Pielou’s evenness index = 0.82) were examined against 70 265 replicate CYP3A4 inhibition measurements. In light of the dependence of microsomal stability on substrate chirality, it is interesting that chirality did not significantly influence CYP3A4 inhibition; the distributions between enantiomeric pairs versus

Figure 5. Enantiomers demonstrate variable rat microsomal ER. Data summary: CDF curves (top) comparing Δ(CYP ER in rat liver microsomes) of enantiomeric pairs (black) with replicate measurements (gray); pie chart of the experimental variation with cutoff of 8% (bottom left); pie chart of enantiomeric pair measurement difference with cutoff of 8% (bottom right). The pie chart cutoff was chosen at around 95% of replicate variations.

Table 1. Enantiomers Demonstrate Variable Rat Microsomal ER

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from the association of enatiomers with our Caco-2, CYP3A4 inhibition, or hERG assay constructs is too small to be detected. These latter properties might therefore be more amenable to QSAR modeling using simpler physicochemical or 2D descriptors that neglect stereochemical information. That Caco-2, CYP3A4 inhibition, and hERG experiments are generally insensitive to chirality supports the rationale for reliance on physicochemical descriptors by medicinal chemists in lead optimization activities. While the value of manipulating physicochemical properties (i.e., reducing log D value) in the interest of mitigating ER has been established by the scientific community, we have quantitatively demonstrated that molecular architecture also has a meaningful impact on this metric.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jmedchem.5b00552. Diversity results, identification protocol, programming source code pertinent to the compilation and statistical analysis of the data presented here, and SMILES strings for compounds 1−7 (PDF)

Figure 6. Enantiomers do not demonstrate variable CYP3A4 inhibition. Data summary: CDF curves (top) comparing Δlog(CYP3A4 (midazolam) human liver microsomes IC50) of enantiomeric pairs (green) with replicate measurements (black); pie chart of the experimental variation with the same cutoff (bottom left); pie chart of enantiomeric pair measurement difference with cutoff of 0.86 (bottom right). The pie chart cutoff was chosen at around 95% of replicate variations.

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AUTHOR INFORMATION

Corresponding Authors

*J.T.B.: e-mail, jeffrey.bagdanoff@novartis.com; phone, +1 (617) 871-5315. *Y.X.: e-mail, [email protected]; phone, +1 (510) 9238292.

the background replicate data were statistically equivalent (p = 0.28). A possible explanation for the lack of chiral discrimination in CYP3A4 inhibition may reside in the mechanistic nuances that define CYP-xenobiotic interactions. Chemical moieties most commonly associated with reversible CYP inhibition are achiral sp2 hybridized systems characterized by electron rich heterocycles including imidazoles,48 thiophenes,49 furan,50 and pyridines.51 Binding of the heteroatom lone-pair with the prosthetic iron(III) is commonly invoked as the CYP inhibition mechanism. Irreversible inhibition primarily occurs following metabolic activation of sp1 and sp2 hybridized centers.52,53 Since neither of these events directly engage chiral carbon centers, chiral recognition may not be relevant to CYP3A4 inhibition.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors thank Julian Levell, Michael Schultz, and Matthew Burger for their thoughtful input. A waiver on ACS data deposition requirements has been granted by the Editors for this study.

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ABBREVIATIONS USED K−S, Kolmogorov−Smirnov; CDF, cumulative distribution function; ER, extraction ratio; ADME, absorption, distribution, metabolism, excretion; IDH, isocitrate dehydrogenase; CYP, cytochrome P450; Cyp3A4, cytochrome P450 isoform 3A4; hERG, human ether-a-go-go-related gene; [A → B], apical to basolateral diffusion; 2D, two-dimensional; 3D, three-dimensional

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CONCLUSION Calculated and measured physicochemical properties provide the foundation for triaging chemical space in the pursuit of druglike molecules. However, these properties are agnostic to the 3D presentation of a compound’s constituent components to a biological system. The problem of defining the structural contribution of discovery phase medicinal chemistry was addressed by analyzing the differential response of enantiomers in a variety of in vitro experiments. Since the physicochemical properties of enantiomers are identical, differences in the outcome of a given in vitro experiment are attributed to molecular architecture. In this analysis, stereochemistry was a statistically meaningful descriptor in rat microsomal ER experiments but not in Caco-2, CYP3A4 inhibition, or hERG experiments. This observation demonstrates that the energy difference resulting from association of enantiomers with a CYP enzyme assay construct is sufficiently large to be detected in an ER measurement. However, the energy difference resulting

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DOI: 10.1021/acs.jmedchem.5b00552 J. Med. Chem. XXXX, XXX, XXX−XXX

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