Computer-Assisted Molecular Modeling of Benzodiazepine and ...

Computer-Assisted Molecular Modeling of Benzodiazepine and Thyromimetic Inhibitors of the HepG2 lodothyronine Membrane Transporter

Laura Kragie, Maureen Mary McCourt

L. Forrester,

Vivian Cody, and

Biological Sciences, Faculty of Natural Sciences and Mathematics (L.K.) State University of New York at Buffalo Amherst, New York 14260 Molecular Biophysics (M.L.F., V.C.) and Electron Diffraction Departments (M.M.) Medical Foundation of Buffalo Buffalo. New York 14203

T3 cellular uptake is inhibited in the presence of benzodiarepines (BZs). The structure-activity relationship of BZ inhibition correlates strongly with halogen substitution of the nonfused phenyl ring and indicates that this ring is required for activity. A structure-activity series of thyromimetic (TH) inhibitors of the HepGP iodothyronine transporter further point out the critical importance of the amino group of the alanine side chain, its L-stereo configuration, and the size of the substituents of the inner and outer phenyl rings. A third series of compounds, reported to interact at related sites, were inactive as HepG2 iodothyronine transport inhibitors, and therefore the potent inhibitors were restricted to the BZ and TH compounds. Using both of these BZ and TH structure-activity series along with computer-assisted molecular modeling techniques, we determined which chemical structural components were important at the transporter interaction site. By superimposing structures from active chemicals, excluding residues from poor inhibitors, and incorporating molecular electropotential data, we developed a five-point model of BZ conformational similarity to the endogenous transporter ligand, L-TJ: the alkyl substitution at the Nl of the BZ ring seems to simulate the alanine side chain of TJ, and the electronegative halogen and oxygen atoms of substituents at M/R7/R2’/R4’ of BZ form a pyrimidal pharmacophore that seems to correspond with the 3-1/5-l/ 3’-l/4’-OH substituents of T3, respectively. These points, suggesting a tilted cross-bow formation, may be sites for ligand interaction with the iodothyronine transporter. (Molecular Endocrinology 8: 382-391, 1994) 0ea9-eae9/94/o3a2~391$03.00/0 Molecular Endocrimy Copyright 0 1994 by TIM Endocrine

INTRODUCTION In this paper we compare the molecular conformations of thyroid hormones to benzodiazepines (BZs), a potential class of membrane iodothyronine transporter antagonists. Thyroid hormones have profound effects on growth, differentiation, maturation of tissues, and the turnover of substrates, vitamins, and hormones. In mammalian species, the active forms are Tq and its more potent deiodinated metabolite, T3 (1). Facilitated carrier-mediated transport allows the hormones to cross the cell membrane and then to interact with cytosolic, enzymatic, mitochondrial, microsomal, and nuclear binding sites (2-4). The iodothyronine membrane transport process has been studied in many tissues, and these data show that membrane transport of TJ is energy dependent, stereo-specific, and critical for cellular nuclear binding and metabolism of T3 (for review see Refs. 5-7). In a pharmacological survey of compounds that may interact with the membrane iodothyronine transporter, it was shown that compounds of the BZ class, like the thyromimetics (THs), are reversible inhibitors of the TJ membrane transporter (8, 9). Because the structureactivity relationship (SAR) describing BZ inhibition of TS uptake in the HepG2 cell line suggests a site that is different from the central and peripheral BZ receptor (BZR) sites, we hypothesized that the BZs and THs may directly interact at the TJ binding site on the membrane transporter. Using both of these SAM, in conjunction with molecular modeling strategies, we developed a model for the structural comparison of iodothyronine transport inhibitors and now propose a pharmacophore for ligand interaction with the TJ transport site.

society

382

lodothyronine

RESULTS

Transporter

Pharmacophore

AND DISCUSSION

in Table 1. This SAR of BZ inhibition reveals that the nonfused phenyl ring is necessary for activity in our series. The strongest correlation occurs at the 2’-position of that ring. Although there are no compounds in our series differing only at R2’, comparisons between similar compounds show that Cl substitutions have the greatest activity, followed by F and H. In addition, a halogen residue at the R4’ and an hydroxyl substitution at R3 increases inhibitory activity. An alkyl group at Rl or on the imidazole/triazole group of the 1,2-annelated BZ series enhances potency. For the R7 group, a Cl is preferable over an NO*. However dihalogenated compounds that are Cl substituted at R7 and R2’ are slightly less potent than the compound that is Cl substituted at R2’ and R4’ (Ro22 8349); monohalogenated compounds Cl substituted at R7 vs. R4’ (Ro5 5115 vs. diazepam) have equal potency. The carbonyl substituent at R2 of the 1,4-BZ series also enhances potency. In the resulting SAR, the importance of the halogensubstituted nonfused phenyl ring is highlighted; this SAR seems unique when compared to those series reported for other BZ sites and/or effects (1 O-l 2). In addition to the BZ SAR series, we studied a limited series of TH inhibitory compounds. TH compounds can act as substrates for the carrier protein and are transported into the ceils. These analogs have different

SARs Figure 1 diagrams the structure and nomenclature of the prototypic classical 1,4-BZ and L-T~ compounds. Table 1 lists the substitutions at sites within the BZ molecules of our BZ SAR series. The BZ potencies for inhibition, illustrated by -Log IC& values, are also listed

R3 I

HO

T3

i Fig. 1. Structure

Table 1. Structural

and Nomenclature

Components

BZ Triazolam Lormetazepam Prazepam (31) Lorazepam (32) Ro22 8349 Midazolam Delorazepam Ro5 4864 Temazepam (33) Flurazepam Ro5 5115 Oxazepam (34) Diazepam (35) PK 11195 Clonazepam (36) Alprazolam Nordiazepam Medazepam (37) Estazolam (38) Chlordiazepoxide (39) Flunitrazepam (40) Nitrazepam (41) Bromazepam (42) Ro5 3663 Flumazenil

of BZ and L-T~

of BZR

Ligands

Rl

fused

methylated CH3 CHPcyclopropyl H CH3 fused methylated H CH3 CHz CH&H2N(CH&H& CH3 H CH3 H H fused methylated H

fused

fused

triazolo ring

imidazo

R2

R3

ring 0 0 0 0 ring 0 0 0 0 0 0 0 b

H OH H OH H H H H OH H H OH H H H H H H H H H H H H H

0 ring 0 H CH3 triazolo ring, without CH3 H NHCHJ 0 CH3 H 0 H 0 H 0 imidazo ring + COO(C2H5) triazolo

Iv

Cl Cl Cl Cl H Cl Cl Cl Cl Cl H Cl Cl H Non Cl Cl Cl Cl Cl Non NOz Br H F

R2'

Cl Cl H Cl Cl F Cl H H F H H H Cl Cl H H H H H F H Nat2’ no C-ring, R5=CH3 no C-ring, R4=CH3,

R4'

H H H H Cl H H Cl H H Cl H H H H H H H H H H H H R5=0

-Log

7.38 7.35 6.69 6.54 6.38 6.25 6.12 5.91 5.83 5.82 5.60 5.53 5.50 5.05 4.95 4.94 4.86 4.44 4.37

I&o

+. SEM

+ 0.17 * 0.07 + 0.09 + 0.05 + 0.12 + 0.06 + 0.03 k 0.08 f 0.14 f 0.10 f 0.14 f 0.08 f 0.10 f 0.14 + 0.33 f 0.16 + 0.16 f 0.14 + 0.05 Br. The presence of large electronegative groups in the outer ring region opposite the BZ’s R2’ group severely decreases potency, as demonstrated by L-T~, rT3, and SKF 905050 (see Fig. 5). These data suggest that the distal end of molecule requires a very precisely oriented and sized fit into the active transporter site. The compounds diazepam, Ro22 8349, Ro5 4864, and Ro5 5115 demonstrate the importance of the halogen sites of BZ in our pharmacophore criteria. Compounds Cl substituted at R7 and R2’ have slightly less potency than compounds Cl substituted at R2’ and

Fig. 8. Stereo Views of the Model for Superimposition of L-T~ and the Active BZ, Prazepam. Revealing the Homology of the CH,-Cyclopropyl Substituent at Rl to the Alanine Side Chain on L-T~ Before fitting structures, the torsion angle of the nonfused phenyl ring of prazepam was rotated from -30” to -55” (these angles have equal heats of formation). Components fitted were (TH to BZ, respectively): 4’.OH to 4’-H, Cl’ to Cl ‘, 5-l to C7. C2 (TH inner ring) to C2 (diazepine ring), alanine CH, to CH? of Rl group, and alanine chiral carbon to base C of the Rl cyclopropyl structure. The resulting RMS fit is 1.02 A.

R4’; compounds Cl substrtuted at R7 vs. R4’ have equal potency. Hence the 2’-halogen may be slrghtly more influential than the R7 halogen In the dihalogenated system, and therefore this position was allowed to be frt less precisely than the other proposed pharmacophore sites.

CONCLUSION

These SAR data and the molecular modelrng of crystallographrcally derived structures suggest a pharmacophore for the T3 site of the putatrve membrane iodothyronine transporter of HepG2 cells. To produce a compound wrth moderate (0.1-l PM) inhibitory activity, at least three of the following structural components are needed (BZ/TH. respectively): 1) R2’-halogen/outer ring iodine; 2) R4’-halogen/outer ring 4’-OH; 3) R3-OH/ inner ring 3-l; 4) R7-halogen/inner ring 5-1, 5) Rl alkyl group or tnazole group/amino acid side chain with Lconfiguration of the primary amine, and 6) no halogen at the 5’-outer ring positron of TH. Potentially good BZs for T3 uptake inhibitors may include modifications of the BZ triazolam such as an additional 4’-hydroxyl group, a halogen (Cl or I) or a hydroxyl at R3, and an amino group on the tnazole’s methyl. To improve on lormetazepam, an alanine side chain could be put at the Rl positton consistent with the L-configuration, as well as adding an OH at the 4’-position of the nonfused phenyl ring. BZs likely to be ineffective would have a nonfused phenyl rrng with a 3’- or 2’-halogen in addition to a 5’or 6’-halogen along with substitution of either a halogen or an OH at R3. These substitutions then would fix the nonfused ring into an orthogonal conformation and thrust the transord-positioned halogen into the restricted zone. As well, an alanine side chain In the Dconfiguration at positron Rl or R2 would render the BZ ineffective. The binding and TH pharmacophorefor the T3 nuclear receptor emphasize the importance of a precise fit of analogs to the receptor actrve site (19-22). Ion pairing to the receptor is suggested by the ionrc side chain on one end and a polar phenolic-OH on the other. The nuclear receptor. though, does not distinguish between the L- or o-configuration of the side chain. The 3,5 substrtuents define the size of the lipophilic pockets as well as the stenc constraints that hold the conformation of the aromatic rings; binding is enhanced by polanzable groups. A lipophilrc residue of limited size IS required at the 3’-site and must be capable of being positioned distal to the nonphenolrc inner ring; here an isopropyl group binds better than an Iodine. The similarities of our model to that of the T3 nuclear receptor pharmacophore include the restrictive zone at the 5’-position (although our SAR seems more restrictive) and increased activity with halogen substttution at 3’,3,5positions. Our differences Include the improvement of inhibitory activity with halogen substitutron at the 3’,3,5sites relative to substitution with alkyl groups and the

lodothyronine

Transporter

Pharmacophore

absolute requirement of L-configuration at the chiral amino acid carbon. We did not test the importance of the 4’OH site or ether linkage substitutions in our brief SAR series, but the outer ring 4’-OH of TS does overlap the 4’-halogen of BZ in our model of BZ/TH homology. As well, our 3’-I is fit to the BZs while in the proximal position, although the distal conformation can just as easily fit BZs with a rotated C-ring. And as the SAR and isopotential map comparisons show, DIMIT and triac are not the best ligands for the iodothyronine site, but they are high affinity ligands for the nuclear receptor (20). Finally, TJ binding in isolated rat hepatic nuclei show no competition from up to 10 @I triazolam and lormetazepam (8). Perhaps the nuclear receptor requires a more precise fit of halogens at the 35 and 3’ positions when compared to the transporter site. BZs are a class of pharmaceutical agents that are primarily used as sedative-hypnotics and anxiolytics. The BZRs for these central nervous system actions are contained in the r-aminobutyric acid receptor chloride ion channel complex (23, 24). At least three subtypes of BZRs have been described that exist in different brain regions and subserve different physiologic functions (25, 26). There is also a BZR that is not linked to the y-aminobutyric acid-BZ-Cl ion channel complex, termed the “peripheral BZ receptor” (10, 23, 24) although it is present in the central nervous system. Several models of the central BZR pharmacophore have been proposed. Common features of these models include two proton-accepting groups separated by approximately 3.0-3.5 A (11). The condensed aromatic ring is also a common feature for all high affinity ligands, but it may not be involved directly in the binding site (11, 12). For inverse agonists of the central BZR, Allen et al. (27) suggest an aromatic ring system constrained to one plane and containing two hydrogen binding sites; the antagonists have long substituents that access regions above or below this aromatic plane. Of the compounds with classic BZ structure, central BZRs show stereoselectivity, are not affected by the R-l alkyl groups, and compounds with 4’-substitutions have diminished affinity (28). The peripheral BZR is distinguished by such varied selective ligands as 4’-Cl diazepam, the isoquinoline carboxamide derivative PK 11195, and protoporphyrins, all of which are devoid of activity at the central BZR (10). Of the classical BZ compounds, the peripheral site prefers an alkyl group of three carbons or less at position 1, a carbonyl at position 2, a 4’-halogen or methoxy group, and halogens at R2’ and R7; substitution at R3 or R4 reduces affinity (28). Our pharmacophore model shares some characteristics from both the central and peripheral BZR pharmacophores. Our model for the iodothyronine transporter includes the planar aromatic ring system with electronegative substituents. However, it requires an additional halogen-substituted phenyl ring oriented perpendicular to the plane of the aromatic system, and our sites for the electronegative substitutions include both the central BZR-preferred (7, 2’) and the peripheral

369

BZR-preferred (4’) positions, as well as at the central/ peripheral BZR-preferred R2, and the peripheral BZRaversive R3 position (although we have not yet determined its stereoselectivity for this substitution). Like the peripheral site, we limit alkyl substitution at Rl to one to three carbons in our model. The alkyl substitution of BZ may simulate the alanine side chain of TJ in our model of fit. Electrostatically, the substitution at the BZ Nl may delocalize the alkyl electrons into the ring system and allow for greater nucleophilicity at substitutions in the aromatic ring system. As well, the necessity of the carbonyl at R2 allows for an amide linkage to Nl and prevents its protonation and the localization of the alkyl electrons (28). The highly electronegative halogen and oxygen atoms at R3/R7/ R2’/R4’ of BZ form a pyrimidal pharmacophore that seems to correspond with the 3-l/5-l/3’-l/4’-OH substitutions of TS. These five points, defining a tilted crossbow formation, may provide proton acceptor sites (such as protonated lysine residues) for interaction with the iodothyronine transporter. The highly sensitive stereospecificity of the TH chiral carbon may suggest hydrogen bonding and/or ion pairing to the active site at the side chain (BZ-Rl) portion of the molecule.

MATERIALS

AND METHODS

Materials Tissue culture media and supplements were from Sigma Chemicals and GIBCO (Grand Island, NY). Our source for [‘251] TB, specific activity of 2200 Ci/mmol. was New England Nuclear, DuPont (Boston, MA). BZs were either purchased from Sigma (St. Louis, MO) or generously supplied from Drs. Peter Sorter (Hoffmann La Roche, Nutley, NJ) or David Triggle (State University of New York, Buffalo, NY). SKF compounds were a generous gift from Dr. Tony Underwood (Smith Kline Beecham, Welwyn, England). The source for the THs (highest purity available) L-T~, L-rT3, L-T~, L-T~, D-T~, and DIMIT was Henning/ Berlin (Berlin, Germany) and for D-T,, 3,5-~-T,, diiodotyrosine, D,L- and L-thyronine was Sigma Chemicals. 3,3,-~-T* was originally from Dr. E. C. Jorgensen. Our HepG2 cell line was a gift from Dr. B. Knowles. All other chemicals were from Sigma and Baker (Phillipsburg, NJ). Uptake

Assay

and lCso Values

for Inhibition

Our procedure to screen for inhibitors of labeled T3 uptake is as described (8,9). HepGP cells were grown in multiwell cluster plates in Dulbecco’s modified Eagle’s media low glucose with 10% fetal bovine serum. Cell suspensions were plated into 12to 24-well plates in equal volumes containing 1 04-1 O5 cells per well and grown to confluence (l-2 x 1 O6 cells/cm*) in 4-14 days. Before assay, wells were filled with Hank’s balanced salt solution plus the drugs or solvent control. To initiate the assay, [‘251]T3 was added to make a final concentration of 15 40 PM. At termination of the assay (30-60 min), wells were washed with a cold glycine buffer, hydrolyzed in NaOH. and then transferred to vials for counting in a r-counter. Each well is one of triplicates for each drug or solvent dose. Temperature-dependent uptake of [‘251]T3 was determined by subtraction of uptake at 4 C from the uptake at 22 C. The nonspecific binding/uptake was defined by uptake at 4 C incubation. I& values were derived from dose-response curves of individual experiments using a standard set of pharmacological statistical programs (29) implemented on an IBM personal computer.

MOL 390

ENDO.

Vol8

1994

Crystallographic

Databases

A search of the Cambridge crystallographic database (Quest 3D, Database 5.04, October 1992, Medical Foundation, Buffalo, NY) revealed crystal structures for 12 of the 25 BZs from our structure-activity series. Structures without available crystal data were created from modifications of the most homologous published BZ crystal structure using the SYBYL software package (Tripos Associates, St. Louis, MO). For example, triazolam was built from estazolam and lormetazepam from lorazepam. We used the thyroid analog crystal structures L-T~ and L-IT3 to build the remaining analogs of our TH series. All structures were energy minimized by using the MOPAC AM1 molecular orbital option in SYBYL on a Silicon Graphics work station (4D70GTB). We also explored the potential energy surface for rotation of the fused diazepine ring (A/B-rings) and the nonfused ring (C-ring). To do this, we started with the minimized structure and rotated the torsion angle between the fused A/B-rings and the nonfused C-ring in 10” increments form 0” to 180”. At each 10” increment a single point energy was calculated, without reoptimization of the structure. Least-Squares

dispensable technical assistance. Dr. Kragie also thanks Drs. Jack Mendelson and Nancy Mello of the Alcohol and Drug Abuse Research Research Center, McLean Hospital, Harvard Medical School, for their current support.

Received October 28, 1993. Revision received December 14,1993. Accepted December 22,1993. Address requests for reprints to: Dr. Laura Kragie, Biological Sciences, Cooke Hall, State University of New York at Buffalo, Buffalo, New York 14260-l 300. This work was supported by NIH Grants DK-01456 (to L.K.) and DK-41009 (to V.C.). Portions of this paper were presented at the 74th Annual Meeting of The Endocrine Society, San Antonio, TX, June 1992, and the 23rd National Medicinal Chemistry Symposium, Division of the American Chemical Society, Buffalo, NY, June 1992.

REFERENCES

Fit and VdW Volumes

Because the fused benzene ring represents the only conformationally stable component in the BZ ring system, conformational comparisons between BZ molecules were made by a least squares fit of the fused benzene ring in each BZ (of the same stereoisomer) using the root mean square (RMS) fit option in SYBYL. The BZs comprised two structural groups, the classical 1,4-BZs (e.g. diazepam) and the 1,2-annelated BZs (e.g. triazolam). The most (A) and least (L) active compounds from each group were superimposed through the RMS fit option. The SYBYL option Compare Volumes subtracted their VdW volumes. For the BZ group, L was subtracted from A to highlight the features unique to the active compounds. The volume in common with the least active compound was subtracted to form the active volume displays (excluded volume analysis). A similar methodology was used for thyroid hormone analogs. These THs were RMS fit by superimposing the ether linkage and both phenyl rings. However, because our TH SAR data suggested that the 5’substituents of the inactive compounds reduced potency, we subtracted A from L to highlight groups unique to the inactive compound. These regions were then avoided in the template for T1 fit. The TS molecule was fit to the VdW surfaces in common to the most active BZ molecules (27). This produced our first approximation of T3/BZ fit. We then went on to RMS fit the following atoms (TH to BZ): Cl’ to Cl’; R4’ to R4’; chiral carbon of the alanine side chain to Rl methyl; 5-l to C7; and 3-l to R3 (see Fig. 1). lsopotential

No. 3

Maps

We created the isopotential maps by using the Isopotential Map option of the SYBYL software package. This option uses the MOPAC geometry and the charges that were created when the structures were energy minimized. It computes and displays the isopotentials at discrete points of the molecule. We calculated the negative isopotential maps of the minimum energy conformers. The level of the surface of -5.0 kcal was used for the comparisons of Fig. 6, A, C, and D. For the comparisons between the charged conformers of L-T~ and DT3 (Fig. 6B), a level of -17 kcal was chosen for display (30).

Acknowledgments The authors thank Dr. Darrell Doyle for his sponsorship of Dr. Kragie and the financial support of Boots Pharmaceuticals (Lincolnshire, IL). Dr. David Smith provided assistance with the SYBYL software programs along with the Medical Foundation graphics department. Rich Smiehorowski provided in-

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