Introduction of C-alkyl branches to L-iminosugars changes their active site binding orientation.

L-ido-Deoxynojirimycin (L-ido-DNJ) itself showed no affinity for human lysosomal acid α-glucosidase (GAA), whereas 5-C-methyl-L-ido-DNJ showed a strong affinity for GAA, comparable to the glucose analog DNJ, with a Ki value of 0.060 µM. This excellent affinity for GAA and enzyme stabilization was observed only when methyl and ethyl groups were introduced. Docking simulation analysis revealed that the alkyl chains of 5-C-alkyl-L-ido-DNJs were stored in three different pockets, depending on their length, thereby the molecular orientation was changed. Comparison of the binding poses of DNJ and 5-C-methyl-L-ido-DNJ showed that they formed a common ionic interaction with Asp404, Asp518, and Asp616, but both the binding orientation and the distance between the ligand and each amino acid residue were different. 5-C-Methyl-L-ido-DNJ dose-dependently increased intracellular GAA activity in Pompe patient fibroblasts with the M519V mutation and also promoted enzyme transport to lysosomes. This study provides the first example of a strategy to design high-affinity ligands by introducing alkyl branches into rare sugars and L-sugar-type iminosugars to change the orientation of binding.

Strategy for Designing Selective Lysosomal Acid α-Glucosidase Inhibitors: Binding Orientation and Influence on Selectivity.

Deoxynojirimycin (DNJ) is the archetypal iminosugar, in which the configuration of the hydroxyl groups in the piperidine ring truly mimic those of d-glucopyranose; DNJ and derivatives have beneficial effects as therapeutic agents, such as anti-diabetic and antiviral agents, and pharmacological chaperones for genetic disorders, because they have been shown to inhibit α-glucosidases from various sources. However, attempts to design a better molecule based solely on structural similarity cannot produce selectivity between α-glucosidases that are localized in multiple organs and tissues, because the differences of each sugar-recognition site are very subtle. In this study, we provide the first example of a design strategy for selective lysosomal acid α-glucosidase (GAA) inhibitors focusing on the alkyl chain storage site. Our design of α-1-C-heptyl-1,4-dideoxy-1,4-imino-l-arabinitol (LAB) produced a potent inhibitor of the GAA, with an IC50 value of 0.44 µM. It displayed a remarkable selectivity toward GAA (selectivity index value of 168.2). A molecular dynamic simulation study revealed that the ligand-binding conformation stability gradually improved with increasing length of the α-1-C-alkyl chain. It is noteworthy that α-1-C-heptyl-LAB formed clearly different interactions from DNJ and had favored hydrophobic interactions with Trp481, Phe525, and Met519 at the alkyl chain storage pocket of GAA. Moreover, a molecular docking study revealed that endoplasmic reticulum (ER) α-glucosidase II does not have enough space to accommodate these alkyl chains. Therefore, the design strategy focusing on the shape and acceptability of long alkyl chain at each α-glucosidase may lead to the creation of more selective and practically useful inhibitors.

Characterizing the selectivity of ER α-glucosidase inhibitors.

The endoplasmic reticulum (ER) contains both α-glucosidases and α-mannosidases which process the N-linked oligosaccharides of newly synthesized glycoproteins and thereby facilitate polypeptide folding and glycoprotein quality control. By acting as structural mimetics, iminosugars can selectively inhibit these ER localized α-glycosidases, preventing N-glycan trimming and providing a molecular basis for their therapeutic applications. In this study, we investigate the effects of a panel of nine iminosugars on the actions of ER luminal α-glucosidase I and α-glucosidase II. Using ER microsomes to recapitulate authentic protein N-glycosylation and oligosaccharide processing, we identify five iminosugars that selectively inhibit N-glycan trimming. Comparison of their inhibitory activities in ER microsomes against their effects on purified ER α-glucosidase II, suggests that 3,7a-diepi-alexine acts as a selective inhibitor of ER α-glucosidase I. The other active iminosugars all inhibit α-glucosidase II and, having identified 1,4-dideoxy-1,4-imino-D-arabinitol (DAB) as the most effective of these compounds, we use in silico modeling to understand the molecular basis for this enhanced activity. Taken together, our work identifies the C-3 substituted pyrrolizidines casuarine and 3,7a-diepi-alexine as promising "second-generation" iminosugar inhibitors.

Design of a New α-1-C-Alkyl-DAB Derivative Acting as a Pharmacological Chaperone for ß-Glucocerebrosidase Using Ligand Docking and Molecular Dynamics Simulation.

Some point mutations in ß-glucocerebrosidase cause either improper folding or instability of this protein, resulting in Gaucher disease. Pharmacological chaperones bind to the mutant enzyme and stabilize this enzyme; thus, pharmacological chaperone therapy was proposed as a potential treatment for Gaucher disease. The binding affinities of α-1-C-alkyl 1,4-dideoxy-1,4-imino-d-arabinitol (DAB) derivatives, which act as pharmacological chaperones for ß-glucocerebrosidase, abruptly increased upon elongation of their alkyl chain. In this study, the primary causes of such an increase in binding affinity were analyzed using protein⁻ligand docking and molecular dynamics simulations. We found that the activity cliff between α-1-C-heptyl-DAB and α-1-C-octyl-DAB was due to the shape and size of the hydrophobic binding site accommodating the alkyl chains, and that the interaction with this hydrophobic site controlled the binding affinity of the ligands well. Furthermore, based on the aromatic/hydrophobic properties of the binding site, a 7-(tetralin-2-yl)-heptyl-DAB compound was designed and synthesized. This compound had significantly enhanced activity. The design strategy in consideration of aromatic interactions in the hydrophobic pocket was useful for generating effective pharmacological chaperones for the treatment of Gaucher disease.

In silico analyses of essential interactions of iminosugars with the Hex A active site and evaluation of their pharmacological chaperone effects for Tay-Sachs disease.

The affinity of a series of iminosugar-based inhibitors exhibiting various ring sizes toward Hex A and their essential interactions with the enzyme active site were investigated. All the Hex A-inhibiting iminosugars tested formed hydrogen bonds with Arg178, Asp322, Tyr421 and Glu462 and had the favorable cation-π interaction with Trp460. Among them, DMDP amide (6) proved to be the most potent competitive inhibitor with a Ki value of 0.041 µM. We analyzed the dynamic properties of both DMDP amide (6) and DNJNAc (1) in aqueous solution using molecular dynamics (MD) calculations; the distance of the interaction between Asp322 and 3-OH and Glu323 and 6-OH was important for stable interactions with Hex A, reducing fluctuations in the plasticity of the active site. DMDP amide (6) dose-dependently increased intracellular Hex A activity in the G269S mutant cells and restored Hex A activity up to approximately 43% of the wild type level; this effect clearly exceeded the border line treatment for Tay-Sachs disease, which is regarded as 10-15% of the wild type level. This is a significantly greater effect than that of pyrimethamine, which is currently in Phase 2 clinical trials. DMDP amide (6), therefore, represents a new promising pharmacological chaperone candidate for the treatment of Tay-Sachs disease.

Docking study and biological evaluation of pyrrolidine-based iminosugars as pharmacological chaperones for Gaucher disease.

We report on the synthesis and biological evaluation of a series of α-1-C-alkylated 1,4-dideoxy-1,4-imino-d-arabinitol (DAB) derivatives as pharmacological chaperones for Gaucher disease. The parent compound, DAB, did not show inhibition of human ß-glucocerebrosidase but showed moderate intestinal α-glucosidase inhibition; in contrast, extension of α-1-C-alkyl chain length gave a series of highly potent and selective inhibitors of the ß-glucocerebrosidase. Our design of α-1-C-tridecyl-DAB (5j) produced a potent inhibitor of the ß-glucocerebrosidase, with IC50 value of 0.77 µM. A molecular docking study revealed that the α-1-C-tridecyl group has a favorable interaction with the hydrophobic pocket and the sugar analogue part (DAB) interacted with essential hydrogen bonds formed to Asp127, Glu235 and Glu340. Furthermore, α-1-C-tridecyl-DAB (5j) displayed enhancement of activity at an effective concentration 10-times lower than isofagomine. α-1-C-Tridecyl-DAB therefore provides the first example of a pyrrolidine iminosugar as a new class of promising pharmacological chaperones with the potential for treatment of Gaucher disease.

Design and Synthesis of Labystegines, Hybrid Iminosugars from LAB and Calystegine, as Inhibitors of Intestinal α-Glucosidases: Binding Conformation and Interaction for ntSI.

This paper identifies the required configuration and orientation of α-glucosidase inhibitors, miglitol, α-1-C-butyl-DNJ, and α-1-C-butyl-LAB for binding to ntSI (isomaltase). Molecular dynamics (MD) calculations suggested that the flexibility around the keyhole of ntSI is lower than that of ctSI (sucrase). Furthermore, a molecular-docking study revealed that a specific binding orientation with a CH-π interaction (Trp370 and Phe648) is a requirement for achieving a strong affinity with ntSI. On the basis of these results, a new class of nortropane-type iminosugars, labystegines, hybrid iminosugars of LAB and calystegine, have been designed and synthesized efficiently from sugar-derived cyclic nitrones with intramolecular 1,3-dipolar cycloaddition or samarium iodide catalyzed reductive coupling reaction as the key step. Biological evaluation showed that our newly designed 3(S)-hydroxy labystegine (6a) inherited the selectivity against intestinal α-glucosidases from LAB, and its inhibition potency was 10 times better than that of miglitol. Labystegine, therefore, represents a promising new class of nortropane-type iminosugar for improving postprandial hyperglycemia.

Docking and SAR studies of calystegines: binding orientation and influence on pharmacological chaperone effects for Gaucher's disease.

We report on the identification of the required configuration and binding orientation of nor-tropane alkaloid calystegines against ß-glucocerebrosidase. Calystegine B2 is a potent competitive inhibitor of human lysosomal ß-glucocerebrosidase with Ki value of 3.3 µM. A molecular docking study revealed that calystegine B2 had a favorable van der Waals interactions (Phe128, Trp179, and Phe246) and the hydrogen bonding (Glu235, Glu340, Asp127, Trp179, Asn234, Trp381 and Asn396) was similar to that of isofagomine. All calystegine isomers bound into the same active site as calystegine B2 and the essential hydrogen bonds formed to Asp127, Glu235 and Glu340 were maintained. However, their binding orientations were obviously different. Calystegine A3 bound to ß-glucocerebrosidase with the same orientations as calystegine B2 (Type 1), while calystegine B3 and B4 had different binding orientations (Type 2). It is noteworthy that Type 1 orientated calystegines B2 and A3 effectively stabilized ß-glucocerebrosidase, and consequently increased intracellular ß-glucocerebrosidase activities in N370S fibroblasts, while Type 2 orientated calystegines B3 and B4 could not keep the enzyme activity. These results clearly indicate that the binding orientations of calystegines are changed by the configuration of the hydroxyl groups on the nor-tropane ring and the suitable binding orientation is a requirement for achieving a strong affinity to ß-glucocerebrosidase.

The antiviral drug acyclovir is a slow-binding inhibitor of (D)-amino acid oxidase.

d-Amino acid oxidase (DAO) is a degradative enzyme that is stereospecific for d-amino acids, including d-serine and d-alanine, which are believed to be coagonists of the N-methyl-d-aspartate (NMDA) receptor. To identify a new class of DAO inhibitor(s) that can be used to elucidate the molecular details of the active site environment of DAO, manifold biologically active compounds of microbial origin and pre-existing drugs were screened for their ability to inhibit DAO activity, and several compounds were identified as candidates. One of these compounds, acyclovir (ACV), a well-known antiviral drug used for the treatment of herpesvirus infections, was characterized and evaluated as a novel DAO inhibitor in vitro. Analysis showed that ACV acts on DAO as a reversible slow-binding inhibitor, and interestingly, the time required to achieve equilibrium between DAO, ACV, and the DAO/ACV complex was highly dependent on temperature. The binding mechanism of ACV to DAO was investigated in detail by several approaches, including kinetic analysis, structural modeling of DAO complexed with ACV, and site-specific mutagenesis of an active site residue postulated to be involved in the binding of ACV. The results confirm that ACV is a novel, active site-directed inhibitor of DAO that can be a valuable tool for investigating the structure-function relationships of DAO, including the molecular details of the active site environment of DAO. In particular, it appears that ACV can serve as an active site probe to study the structural basis of temperature-induced conformational changes of DAO.

Design and Pharmacological Chaperone Effects of N-(4'-Phenylbutyl)-DAB Derivatives Targeting the Lipophilic Pocket of Lysosomal Acid α-Glucosidase.

This study provides the first example of a strategy to design a practical ligand toward lysosomal acid α-glucosidase (GAA) focusing on N-alkyl derivatives of 1,4-dideoxy-1,4-imino-d-arabinitol (DAB). The optimized N-4'-(p-trifluoromethylphenyl)butyl-DAB (5g) showed a Ki value of 0.73 µM, which was 353-fold higher affinity than N-butyl-DAB (3f) without a terminal phenyl group. Docking analysis showed that the phenyl part of 5g was accommodated in a lipophilic pocket. Furthermore, the p-trifluoromethyl group effectively suppresses the fluctuation of the phenyl group, allowing it to produce a stable bonding form with GAA. 5g increased the midpoint of the protein's protein denaturation temperature (Tm) by 6.6 °C above that in the absence of the ligand and acted as a "thermodynamic stabilizer" to improve the thermal stability of rhGAA. 5g dose-dependently increased intracellular GAA activities in Pompe patient's fibroblasts with the M519V mutation; its effect was comparable to that of DNJ, which is under clinical trials.

Brazilian propolis-derived components inhibit TNF-α-mediated downregulation of adiponectin expression via different mechanisms in 3T3-L1 adipocytes.

BACKGROUND: Previous reports suggest that Brazilian propolis has multiple biological functions and may help to restore adiponectin expression and insulin sensitivity. However, little is known about the molecular mechanisms by which these compounds inhibit the downregulation of adiponectin. METHODS: The effect of various Brazilian propolis-derived components on inhibition of tumor necrosis factor-α (TNF-α)-mediated downregulation of adiponectin expression in 3T3-L1 adipocytes and molecular mechanism was investigated. RESULTS AND CONCLUSIONS: Pretreatment with either artepillin C (C3) or its derivative (C4) significantly inhibited TNF-α-mediated downregulation of adiponectin expression in 3T3-L1 adipocytes. Interestingly, C3 strongly activated peroxisome proliferator-activated receptor γ (PPARγ) transcriptional activity. Treatment of adipocytes with C3 resulted in the upregulation of adiponectin and fatty acid-binding protein 4 expression, but C4 did not significantly induce PPARγ transactivation. C4 did, however, inhibit the TNF-α-induced c-Jun-NH(2)-terminal kinase (JNK) signaling that is involved in adiponectin expression. Molecular docking studies based on hPPARγ with C3 and JNK1 with C4 clearly supported our experimental results. These data demonstrate that 1) both C3 and C4 significantly inhibit the TNF-α-mediated downregulation of adiponectin in adipocytes, 2) C3 functions as a PPARγ agonist, and its inhibition of the effect of TNF-α is due to this PPARγ transactivation, and 3) C4 is an effective inhibitor of JNK activation, thus inhibiting the TNF-α-mediated downregulation of adiponectin. GENERAL SIGNIFICANCE: Brazilian propolis-derived components (C3 and C4) can significantly inhibit TNF-α-mediated downregulation of adiponectin in adipocytes, although they do so via different mechanisms.

Design, synthesis and in vitro evaluation of a series of α-substituted phenylpropanoic acid PPARγ agonists to further investigate the stereochemistry-activity relationship.

We previously demonstrated that the α-benzylphenylpropanoic acid-type PPARγ-selective agonist 6 exhibited a reversed stereochemistry-activity relationship, that is, the (R)-enantiomer is a more potent PPARγ agonist than the (S)-enantiomer, compared with structurally similar α-ethylphenylpropanoic acid-type PPAR agonists. Here, we designed, synthesized and evaluated the optically active α-cyclohexylmethylphenylpropanoic acid derivatives 7 and α-phenethylphenylpropanoic acid derivatives 8, respectively. Interestingly, α-cyclohexylmethyl derivatives showed reversal of the stereochemistry-activity relationship [i.e., (R) more potent than (S)], like α-benzyl derivatives, whereas α-phenethyl derivatives showed the 'normal' relationship [(S) more potent than (R)]. These results suggested that the presence of a branched carbon atom at the ß-position with respect to the carboxyl group is a critical determinant of the reversed stereochemistry-activity relationship.

5-C-Branched Deoxynojirimycin: Strategy for Designing a 1-Deoxynojirimycin-Based Pharmacological Chaperone with a Nanomolar Affinity for Pompe Disease.

In recent years, the function of pharmacological chaperones as a "thermodynamic stabilizer" has been attracting attention in combination therapy. The coadministration of a pharmacological chaperone and recombinant human acid α-glucosidase (rhGAA) leads to improved stability and maturation by binding to the folded state of the rhGAA and thereby promotes enzyme delivery. This study provides the first example of a strategy to design a high-affinity ligand toward lysosomal acid α-glucosidase (GAA) focusing on alkyl branches on 1-deoxynojirimycin (DNJ); 5-C-heptyl-DNJ produced a nanomolar affinity for GAA with a Ki value of 0.0047 µM, which is 13-fold more potent than DNJ. The protein thermal shift assay revealed that 10 µM 5-C-heptyl-DNJ increased the midpoint of the protein denaturation temperature (Tm) to 73.6 °C from 58.6 °C in the absence of the ligand, significantly improving the thermal stability of rhGAA. Furthermore, 5-C-heptyl-DNJ dose dependency increased intracellular GAA activities in Pompe patient's fibroblasts with the M519V mutation. The introduction of C5 alkyl branches on DNJ provides a new molecular strategy for pharmacological chaperone therapy for Pompe disease, which may lead to the development of higher-affinity and practically useful chaperones.

Structure-based design, synthesis, and nonalcoholic steatohepatitis (NASH)-preventive effect of phenylpropanoic acid peroxisome proliferator-activated receptor (PPAR) α-selective agonists.

A series of α-ethylphenylpropanoic acid derivatives was prepared as candidate peroxisome proliferator-activated receptor (PPAR) α-selective agonists, based on our PPARα/δ dual agonist 3 as a lead compound. Structure-activity relationship studies clearly indicated that the steric bulkiness and position of the distal hydrophobic tail part are critical for PPARα agonistic activity and PPARα selectivity, as had been predicted from a molecular-modeling study. A representative compound blocked the progression of nonalcoholic steatohepatitis (NASH) in an animal model.

Docking and SAR studies of D- and L-isofagomine isomers as human ß-glucocerebrosidase inhibitors.

We report the structure-activity relationship of a series of D-, and L-isofagomine and fagomine isomers as glycosidase inhibitors. Our study revealed that a positive charge at the anomeric position of d-isofagomines enhanced the potency toward ß-glycosidases, while the epimerization at the C3 OH group drastically reduced their inhibitory potency by over three orders of magnitude. Furthermore, d-3,4-di-epi-isofagomine abolished their inhibition activities against all enzymes. L-Isofagomine was also a fairly potent inhibitor of human ß-glucocerebrosidase, with an IC50 value of 8.7 µM. A molecular docking study revealed that the positions and orientations of the piperidine ring of D-3-epi-isofagomine in the binding site was similar to that of D-isofagomine, while D-3-epi-isofagomine missed the hydrogen bond interactions between Asp127 and the 3-OH group and between Trp179 and the 3-OH group. Furthermore, the top 10 docking models ranked by IFDscore suggested that D-3,4-di-epi-isofagomine can not bind to ß-glucocerebrosidase at a stable interaction mode. These results provide an insight into the structural requirements of isofagomine isomers for developing a new type of pharmacological chaperone for Gaucher disease.

[Design and synthesis of peroxisome proliferator-activated receptor (PPAR) delta agonists and its implication to the driving force to elicit PPAR delta selectivity].

A series of 3-(4-alkoxypheny)propanoic acid derivatives was prepared as candidate peroxisome proliferator-activated receptor (PPAR) delta-selective agonists, based on our previously discovered potent human PPARalpha/delta dual agonist TIPP-401 as a lead compound. Structure-activity relationship studies clearly indicated the importance of the chain length of the alkoxy group at the 4-position, and the n-butoxy compound exhibited the most potent PPARdelta transactivation activity and highest PPARdelta selectivity. The (S)-enantiomer of a representative compound (TIPP-204) exhibited extremely potent PPARdelta transactivation activity, comparable to that of the known PPARdelta-selective agonist GW-501516. To understand why TIPP-204 shows high selectivity for hPPARdelta among hPPAR subtypes, and why TIPP-401, a structurally related compound, is a hPPARalpha/delta dual agonist, computational docking of TIPP-401 to the ligand binding domains of hPPARalpha and hPPARdelta and X-ray structure analysis of TIPP-204-hPPARdelta ligand binding domain were carried out. The results allowed identification of certain amino acids as putative determinants of the hPPARdelta selectivity of TIPP-204. To confirm the significance of these amino acids, GAL4-fusion proteins of mutated hPPARdeltas and hPPARalphas were prepared, and the transactivation activity of TIPP-204 toward the mutants was evaluated. The amino acid(s) that predominantly influence the potency and selectivity of TIPP-204 are different from that of the well-known PPARdelta-selective agonist GW-501516, which belongs to a different chemical class. The significance of these amino acids was confirmed by the examination of the complex structure between TIPP-204 and hPPARdelta. The results revealed several interactions relevant to the hPPARdelta-selectivity of the two ligands and will be useful for logical hPPARdelta ligand design.

[Identifying Inhibitors of USP7-HDM2 Protein-Protein Interaction (PPI) by the in Silico Fragment-mapping Method].

Proteolysis mediated by the ubiquitin-proteome system plays an important role in cancer. Recently, a deubiquitinating enzyme, ubiquitin-specific protease 7 (USP7) has attracted attention as a key regulator of the p53-human double minute 2 (HDM2) pathway in cancer cells. Although some USP7 enzyme inhibitors have been identified, issues related to activity and selectivity prevent their therapeutic application. In this study, we aimed to search for novel USP7-HDM2 protein-protein interaction (PPI) inhibitors that do not affect the USP7 enzyme activity. Using the fragment-mapping program Fsubsite and the canonical subsite-fragment database (CSFDB) developed in our laboratory, we mapped a variety of fragments onto USP7 protein and constructed 3D-pharmacophore models based on the arrangement patterns of the mapped fragments. Finally, we performed 3D pharmacophore-based virtual screening of a commercial compound database and successfully selected promising USP7-HDM2 PPI inhibitor candidates.

In silico analyses of the effects of a point mutation and a pharmacological chaperone on the thermal fluctuation of phenylalanine hydroxylase.

Phenylketonuria (PKU) is an inborn error of phenylalanine metabolism due to mutations in phenylalanine hydroxylase (PAH). Recently, small compounds, known as pharmacological chaperones (PhCs), have been identified that restore the enzymatic activity of mutant PAHs. Understanding the mechanism of the reduction in enzymatic activity due to a point mutation in PAH and its restoration by PhC binding is important for the design of more effective PhC drugs. Thermal fluctuations of an enzyme can alter its activity. Here, molecular dynamics simulation show the thermal fluctuation of PAH is increased by introduction of the A313T mutation. Moreover, a simulation using the A313T-PhC complex model was also performed. Thermal fluctuation of the mutant was found to be reduced upon PhC binding, which contributes to restoring its enzymatic activity.

Multi-step virtual screening to develop selective DYRK1A inhibitors.

Developing selective inhibitors for a particular kinase remains a major challenge in kinase-targeted drug discovery. Here we performed a multi-step virtual screening for dual-specificity tyrosine-phosphorylation-regulated kinase 1A (DYRK1A) inhibitors by focusing on the selectivity for DYRK1A over cyclin-dependent kinase 5 (CDK5). To examine the key factors contributing to the selectivity, we constructed logistic regression models to discriminate between actives and inactives for DYRK1A and CDK5, respectively, using residue-based binding free energies. The residue-based parameters were calculated by molecular mechanics-generalized Born surface area (MM-GBSA) decomposition methods for kinase-ligand complexes modeled by computer ligand docking. Based on the findings from the logistic regression models, we built a three-dimensional (3D) pharmacophore model and chose filter criteria for the multi-step virtual screening. The virtual hit compounds obtained from the screening were assessed for their inhibitory activities against DYRK1A and CDK5 by in vitro assay. Our screening identified two novel selective DYRK1A inhibitors with IC50 values of several µM for DYRK1A and >100µM for CDK5, which can be further optimized to develop more potent selective DYRK1A inhibitors.

Three-dimensional quantitative structure-activity relationship analysis for human pregnane X receptor for the prediction of CYP3A4 induction in human hepatocytes: structure-based comparative molecular field analysis.

The pregnane X receptor [PXR (NR1I2)] induces the expression of xenobiotic metabolic genes and transporter genes. In this study, we aimed to establish a computational method for quantifying the enzyme-inducing potencies of different compounds via their ability to activate PXR, for the application in drug discovery and development. To achieve this purpose, we developed a three-dimensional quantitative structure-activity relationship (3D-QSAR) model using comparative molecular field analysis (CoMFA) for predicting enzyme-inducing potencies, based on computer-ligand docking to multiple PXR protein structures sampled from the trajectory of a molecular dynamics simulation. Molecular mechanics-generalized born/surface area scores representing the ligand-protein-binding free energies were calculated for each ligand. As a result, the predicted enzyme-inducing potencies for compounds generated by the CoMFA model were in good agreement with the experimental values. Finally, we concluded that this 3D-QSAR model has the potential to predict the enzyme-inducing potencies of novel compounds with high precision and therefore has valuable applications in the early stages of the drug discovery process.