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Background Cyclopyrimorate is a highly effective bleaching herbicide discovered by Mitsui Chemicals Agro, Inc. The target site was recently reported to be homogentisate solanesyltransferase (HST) in the plastoquinone (PQ) biosynthesis pathway on the basis of the number of intermediates in cyclopyrimorate-treated plants and in vitro HST assays. Here, the target site of cyclopyrimorate was further explored using both in vivo and in vitro experiments. Results The cyclopyrimorate-dependent bleaching effect on Arabidopsis thaliana was reversed by decyl PQ, suggesting that this symptom is attributable to the inhibition of PQ biosynthesis. Furthermore, homogentisate (HGA), a substrate of HST, weakly reversed the bleaching effect of cyclopyrimorate in a dose-dependent manner. We expected that the weak reversal by HGA was due to competitive inhibition by cyclopyrimorate or des-morpholinocarbonyl cyclopyrimorate (DMC), a metabolite of cyclopyrimorate in plants that exhibit higher HST-inhibitory activity as compared to cyclopyrimorate. Kinetic analysis was therefore conducted using DMC. DMC inhibited HST competitively with respect to HGA, and was a mixed non-competitive inhibitor with respect to the other substrate, farnesyl diphosphate. Moreover, neither cyclopyrimorate nor DMC inhibited 2-methyl-6-phytyl-1,4-benzoquinone/2-methyl-6-solanesyl-1,4-benzoquinone methyltransferase, which is located downstream of HST in the PQ biosynthesis pathway. Conclusions The target site of cyclopyrimorate and DMC is HST, which is a novel target site for commercial herbicides. © 2019 Society of Chemical Industry.

Imidacloprid has been used to control one of most serious pests, Bemisia tabaci. However, B. tabaci has developed imidacloprid resistance mainly by over-expressing CYP6CM1. It was reported that imidacloprid-resistant B. tabaci showed no or low level of cross-resistance against dinotefuran. Here, we expressed CYP6CM1 variants using Sf9/baculovirus and/or Drosophila S2 cells and showed that CYP6CM1 variants metabolized imidacloprid but not dinotefuran. In addition, we demonstrated that imidacloprid and pymetrozine competed for a CYP6CM1 variant more efficiently than dinotefuran, using a luminescent substrate competition assay. These results suggest that lack of metabolic activity of CYP6CM1 variants against dinotefuran caused no or low level of cross-resistance.

The peach-potato aphid, Myzus persicae, is a serious crop pest that has developed imidacloprid resistance, mainly through overexpression of CYP6CY3. Here, we established a metabolic assay using Drosophila S2 cells that stably expressed CYP6CY3. We found that CYP6CY3 showed metabolic activity against imidacloprid, as well as acetamiprid, clothianidin, and thiacloprid, but had no activity against dinotefuran. Our study suggested that stable gene expression in Drosophila S2 cells is useful for examining which insecticide is metabolized by P450 monooxygenases.

Meta-diamide insecticides including broflanilide have a high insecticidal activity by acting on RDL GABA receptors. Both membrane potential assays and docking studies suggest that the target site of meta-diamides is different from that of conventional noncompetitive inhibitors, such as fipronil. In fact, meta-diamides are effective against cyclodiene- and fipronil-resistant pests that carry target-site mutations. Dinotefuran uniquely possesses a tetrahydrofuran ring, whereas other neonicotinoids possess aromatic rings. Moreover, dinotefuran has been reported to be effective against imidacloprid-resistant strains. A docking study predicted the weak binding of dinotefuran to cytochrome P450s which are associated with imidacloprid resistance. Metabolic assays revealed that dinotefuran was not metabolized by these cytochrome P450s. These findings suggest that the lack of metabolic activity of P450s against dinotefuran causes a low level of cross-resistance.

Cyclopyrimorate invented by Mitsui Chemicals Agro, Inc. is a novel bleaching herbicide effective against a broad range of rice weeds, including biotypes resistant to acetolactate synthase inhibitors. It was discovered through structural modifications of a lead compound, which was designed from partial substructures of pyridazine herbicides, such as credazine and pyridafol. The target site of cyclopyrimorate was demonstrated to be homogentisate solanesyltransferase, a novel target site for commercial herbicides. Cyclopyrimorate will be on the market, thus offering a new strategy for the management of herbicide resistance. In this chapter the discovery, optimization, and mode of action of cyclopyrimorate are reviewed.

Broflanilide shows high insecticidal activity against various pests, including lepidopteran, coleopteran, and thysanopteran. The meta-diamide structure of broflanilide was discovered by performing major modifications of the lead compound, flubendiamide, which is known to act as ryanodine receptor modulator. After the optimization of meta-diamides, broflanilide was selected as a development compound. In contrary to flubendiamide, broflanilide acts as a GABA-gated chloride channel allosteric modulator. Its action site was suggested to be different from that of conventional GABA-gated chloride channel blockers, such as dieldrin and fipronil. Broflanilide controls pest susceptible and resistant to fipronil, furthermore it is effective against flubendiamide-resistant diamondback moths indicating that there is no cross-resistance concern for broflanilide with other conventional insecticides.

BACKGROUND Insect Rdl GABA receptor is an important insecticide target. To design a novel insecticide, studies on the structures of homologous pentameric ligand-gated ion channels provide information about important amino acids that are necessary for the function of insect Rdl GABA receptors. RESULTS L9'A, T12'A, T13'A, T13'S, M15'S, and M15'N mutations in the Drosophila Rdl GABA receptor subunit caused the protein to spontaneously adopt the open state conformation. In contrast, the S16'A, S16'T, S17'A, and S17'H mutant homomers showed the same levels of agonist and antagonist sensitivity as the wild-type receptor. The G336M mutation in the Drosophila Rdl GABA receptor abolished the agonist activities of ivermectin and milbemectin, but the F339M mutation did not. Additionally, the F339M mutation caused spontaneous opening of the receptor. In the Drosophila Rdl model, the hydrophobic girdle plays an important role in stabilization of the closed state. Mutations which decrease hydrophobic interactions resulted in spontaneous opening, supporting the importance of the hydrophobic girdle for keeping the channel closed. Through a mutational study of transmembrane 3 (TM3) cytoplasmic domain and Rdl GABA receptor modeling, hydrophobic interactions between TM3 and TM4 and intersubunit interaction were demonstrated to be important for channel gating. Alternatively, the intrasubunit interaction between TM2 and TM3 domains were less important for channel gating in case of Drosophila Rdl GABA receptor. CONCLUSION This study demonstrates important amino acids critical to the function of the Drosophila Rdl GABA receptor based on the mutational studies and Drosophila Rdl GABA receptor modeling approach. © 2020 Society of Chemical Industry.

BACKGROUND Meta-diamides [3-benzamido-N-(4-(perfluoropropan-2-yl)phenyl)benzamides] show high insecticide activity by acting as antagonists to the insect resistance to dieldrin (RDL) γ-aminobutyric acid (GABA) receptors. In contrast, low-level antagonist activities of meta-diamides have been demonstrated against the human GABA type A receptor (GABAA R) α1β2γ2S, mammalian GABAA R α1β3γ2S, and the human glycine receptor (GlyR) α1β. Glycine residue 336 in the membrane-spanning region M3 of the Drosophila RDL GABA receptor is essential for its high sensitivity to meta-diamide 7, [3-benzamido-N-(2-bromo-4-(perfluoropropan-2-yl)-6-(trifluoromethyl)phenyl)-2-fluorobenzamide]. RESULTS We examined the effects of an equivalent mutation (M288G) in spontaneously opened human GABAA R β3 homomers using membrane potential assay. Picrotoxin and fipronil blocked spontaneously opened human GABAA Rs β3 and β3-M286G in a concentration-dependent manner. In contrast, meta-diamide 7 did not block spontaneously opened GABAA R β3 homomers, although meta-diamide 7 blocked spontaneously opened GABAA R β3-M286G homomers. In addition, inhibitory potency of meta-diamide 7 for GABA-induced membrane potential change in cells expressing GABAA R α1β3-M286G was much higher than that in cells expressing GABAA R α1β3. In the same way, the equivalent mutation (A288G) in GlyR α1 increased the inhibitory potency of meta-diamide 7 for GlyRs α1 and α1β. CONCLUSION Studies substituting an equivalent mutation (M288G) in spontaneously opening human GABAA R β3 homomers and human GABAA Rs α1β3 heteromers suggest that M286 in human GABAA R β3 is important for the low sensitivity to meta-diamide 7. In this study, we summarize the mechanisms underlying the selectivity of meta-diamides between insect RDL and human GABA and glycine receptors. © 2020 Society of Chemical Industry.

Imidacloprid is very effective in controlling Nilaparvata lugens Stal, which severely damages rice plants. Following heavy imidacloprid use, imidacloprid-resistant N. lugens, which showed cross-resistance to other neonicotinoids, appeared. We used the baculovirus/Sf9 expression system to express CYP6ER1 variants carrying A375del + A376G (del3) mutations, either with or without T318S mutation, which confer imidacloprid resistance in N. lugens. These CYP6ER1 variants metabolized imidacloprid but did not metabolize dinotefuran. Moreover, Drosophila expressing a CYP6ER1 variant carrying T318S + del3 mutations were resistant to imidacloprid, with a resistance ratio of 288.7, whereas the resistance ratio to dinotefuran was 3.6. These findings indicate that N. lugens has a low level of resistance to dinotefuran, and the increase of resistance is slow. We also studied the metabolism of other neonicotinoids, as well as sulfoxaflor and flupyradifurone, by CYP6ER1 variants carrying del3 mutations, either with or without the T318S mutation. Sulfoxaflor, was not metabolized by either CYP6ER1-del3 or CYP6ER1-T318Sdel3 variants. However, these variants did metabolize flupyradifurone. This study sheds light on the substrate selectivity of CYP6ER1 variants.

Dinotefuran (DTF) is a systemic neonicotinoid insecticide characterized by a tetrahydrofuran ring. In the present study, we examined the characteristics of DTF binding to native nicotinic acetylcholine receptors (nAChRs) expressed in the American cockroach Periplaneta americana using radioligand-binding methods. The Scatchard analysis, using [3H]imidacloprid (IMI), indicated that IMI has a single class of high-affinity binding sites in the P. americana nerve cord. In contrast, the Scatchard analysis using [3H]DTF indicated that DTF has two different classes of binding sites. Both DTF and IMI were found to bind to one of the classes, for which DTF showed low affinity. The other class, for which DTF showed high affinity, was localized in the abdominal nerve cord but not in the thoracic nerve cord. IMI showed low affinity for the high-affinity DTF binding sites. Our data suggest that DTF binds with high affinity to a nAChR subtype distinct from the high-affinity subtype for IMI. This difference might be responsible, at least in part, for the difference in resistance development to DTF and IMI in P. americana.

Broflanilide (1), discovered by Mitsui Chemicals Agro, Inc., has a unique chemical structure characterized as a meta-diamide and exhibits high activity against various pests, including Lepidopteran, Coleopteran, and Thysanopteran pests. Because broflanilide has a novel mode of action, the Insecticide Resistance Action Committee (IRAC) categorized it as a member of a new group: Group 30. The meta-diamide structure was generated via drastic structural modification of a lead compound, flubendiamide (2), and the subsequent structural optimization of meta-diamides on each of its three benzene rings led to the discovery of broflanilide. In the present study, the details of the generation of meta-diamides from the lead compound and the structural optimization of meta-diamides are described.

Insecticide resistance has been and continues to be a significant problem for invertebrate pest control. As such, effective insecticide resistance management (IRM) is critical to maintain the efficacy of current and future insecticides. A technical group within CropLife International, the Insecticide Resistance Action Committee (IRAC) was established 35 years ago (1984) as an international association of crop protection companies that today spans the globe. IRAC's focus is on preserving the long-term utility of insect, mite, and most recently nematode control products through effective resistance management to promote sustainable agriculture and improved public health. A central task of IRAC has been the continual development and documentation of the Mode of Action (MoA) Classification scheme, which serves as an important tool for implementing IRM strategies focused on compound rotation / alternations. Updates to the IRAC MoA Classification scheme provide the latest information on the MoA of current and new insecticides and acaricides, and now includes information on biologics and nematicides. Details for these new changes and additions are reviewed herein.

The action mechanism of cyclopyrimorate, a novel herbicide for weed control in rice fields, was investigated. Cyclopyrimorate caused bleaching symptoms in Arabidopsis thaliana similar to those caused by existing carotenoid biosynthesis inhibitors, mesotrione and norflurazon. However, cyclopyrimorate treatment resulted in significant accumulation of homogentisate and a reduction in the level of plastoquinone. A metabolite of cyclopyrimorate, des-morpholinocarbonyl cyclopyrimorate (DMC), was detected in plants. These data suggested that cyclopyrimorate and/or DMC inhibit homogentisate solanesyltransferase (HST), a downstream enzyme of 4-hydroxyphenylpyruvate dioxygenase in the plastoquinone biosynthesis pathway. In vitro assays showed that A. thaliana HST was strongly inhibited by DMC and weakly by cyclopyrimorate, whereas other commercial bleaching herbicides did not inhibit HST. DMC derivatives showed a positive correlation between HST inhibition and in vivo bleaching activities. These results indicate that the target site of cyclopyrimorate and DMC is HST, a novel target site of commercial herbicides.

Broflanilide is a meta-diamide [3-benzamido-N-(4-(perfluoropropan-2-yl)phenyl)benzamide] that exhibits high larvicidal activity against Spodoptera litura. It has been suggested that broflanilide is metabolized to desmethyl-broflanilide and that it acts as a noncompetitive resistant-to-dieldrin (RDL) γ-aminobutyric acid (GABA) receptor antagonist. The binding site of desmethyl-broflanilide was demonstrated to be distinct from that of conventional noncompetitive antagonists such as fipronil. It has been proposed that the site of action for desmethyl-broflanilide is close to G336 in the M3 region of the Drosophila RDL GABA receptor. However, although the site of action for desmethyl-broflanilide appears to overlap with that of macrocyclic lactones, different modes of actions have been demonstrated for desmethyl-broflanilide and the macrocyclic lactones. The mechanisms underlying the high selectivity of meta-diamides are also discussed in this review. Broflanilide is expected to become a prominent insecticide because it is effective against pests with resistance to cyclodienes and fipronil.

Meta-diamides [3-benzamido-N-(4-(perfluoropropan-2-yl)phenyl)benzamides] are a distinct class of RDL GABA receptor noncompetitive antagonists showing high insecticidal activity against Spodoptera litura. The mode of action of the meta-diamides was demonstrated to be distinct from that of conventional noncompetitive antagonists (NCAs) such as fipronil, picrotoxin, lindane, dieldrin, and α-endosulfan. It was suggested that meta-diamides act at or near G336 in the M3 region of the Drosophila RDL GABA receptor. Although the site of action of the meta-diamides appears to overlap with that of macrocyclic lactones including avermectins and milbemycins, differential effects of mutations on the actions of the meta-diamides and the macrocyclic lactones were observed. Molecular modeling studies revealed that the meta-diamides may bind to an inter-subunit pocket near G336 in the Drosophila RDL GABA receptor better when in the closed state, which is distinct from the NCA-binding site, which is in a channel formed by M2s. In contrast, the macrocyclic lactones were suggested to bind to an inter-subunit pocket near G336 in the Drosophila RDL GABA receptor when in the open state. Furthermore, mechanisms underlying the high selectivity of meta-diamides are discussed. This minireview highlights the unique features of novel meta-diamide insecticides and demonstrates why meta-diamides are anticipated to become prominent insecticides that are effective against pests resistant to cyclodienes and fipronil.

The target site of tolprocarb has been reported to be polyketide synthase (PKS). Here, we evaluated the activities for Pyricularia oryzae PKS and melanin biosynthesis as well as the control efficacy of rice blast using a series of tolprocarb derivatives. A comparison of the inhibitory activities of PKS and melanin biosynthesis revealed a linear relationship (r2=0.90), confirming PKS as the target site of tolprocarb. A compound beyond this relationship was metabolized by P. oryzae to an inactive compound. The control efficacy of rice blast was explained using the melting point and either the inhibitory activity of PKS or melanin biosynthesis. Structure-activity analysis revealed that both end parts of tolprocarb preferred hydrophobic groups, and the chirality of the substituent in the middle part significantly influenced the activities. These relationships will provide useful information for the development of novel PKS inhibitors.

Novel tricyclic 3,3a,5,9b-tetrahydro-2H-furo[3,2-c][2]benzopyran (TFB) derivatives were synthesized, and their herbicidal activities were elucidated. They were synthesized from D-glucose as a natural chiral source. The formation of the TFB skeleton was achieved by a Friedel-Crafts type intramolecular cyclization of methyl 5-deoxy-2,3-O-dibenzyl-5-C-methyl-D-xylofranosides. The intramolecular cyclization was dependent upon the electronic effects of the substituents at the C-2 benzyloxy group of methyl xylofranosides. Some TFBs exhibited a remarkable herbicidal activity to annual paddy weeds, such as Echinochloa sp, without injury to the rice.

The λ-dynamics approach to free energy simulations has been applied the host−guest system of β-cyclodextrin interacting with a series of monosubstituted benzenes. Each ligand was explicitly represented, and restraining potentials [Banba, S.; Brooks, C. L., III, J. Chem. Phys. 2000, 113, 3423] were used to restrain the unselected ligands in nearly binding conformations. Using biasing potentials to enhance convergence, excellent correlation between the free energies derived from λ-dynamics and free energy perturbation results is obtained. Effects of the strength of the restraining potentials and bias potentials on the sampling of the conformational space have been examined. Ligands with smaller substituents explored alternate binding orientations in the presence of weak and moderate restraining potentials.

The λ-dynamics simulation method was used to study the binding of 10 five-member ring heterocycle derivatives to an artificial cavity created inside cytochrome C peroxidase by mutagenesis. Application of λ dynamics using a multiple topology approach resulted in trapping in local minima. To extend the method to these cases, a new restraining potential was devised and added to the extended Hamiltonian. Two approximations were introduced in order to estimate the binding free energy within small simulation times using this potential: (a) The entropy terms related to the restraining potential are assumed to cancel, due to the similarity of the ligands. (b) The restraining potential calculated from the coordinates of the environmental atoms during a λ-dynamics simulation is assumed to be equal to that of the average coordinates. Relatively short λ-dynamics simulations with this restraining potential successfully yielded reasonable estimates of the binding affinity of the ligands as compared with both experimental data and free energy perturbation calculations. Long time λ-dynamics simulations with a ten-ligand system revealed that better ligands tend to have small statistical errors, which is appropriate for screening out the plausible ligands from all candidates. Furthermore, short time λ-dynamics simulations with ten identical ligands demonstrated that sufficient precision was achieved for putative discovery of tight binding ligands or guests.

The recently developed λ-dynamics free-energy based simulation method was used to study the binding of 10 five-member ring heterocycle derivatives to an artificial cavity created by mutagenesis inside cytochrome c peroxidase. Application of λ-dynamics to this system gives a reasonable estimate of the binding affinity of the ligands. This methodology also provides a means to explore the binding orientations and conformations of the ligands inside the binding pocket much better than does conventional MD. This is due to the scaling of forces inherent in the λ-dynamics method, which lowers the barriers separating different binding modes and conformations. Examination of the λ-dynamics trajectory of the ligands revealed alternative binding orientations and conformations not detected by crystallography. Furthermore, a λ-dynamics simulation starting from random initial orientations, in which some ligands take significantly different orientations as compared with those from the X-ray structure, successfully sampl...

The design, synthesis, and pharmacological evaluation of (S)-(-)-ethyl [6-[4-(morpholinoformimidoyl)benzamido]-3, 4-dihydro-2H-1-benzopyran-3-yl]acetate hydrochloride ((S)-4.HCl, MS-180), an orally active glycoprotein IIb-IIIa (GPIIb-IIIa) antagonist, are reported. Pharmacophore mapping of amidino and carboxyl groups of already known GPIIb-IIIa antagonists led to the synthesis of nine amidino acids containing 6,6-bicyclic ring skeletons (10a-i). Among them, the compounds 10a,c,e having an amide bond and 1,2,3,4-tetrahydronaphthalene or 3, 4-dihydro-2H-1-benzopyran skeleton showed marked inhibitions with IC50 values of 46-57 nM in human platelet aggregation assay in vitro, but low oral activities. N-Alkylation of the amidino group coupled with the ester prodrug approach afforded MS-180 ((S)-4.HCl), which generates in vivo the corresponding carboxylic acid (S)-3 as an active species. In vitro, (S)-3 inhibited ADP-induced aggregation of guinea pig, dog, and human platelets (IC50 = 110, 253, and 35 nM, respectively) and inhibited the binding of fibrinogen to immobilized GPIIb-IIIa of human platelets (IC50 = 0.12 nM). After oral administration of MS-180 ((S)-4.HCl) to fasted beagle dog, ex vivo inhibition of platelet aggregation was observed. The maximal inhibitions were observed 2-4 h after dosing with dose dependency (60% inhibition at a dose of 1 mg/kg, 85% at 3 mg/kg, and 100% at 10 mg/kg, respectively) and the extent of the inhibitions paralleled the plasma concentration of the active species (S)-3. On the basis of these studies, we selected MS-180 ((S)-4.HCl) as a candidate for clinical evaluation as a drug for the treatment and prevention of thrombosis in patients.

In spite of recent advance in understanding of the stoichiometry of 22-kDa human GH (22K-hGH) with cell surface hGH receptor (hGHR) and hGH-binding protein (hGH-BP) circulating in human plasma, that of 20-kDa hGH (20K-hGH) is poorly understood. To clarify this, mouse pro-B Ba/F3 cells stably expressing the full-length hGHR (Ba/F3-hGHR) and both recombinant and native hGH-BP were used in this study. Cell proliferation assay revealed that the two hGH isoforms increased Ba/F3-hGHR cells to the same extent in a dose-dependent manner at 0.1 pM-10 nM. However, the self-inhibition observed in 20K-hGH at 5 microM was significantly less than that in 22K-hGH. Furthermore, addition of 1 and 10 nM recombinant hGH-BP caused a slight inhibition in 20K-hGH, but a drastic inhibition in 22K-hGH. Gel filtration chromatography of mixtures of 20K-hGH with recombinant hGH-BP clearly demonstrated that 20K-hGH formed a 1:2 (hGH:hGH-BP) complex efficiently but no detectable 1:1 complex in any conditions. Supporting data were also obtained with native hGH-BP. Computer-aided homology modeling of 20K-hGH has provided speculative data that the conformational change caused by deletion of 15 residues may occur only in the loop between helix 1 and helix 2, resulting in the reduction of its site 1 affinity. In conclusion, 20K-hGH possesses a unique property for forming a 1:2 complex to the same extent as 22K-hGH but has difficulty in forming a 1:1 complex, which might be attributed to the conformational change restricted to its site 1 region.

Despite the lower site 1 affinity of the 20-kDa human growh hormone (20K-hGH) for the hGH receptor (hGHR), 20K-hGH has the same hGHR-mediated activity as 22-kDa human GH (22K-hGH) at low hGH concentration and even higher activity at high hGH concentration. This study was performed to elucidate the reason why 20K-hGH can activate hGHR to the same level as 22K-hGH. To answer the question, we hypothesized that the binding between the stem regions of hGHR could compensate for the weaker site 1 binding of 20K-hGH than that of 22K-hGH in the sequential binding with hGHR. To demonstrate it, we prepared 15 types of alanine-substituted hGHR gene at the stem region and stably transfected them into Ba/F3 cells. Using these cells, we measured and compared the cell proliferation activities between 20K- and 22K-hGH. As a result, the activity of 20K-hGH was markedly reduced than that of 22K-hGH in three types of mutant hGHR (T147A, H150A, and Y200A). Regarding these mutants, the dissociation constant of hGH at the first and second step (KD1 and KD2) in the sequential binding with two hGHRs was predicted based on the mathematical cell proliferation model and computational simulation. Consequently, it was revealed that the reduction of the activity in 20K-hGH was attributed to the change of not KD1 but KD2. In conclusion, these findings support our hypothesis, which can account for the same potencies for activating hGHR between 20K- and 22K-hGH, although the site 1 affinity of 20K-hGH is lower than that of 22K-hGH.

Owing to the dramatically increased number of 3D structures of pharmaceutical targets, structure based approaches play an important role in drug design. Especially, rapid and accurate estimation of binding affinities is essential for efficient screening of drugs. For this purpose, the free energy based screen methods have been developed by some groups. These methods are much faster than the free energy perturbation (FEP) method or thermodynamic integration method, and give more accurate relative free energies than the empirical scoring function methods. The λ-dynamics method, one of these methods, has been recently developed by Brooks and co-workers. The λ-dynamics method is an extension of the FEP method. It differs from the FEP method in the following aspects: (1) In the FEP method, a single coupling parameter λ is used to transform one ligand to another. While in the λ-dynamics method, multiple λs, each corresponding to a given ligand, are used. Because of this feature, the binding free energies of multiple ligands are evaluated simultaneously. (2) In the FEP method λ is fixed during the simulation. While in λ-dynamics, the λs evolve according to their equations of motion via use of an extended system. The free energy difference can be obtained from the probability of the ligand i having the dominant state (i.e. λi=1, {λm≠i=0}). In this method, the total computation time is not expected to increase with the total number of ligands because only the few favorable binders are able to compete for the λ=1 state. This is in coutrast to the conventional free energy calculation methods. The λ-dynamics method was used to study the binding of 10 heterocycle derivatives to an artificial cavity created inside cytochrome c peroxidase and the host guest system of β- cyclodextrin-monosubstituted benzene derivatives. Straightforward application of λ-dynamics using a multiple topology approach resulted in trapping in local minima. To extend the λ- dynamics method to multiple topology model, a new restraining potential, which keeps the ligands in lower-energy states, is added to the λ-dynamics Hamiltonian. Relatively short time λ- dynamics simulations with the restraining potential successfully identified the best binders as compared with both experimental data and FEP calculations. Using the iterative procedure with biasing potentials to enhance convergence, λ-dynamics method successfully yielded reasonable estimates of the binding affinity of all ligands. Furthermore, long time λ-dynamics simulations revealed that better ligands tend to have small statistical errors, which is appropriate for screening out the plausible ligands from all candidates. This methodology also provides a means to explore the binding orientations and conformations of the ligands inside the binding pocket much better than does conventional MD. A λ-dynamics simulation starting from random initial orientations, in which some ligands take significantly different orientations as compared with those from the X-ray structure successfully sample the X-ray crystallographic orientations in all ligands, even though conventional MD starting from the same initial structures remain trapped in the local minima from which they start. Such an efficient sampling of ligand orientation and conformation is expected to diminish the limitation that an initial ligand structure must be close to its true bound orientation in order to get a reasonable estimate of binding free energy. The incorporation of the generalized Born (GB) approach into free energy simulation methods (e.g. FEP or λ-dynamics) using thermodynamic cycles was introduced and applied to the trypsin-benzamidine derivatives system and seven mono-substituted benzen derivatives bound to β-cyclodextrin. Since the GB model is fully analytical continuum solvation representation, derivatives of the energy with respect to individual atoms are available and allow the effect of solvation to be efficiently included in molecular dynamics, The GB energies for the intermediate states, in which more than one ligand obtained competitive λ values, were defined by two ways. Both GB coupling schemes have been incorporated into the program CHARMM. The free energy simulations using both definitions of the GB implicit solvent model gave consistent binding free energy differences (ΔΔG) as compared to those using an explicit solvation model. Non- electrostatic solvation energy contributions, which are not included in the GB energies and approximately related to the solvent-accessible surface area, were successfully included using umbrella sampling techniques. Furthermore, a variant of the λ-dynamics approach, Chemical Monte Carlo / molecular dynamics method (CMC/MD), was implemented in CHARMM and compared with FEP and λ-dynamics methods. In the CMC/MD method, the Metropolis Monte Carlo criterion is used to evolve the λ-space and molecular dynamics is used to evolve the atomic coordinates. Free energy differences calculated using the GB energy agreed well among FEP, λ-dynamics, and CMC/MD. The λ dependent partial charge model was also introduced for the incorporation of hybrid topology model into the λ-dynamics method. In this model, the invariable ligand atoms are represented by the single topology and their partial charges are altered according to the movement of the coupling parameters as they would be the same as those of the end points. The hybrid topology λ-dynamics/GB simulations successfully converged without any restraining potential, however, the sampling configurational space was restricted as compared with that of multiple topology model. They have also applied the λ-dynamics method for the stability analysis of the DNA- binding domain of the Myb transcriptional regulator. In this case, seven different side-chain mutants simultaneously compete to make the protein stabilize, whereas, multiple ligands compete in the previous λ-dynamics simulations. A single short (300 ps) λ-dynamics simulation successfully identified the best stabilized mutant. Furthemore, a series of λ-dynamics trajectories generated by the iterative techniques based on Weighted Histogram Analysis Method (WHAM) showed excellent correlation with data obtained from the conventional FEP simulations or experiments. The additional biasing potentials along λ coordinates successfully increased the ratio of the end points without wasting time by sampling unphysical intermediate states in the λ- dynamics simulations. Furthermore, a hybrid Monte Carlo and Langevin dynamics method (MC/LD) was introduced to study the binding orientations of toluene in β-cyclodextrin. In this method, the guest atoms are replicated. One of the replicas is sampled with the full force of the host, while the rest of the replicas are sampled with scaled "ghost force" to find the other local minima. In constant time intervals of Langevin dynamics (LD) simulation, Monte Carlo method is applied to choose a new guest among all replicas, which helps to jump the barrier quickly. The trajectory of the MC/LD simulation successfully gave the broader set of binding orientations as compared with the conventional MD trajectory. The potential of mean force calculated using its trajectory gave the good agreement with that calculated by intensive computational use of umbrella sampling method and WHAM. Therefore, MC/LD can succeed in both exploring the free energy surface much more efficiently and yielding the canonical ensemble of the system. In conclusion, they have developed and applied the free energy based screening methods such as λ-dynamics and CMC/MD. These methods will be used either to rapidly identify ligands with favorable binding free energy or to estimate specific clange in free energy using the iterative procedure with WHAM. Since they screen the binding free energy of the ligands instead of interaction energy, they provide accurate assessment of binding affinity. The restraining potential is very effective and important for the application of the λ-dynamics method to a multiplc topology model. It successfully enhanced both sampling of λ-space and binding configurations of the ligands. The combination of the GB model with λ-dynamics or CMC/MD has a great potential in the application for drug lead optimization. These combinations may fill the gap between the empirical methods using a single minimized complex structure and the theoretically rigorous methods like FEP or thermodynamic integration. The hybrid topology λ-dynamics representation with λ dependent partial charge model will be the promising representation when one investigates free energy changes for an ensemble of slightly varying ligands. Moreover, the MC/LD method can be applied efficiently to explore the free energy surface which will be useful in many purposes such as the protein folding studies, the loop search, or the conformational search of side chains.