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Structure-based site-directed affinity maturation of antibodies can be expanded by multiple-point mutations to obtain various mutants. However, selecting the appropriate number of promising mutants for experimental evaluation from the vast number of combinations of multiple-point mutations is challenging. In this report, we describe how to narrow candidate mutants using the so-called weak interaction analysis such as CH-π and CH-O in addition to widely recognized interactions such as hydrogen bonds.

We have previously shown that the oral administration of the small molecule hPTHR1 agonist PCO371 and its lead compound, 1 (CH5447240) results in PTH-like calcemic and hypophostemic activity in thyroparathyroidectomized rats. However, 1 was converted to a reactive metabolite in a human liver microsome assay. In this article, we report on the modification path that led to an enhancement of PTHR1 agonistic activity and reduction in the formation of a reactive metabolite to result in a potent, selective, and orally active PTHR1 agonist 1-(3,5-dimethyl-4-(2-((4-oxo-2-(4-(trifluoromethoxy)phenyl)-1,3,8-triazaspiro[4.5]dec-1-en-8-yl)sulfonyl)ethyl)phenyl)-5,5-dimethylimidazolidine-2,4-dione (PCO371, 16c). This compound is currently being evaluated in a phase 1 clinical study for the treatment of hypoparathyroidism.

Low-resolution electron density maps can pose a major obstacle in the determination and use of protein structures. Herein, we describe a novel method, quality assessment based on an electron density map (QAEmap), that evaluates local protein structures determined by X-ray crystallography and corrects structural errors using low-resolution maps. QAEmap uses a three-dimensional deep convolutional neural network with electron density maps and their corresponding coordinates as input and predicts the correlation between the local structure and the putative high-resolution experimental electron density map. This estimates how well the structure fits the high-resolution map. Further, we propose that this method may be applied to evaluate ligand binding, which can be difficult to determine at low resolution.

The generation of a wide range of candidate antibodies is important for the successful development of drugs that simultaneously satisfy multiple requirements. To find cooperative mutations and increase the diversity of mutants, an in silico double-point mutation approach, in which 3D models of all possible double-point mutant/antigen complexes are constructed and evaluated using interaction analysis, was developed. Starting from an antibody with very high affinity, four double-point mutants were designed in silico. Two of the double-point mutants exhibited improved affinity or affinity comparable to that of the starting antibody. The successful identification of two active double-point mutants showed that a cooperative mutation could be found by utilizing information regarding the interactions. The individual single-point mutants of the two active double-point mutants showed decreased affinity or no expression. These results suggested that the two active double-point mutants cannot be obtained through the usual approach i.e. a combination of improved single-point mutants. In addition, a triple-point mutant, which combines the distantly located active double-point mutation and an active single-point mutation collaterally obtained in the process of the double-point mutation strategy, was designed. The triple-point mutant showed improved affinity. This finding suggested that the effects of distantly located mutations are independent and additive. The double-point mutation approach using the interaction analysis of 3D structures expands the design repertoire for mutants, and hopefully paves a way for the identification of cooperative multiple-point mutations.

Deep learning is developing as an important technology to perform various tasks in cheminformatics. In particular, graph convolutional neural networks (GCNs) have been reported to perform well in many types of prediction tasks related to molecules. Although GCN exhibits considerable potential in various applications, appropriate utilization of this resource for obtaining reasonable and reliable prediction results requires thorough understanding of GCN and programming. To leverage the power of GCN to benefit various users from chemists to cheminformaticians, an open-source GCN tool, kGCN, is introduced. To support the users with various levels of programming skills, kGCN includes three interfaces: a graphical user interface (GUI) employing KNIME for users with limited programming skills such as chemists, as well as command-line and Python library interfaces for users with advanced programming skills such as cheminformaticians. To support the three steps required for building a prediction model, i.e., pre-processing, model tuning, and interpretation of results, kGCN includes functions of typical pre-processing, Bayesian optimization for automatic model tuning, and visualization of the atomic contribution to prediction for interpretation of results. kGCN supports three types of approaches, single-task, multi-task, and multi-modal predictions. The prediction of compound-protein interaction for four matrixmetalloproteases, MMP-3, -9, -12 and -13, in the inhibition assays is performed as a representative case study using kGCN. Additionally, kGCN provides the visualization of atomic contributions to the prediction. Such visualization is useful for the validation of the prediction models and the design of molecules based on the prediction model, realizing “explainable AI” for understanding the factors affecting AI prediction. kGCN is available at https://github.com/clinfo.

During the course of derivatization of HTS hit 4a, we have identified a novel small-molecule hPTHR1 agonist, 1-(3,5-dimethyl-4-(2-((2-((1R,4R)-4-methylcyclohexyl)-4-oxo-1,3,8-triazaspiro[4.5]dec-1-en-8-yl)sulfonyl)ethyl)phenyl)-1-methylurea (CH5447240, 14l). Compound 14l exhibited a potent in vitro hPTHR1 agonist effect with EC20 of 3.0 μM and EC50 of 12 μM and showed excellent physicochemical properties, such as high solubility in fasted state simulated intestinal fluid and good metabolic stability in human liver microsomes. Importantly, 14l showed 55% oral bioavailability and a significantly elevated serum calcium level in hypocalcemic model rats.

The accumulation of epigenetic alterations is one of the major causes of tumorigenesis. Aberrant DNA methylation patterns cause genome instability and silencing of tumor suppressor genes in various types of tumors. Therefore, drugs that target DNA methylation-regulating factors have great potential for cancer therapy. Ubiquitin-like containing PHD and RING finger domain 1 (UHRF1) is an essential factor for DNA methylation maintenance. UHRF1 is overexpressed in various cancer cells and down-regulation of UHRF1 in these cells reactivates the expression of tumor suppressor genes, thus UHRF1 is a promising target for cancer therapy. We have previously shown that interaction between the tandem Tudor domain (TTD) of UHRF1 and DNA ligase 1 (LIG1) di/trimethylated on Lys126 plays a key role in the recruitment of UHRF1 to replication sites and replication-coupled DNA methylation maintenance. An arginine binding cavity (Arg-binding cavity) of the TTD is essential for LIG1 interaction, thus the development of inhibitors that target the Arg-binding cavity could potentially repress UHRF1 function in cancer cells. To develop such an inhibitor, we performed in silico screening using not only static but also dynamic metrics based on all-atom molecular dynamics simulations, resulting in efficient identification of 5-amino-2,4-dimethylpyridine (5A-DMP) as a novel TTD-binding compound. Crystal structure of the TTD in complex with 5A-DMP revealed that the compound stably bound to the Arg-binding cavity of the TTD. Furthermore, 5A-DMP inhibits the full-length UHRF1:LIG1 interaction in Xenopus egg extracts. Our study uncovers a UHRF1 inhibitor which can be the basis of future experiments for cancer therapy.

Resistance of prostate cancer to castration is currently an unavoidable problem. The major mechanisms underlying such resistance are androgen receptor (AR) overexpression, androgen-independent activation of AR, and AR mutation. To address this problem, we developed an AR pure antagonist, CH5137291, with AR nuclear translocation-inhibiting activity, and compared its activity and characteristics with that of bicalutamide. Cell lines corresponding to the mechanisms of castration resistance were used: LNCaP-BC2 having AR overexpression and LNCaP-CS10 having androgen-independent AR activation. VCaP and LNCaP were used as hormone-sensitive prostate cancer cells. In vitro functional assay clearly showed that CH5137291 inhibited the nuclear translocation of wild-type ARs as well as W741C- and T877A-mutant ARs. In addition, it acted as a pure antagonist on the transcriptional activity of these types of ARs. In contrast, bicalutamide did not inhibit the nuclear translocation of these ARs, and showed a partial/full agonistic effect on the transcriptional activity. CH5137291 inhibited cell growth more strongly than bicalutamide in VCaP and LNCaP cells as well as in LNCaP-BC2 and LNCaP-CS10 cells in vitro. In xenograft models, CH5137291 strongly inhibited the tumor growth of LNCaP, LNCaP-BC2, and LNCaP-CS10, whereas bicalutamide showed a weaker effect in LNCaP and almost no effect in LNCaP-BC2 and LNCaP-CS10 xenografts. Levels of prostate-specific antigen (PSA) in plasma correlated well with the antitumor effect of both agents. CH5137291 inhibited the growth of LNCaP tumors that had become resistant to bicalutamide treatment. A docking model suggested that CH5137291 intensively collided with the M895 residue of helix 12, and therefore strongly inhibited the folding of helix 12, a cause of AR agonist activity, in wild-type and W741C-mutant ARs. In cynomolgus monkeys, the serum concentration of CH5137291 increased dose-dependently and PSA level decreased 80% at 100 mg/kg. CH5137291 is expected to offer a novel therapeutic approach against major types of castration-resistant prostate cancers.

A series of nonsecosteroidal vitamin D 3 analogs with carboxylic acid were explored. Through our systematic SAR studies on the side chain moiety, compound 6b was identified as the optimal compound showing excellent vitamin D receptor (VDR) agonistic activity. Compound 6b had the diethyl group in the terminal which was bound by ( E )-olefin linker to the bisphenyl core. Calculating the volume of the side chain showed that the diethyl group in 6b filled the hydrophobic region of VDR with the ideal packing coefficient based on the 55% rule, and that this resulted in the most potent in vitro activity.

Purpose: We investigated the antitumor activity of the combination of two different humanized monoclonal human epidermal growth factor receptor (HER) 2 antibodies, pertuzumab and trastuzumab, for gastric cancer. Experimental Design: Tumor mouse xenograft models were used to examine antitumor activity. Cell proliferation was examined using crystal violet staining. HER family proteins' expression was analyzed by ELISA and immunohistochemistry. Phosphorylated proteins and heterodimers were detected by Western blotting and in situ proximity ligation assay (PLA), respectively. Apoptosis activity was examined by caspase 3/7 activity. Antibody-dependent cellular cytotoxicity (ADCC) activity was detected by xCELLigence. Microvessel density was examined by CD31 staining. Results: Pertuzumab in combination with trastuzumab showed significant antitumor activity compared with each monotherapy in NCI-N87, an HER2-positive human gastric cancer xenograft model. The efficacy was stronger than that of the maximum effective dose with each monotherapy. Similar antitumor activity was shown in 4-1ST, another HER2-positive gastric cancer model, but not in MKN-28, an HER2-negative model. Combining pertuzumab with trastuzumab enhanced cell growth inhibition and apoptosis activity by inhibiting EGFR-HER2 heterodimerization and the phosphorylation of these receptors and their downstream factors. This effect was also seen in HER2-HER3 signaling. Furthermore, pertuzumab in combination with trastuzumab potentiated the ADCC activity of those antibodies and reduced tumor microvessel density. Conclusions: We showed the significantly enhanced efficacy of pertuzumab combining with trastuzumab for HER2 overexpressing gastric cancer through the potentiation of cell growth inhibition, apoptosis activity, cell killing activity by ADCC, and antiangiogenic activity. This study suggests the clinical benefit of combination therapy with pertuzumab and trastuzumab for patients with HER2-positive gastric cancers. Clin Cancer Res; 17(15); 5060–70. ©2011 AACR.

A series of 5,5-dimethylthiohydantoin derivatives were synthesized and evaluated for androgen receptor pure antagonistic activities for the treatment of castration-resistant prostate cancer. Since CH4933468, which we reported previously, had a problem with agonist metabolites, novel thiohydantoin derivatives were identified by applying two strategies. One was the replacement of the alkylsulfonamide moiety by a phenylsulfonamide to avoid the production of agonist metabolites. The other was the replacement of the phenyl ring with a pyridine ring to improve in vivo potency and reduce hERG affinity. Pharmacological assays indicated that CH5137291 (17b) was a potent AR pure antagonist which did not produce the agonist metabolite. Moreover, CH5137291 completely inhibited in vivo tumor growth of LNCaP-BC2, a castration-resistant prostate cancer model.

A series of 5,5-dimethylthiohydantoin derivatives were synthesized and evaluated for androgen receptor pure antagonistic activities for the treatment of hormone refractory prostate cancer. CH4933468 (32d) with a sulfonamide side chain not only exhibited antagonistic activity with no agonistic activity in the reporter gene assay but also inhibited the growth of bicalutamide-resistant cell lines. This compound also inhibited tumor growth of the LNCaP xenograft in mice dose-dependently.

Selective factor VIIa-tissue factor complex (FVIIa/TF) inhibition is regarded as a promising target for developing new anticoagulant drugs. Compound 1 was discovered from focused screening of serine protease-directed compounds from our internal collection. Using parallel synthesis supported by structure-based drug design, we identified peptidemimetic FVIIa/TF inhibitors (compounds 4-11) containing L-Gln or L-Met as the P2 moiety. However, these compounds lacked the selectivity of other serine proteases in the coagulation cascade, especially thrombin. Further optimization of these compounds was carried out with a focus on the P4 moiety. Among the optimized compounds, 12b-f showed improved selectivity.

The 3D structure of human factor VIIa/soluble tissue factor in complex with a peptide mimetic inhibitor, propylsulfonamide-D-Thr-Met-p-aminobenzamidine, is determined by X-ray crystallography. As compared with the interactions between thrombin and thrombin inhibitors, the interactions at S2 and S3 sites characteristic of factor VIIa and factor VIIa inhibitors are revealed. The S2 site has a small pocket, which is filled by the hydrophobic methionine side chain in P2. The small S3 site fits the small size residue, D-threonine in P3. The structural data and SAR data of the peptide mimetic inhibitor show that these interactions in the S2 and S3 sites play an important role for the improvement of selectivity versus thrombin. The results will provide valuable information for the structure-based drug design of specific inhibitors for FVIIa/TF.

Compounds with a medium-sized flexible ring often show atropisomerism that is caused by the high-energy barriers between long-lived conformers that can be isolated and often have different biological properties to each other. In this study, the frequency of the transition between the two stable conformers, aS and aR, of thienotriazolodiazepine compounds with flexible 7-membered rings was estimated computationally by Monte Carlo (MC) simulations and validated experimentally by NMR experiments. To estimate the energy barriers for transitions as precisely as possible, the potential energy (PE) surfaces used in the MC simulations were calculated by molecular orbital (MO) methods. To accomplish the MC simulations with the MO-based PE surfaces in a practical central processing unit (CPU) time, the MO-based PE of each conformer was pre-calculated and stored before the MC simulations, and then only referred to during the MC simulations. The activation energies for transitions calculated by the MC simulations agreed well with the experimental ΔG determined by the NMR experiments. The analysis of the transition trajectories of the MC simulations revealed that the transition occurred not only through the transition states, but also through many different transition paths. Our computational methods gave us quantitative estimates of atropisomerism of the thienotriazolodiazepine compounds in a practical period of time, and the method could be applicable for other slow-dynamics phenomena that cannot be investigated by other atomistic simulations.

Purpose. Canalicular multispecific organic anion transporter (cMOAT/MRP2) is known to exhibit a broad substrate specificity toward amphiphatic organic anions, including methotrexate (MTX). The present study aims to identify the physicochemical properties of MTX derivatives that correlate with recognition specificity by cMOAT/MRP2.

Two proteins play important roles in the expression of vitamin D function: the specific nuclear receptor protein (vitamin D receptor, VDR) and the transport protein (vitamin D binding protein, DBP). This study was conducted to clarify the conformation of vitamin D responsible for binding to those proteins. For the purpose, the side chain mobility of 1,25(OH)2D3 (1) and its 20-epimer, 20-epi-1,25(OH)2D3 (2), was analyzed by a systematic conformational search. The results were depicted as a three-dimensional dot map, which indicates that the side chains of the two vitamins (1 and 2) occupy different spatial regions that are separated in two areas. We denoted these areas as A and G for 1 and EA and EG for 2. Four analogs, the diastereomers at C(20) and C(22) (3-6) of 22-methylated 1,25(OH)2D3 whose side chains were confined to occupy G, A, EA, and EG, respectively, were designed. These analogs (3-6) were synthesized efficiently by a stereoselective conjugate addition of organocuprate to steroidal E- and Z-22-en-24-ones as the key step. In binding to the VDR the affinities of the analogs (3-6) relative to 1,25-(OH)2D3 (1) were 1/60, 1/3, 20, and 1/100, respectively. These results indicate that the A region is responsible for binding of 1 to VDR and the EA region for binding of 2. Only isomer 4 showed significant affinity for DBP, indicating only the A region is responsible for binding to DBP. Thus, 5 showed clear separation of binding affinities for two proteins, VDR and DBP. Having the highest known VDR affinity, (22R)-22-methyl-20-epi-1,25(OH)2D3 (5) has potential both as a therapeutic agent and as a tool to study the molecular mechanism of vitamin D-mediated gene transcription.

Side chain mobility of 1,25(OH)2D3 (1) and its 20-epimer (2) was analyzed and shown in a three-dimensional dot map that indicates the two distinct spatial regions accessible by the terminal 25-hydroxyl group for each vitamin. The biological activities of two analogs (3 and 4) of 1, which possess restricted side chain flexibility, were tested and the results suggest the spatial region of 1 responsible for binding to VDR and DBP.

In an extension of our study on gamma hydroxy carboxylic acid analogs, we explored a series of nonsecosteroidal vitamin D receptor (VDR) agonists in which 1,3-diol of 1,25(OH)2D3 had been replaced by aryl acetic acid. These analogs showed very potent activity in vitro compared with 1,25(OH)2D3. An X-ray analysis of 8d showed that the inserted phenyl ring well mimicked the folded methylene linker of the gamma hydroxy carboxylic acid moiety but the carboxylic acid of 8d interacted with VDR in a different manner from gamma hydroxy carboxylic acids. Through our in vivo screening in an osteoporosis rat model using immature rats, we identified a potent active vitamin D3 analog, compound 7e. In mature rats of the same model, compound 7e also showed good PK profiling and excellent ability to prevent bone mineral density loss without severe hypercalcemia. Our nonsecosteroidal VDR agonist 7e (CH5036249) could be a possible new drug candidate for treating osteoporosis in human.

Comparative molecular field analysis (CoMFA) was applied to 6-substituted benzopyran-4-carbothioamide potassium channel openers 1. CoMFA suggested that the electrostatic, steric, and hydrophobic factors of the 6-substituent were correlated with the vasorelaxant activity, among which the contribution of electrostatic factor was the most important. Comparative molecular field analysis (CoMFA) was applied to 6-substituted benzopyran-4-cabothioamide potassium channel openers 1. CoMFA suggested that the electrostatic, steric, and hydrophobic factors of the 6-substituents were correlated with the vasorelaxant activity.

QSAR study of 6-substituted benzopyran-4-carbothioamides 1 showed that vasorelaxant activity is linearly correlated with the electronic parameter (σm) and parabolically correlated with steric (L) and hydrophobic (π) parameters of the 6-substituent.

A pharmacophore model which explains rationally structure-activity relationships of chemically diverse potent K + channel openers, has been constructed. Potent benzopyran derivatives with thioamide, amide, and ( N -cyano)amidine groupa at the 4-position have been designed using the model.

Selective factor VIIa-tissue factor complex (FVIIa/TF) inhibition is regarded as a promising target for developing new anticoagulant drugs. In previous reports, we described a S3 subsite found in the X-ray crystal structure of compound 2 that bound to FVIIa/soluble tissue factor (sTF). Based on the X-ray crystal structure information and with the aim of improving the inhibition activity for FVIIa/TF and selectivity against other serine proteases, we synthesized derivatives by introducing substituents at position 5 of the indole ring of compound 2. Among them, compound 16 showed high selectivity against other serine proteases. Contrary to our expectations, compound 16 did not occupy the S3-subsite; X-ray structure analysis revealed that compound 16 improved selectivity by forming hydrogen bonds with Gln217, Thr99 and Asn100.

Quantitative structure-activity relationships for quinolone antibacterials have been previously examined and a steric parameter L for the N1-substituents found to be important in QSAR equations. But some compounds for which previous QSAR equations could not predict the activity have appeared recently. In this study, conformations of a variety of N1-substituents of quinolone antibacterials were analyzed by a molecular modeling method. An active conformation of each of the compounds was estimated with information of the energy profile calculated by molecular orbital methods and of its biological activity. A model of a receptor corresponding to the N1-substituents was constructed by superposing van der Waals volumes of active conformer of highly active compounds. As a result of these conformational analyses and receptor mapping, it is proposed that there are two different optimum volumes for increasing the activity of quinolone antibacterials and two unfavorable regions for reducing the activity. It is suggested that the steric parameter L which appeared in previous QSAR equations corresponds to one of the optimum volumes of the proposed receptor model. With this receptor model, a relation between structure and activity of the compounds, including those mispredicted compounds in previous QSAR equations is able to be rationalized qualitatively and elegantly. We believe that this receptor model is useful for a prediction of the activity of compounds not yet synthesized as well as for designing new quinolone antibacterials.

The 3-(4-cyano-3-trifluoromethylphenyl)-5,5-dimethylthiohydantoin derivatives which have carboxy-terminal side chains were synthesized and their agonistic/antagonistic activities against androgen receptor (AR) measured. Among them, compound 13b showed antagonistic activity (IC50=130 nM) with no agonistic activity even at 10000 nM. This compound exhibited significant metabolic stability and oral antiandrogenic activity (ED50=7 mg/kg).

Lead optimization of CH4892280 (4), an androgen receptor (AR) pure antagonist, was investigated. Compounds 6 and 7, which have a carboxylic acid at the end of the side chain at the position 7alpha of dihydrotestosterone (DHT), showed partial agonistic activities in reporter gene assay (RGA). Conversion of the steroidal core structure to 17alpha-methyltestosterone gave compound 14, which showed weak pure antagonistic activity. Optimization of the side chain by the insertion of a phenyl ring led to compounds 22 and 28-30, which showed pure antagonistic activities at submicromolar concentrations. The structure-activity relationships were clarified.

A series of 7α-substituted dihydrotestosterone derivatives were synthesized and evaluated for androgen receptor (AR) pure antagonistic activity. From reporter gene assay (RGA), the compound with a side chain containing N - n -butyl- N -methyl amide ( 19a ) showed pure antagonistic activity (IC 50 = 340 nM, FI 5 > 10,000 nM), whereas known AR antagonists showed partial agonistic activities. The optimization of 19a led to compound 23 ( CH4892280 ), which showed more potent pure antagonistic activity (IC 50 = 190 nM, FI 5 > 10,000 nM). The SARs of tested compounds suggested that the length of the side chain and the substituents on the amide nitrogen are important for pure antagonistic activities.

Selective factor VIIa-tissue factor complex (FVIIa/TF) inhibition is seen as a promising target for developing new anticoagulant drugs. Structure-based designs of the P3 moiety in the peptide mimetic factor VIIa inhibitor successfully lead to novel inhibitors with selectivity for FVIIa/TF and extrinsic coagulation the same as or even higher than those of previously reported peptide mimetic factor VIIa inhibitors. X-ray crystal structure analysis reveals that one of the novel inhibitors shows improved selectivity by forming interactions between the inhibitor and FVIIa as expected. Another of the novel inhibitors achieves improved selectivity through an unexpected hydrogen bond with Gln217, with a unique bent conformation in FVIIa/TF accompanied by conformational changes of the inhibitor and the protein.

The crystal structure of human factor VIIa/soluble tissue factor (FVIIa/sTF) in complex with a highly selective peptide-mimetic FVIIa inhibitor which shows 1670-fold selectivity against thrombin inhibition has been solved at 2.6 A resolution. The inhibitor is bound to FVIIa/sTF at the S1, S2 and S3 sites and at the additional S1 subsite. Two charged groups, the amidino group in P2 and the carboxylate group in P4, form ionic interactions with Asp60 and Lys192 of FVIIa, respectively. Structural comparisons between factor VIIa and thrombin show that thrombin has oppositely charged residues, Lys60F and Glu192, in the S2 site and the S1 subsites, respectively. These data suggest that the utilization of the differences of charge distribution in the S2 site and the S1 subsites between FVIIa and thrombin is critical for achieving high selectivity against thrombin inhibition. These results will provide valuable information for the structure-based drug design of specific inhibitors for FVIIa/TF.

Selective factor VIIa-tissue factor complex (FVIIa/TF) inhibition is seen as a promising target for developing new anticoagulant drugs. A novel peptide mimetic factor VIIa inhibitor, ethylsulfonamide-d-biphenylalanine-Gln-p-aminobenzamidine, shows 100-fold selectivity against thrombin in spite of its large P3 moiety, unlike previously reported FVIIa/TF selective inhibitors. X-ray crystal structure analysis reveals that the large P3 moiety, d-biphenylalanine, and the small P4 moiety, ethylsulfonamide, make novel interactions with the 170-loop and Lys192 of FVIIa/TF, respectively, accompanying ligand-induced conformational changes of the 170-loop, Gln217, and Lys192. Structural comparisons of FVIIa with thrombin and amino acid sequence comparisons among coagulation serine proteases suggest that these interactions play an important role in achieving selective inhibition for FVIIa/TF.

In previous studies, we have grouped regions in space occupied by the vitamin D side chain into four: A, G, EA, and EG. We showed that the receptor (VDR) affinity of 1alpha,25-dihydroxyvitamin D3 derivatives increases, in terms of side-chain region, in the order EG, G, A, and EA. We called this the active space group concept. In the present study, we used this active space group concept to analyze the conformation-activity relationship of about 40 representative potent 1alpha,25-dihydroxyvitamin D3 analogues. We initially listed structural modifications in the side chain of potent vitamin D analogues and estimated their potency factor. Possible side-chain conformations of representative analogues were calculated by the molecular mechanics method and plotted on a dot map compared with the regions A, G, EA, and EG. The cell-differentiating potency of the analogues was correlated with our active space group concept with few exceptions. Among potent analogues with a natural configuration at C(20), the side chains of those with a 22-oxa, 22-ene, 16-ene, or a 18-nor modification were located in front of region EA (termed F). The side chains of the most potent 20-epi-22-oxa-24-homovitamin D analogues were concentrated at the left side of the EA region (L-EA). Thus, the side chains of almost all potent analogues were distributed around the EA region, and potency increased in the order A, F, EA, and L-EA.

Various structural transformation processes observed in a number of past developmental examples of pharmaceuticals and agrochemicals are regarded as being invaluable precedents for the prospective analog design. In certain cases, (sub)structural transformation patterns are interchangeable among various compound series in spite of differences in their pharmacological category. Thus, the patterns extracted with a computer-readable format could be accumulated and integrated as a database for potential “rules” for bioanalogous molecular transformations. EMIL is a system that incorporates the database and a data-processing engine constructed to release “higher-ordered” candidate structures from a “lower-ordered” input structure “automatically”. Conceptual background for the database construction and the procedure for the database collection are presented on the basis of some lead evolution examples among pharmaceutical and agrochemical series of compounds.

Background: Prostate cancer resistant to total androgen depletion therapies is termed castration-resistant prostate cancer and novel therapeutic approaches are in demand. The mechanisms of castration resistance in prostate cancer are reported to be: 1) hypersensitivity to androgen derived from overexpression of androgen receptor (AR), 2) androgen-independent activation of AR, termed “outlaw”, and 3) loss of ligand specificity derived from mutation of AR. To address this demand, we screened and developed an AR antagonist without agonist activity, a so-called AR pure antagonist, CH5137291. Methods: We compared the inhibitory effects of CH5137291 with bicalutamide on the growth of hormone-sensitive and castration-resistant prostate cancer cells. To confirm the in vivo efficacy of CH5137291, we used xenograft models. The antagonist/agonist effect of CH5137291 on AR transcriptional activity was analyzed using reporter gene assays. To elucidate the mechanisms of CH5137291, we examined the effect of CH5137291 on the protein level of nuclear AR in LNCaP-CS10 and the effect of CH5137291 on the subcellular localization of AR in LNCaP cells. Finally, the exposure of CH5137291 and its effect on serum prostate-specific antigen (PSA) level were investigated in cynomolgus monkey. Results: CH5137291 showed in vitro inhibitory effects superior to bicalutamide in all models tested including hormone-sensitive VCaP, LNCaP and castration-resistant, LNCaP-BC2 (overexpression model) and LNCaP-CS10 (outlaw model). CH5137291 treatment (10, 100 mg/kg) inhibited the tumor growth to initial levels and PSA production to lower than initial levels in castration-resistant LuCaP35V, LNCaP-BC2 and LNCaP-CS10 xenograft models. In contrast, bicalutamide (10, 100 mg/kg) did not inhibit tumor growth or PSA production in any of the xenograft models. In addition, CH5137291 exhibited prolongation of the time to progression compared with bicalutamide in the LNCaP xenograft model. In vitro functional assays clarified that CH5137291 showed pure antagonist activity against wild type, bicalutamide-resistant type (W741C), and flutamide-resistant type (T877A) AR in transcription and inhibited the nuclear translocation of all types of AR tested. CH5137291 also exhibited a dose-dependent increase in serum concentration and inhibited PSA production in cynomolgus monkey. Conclusion: Our novel AR pure antagonist CH5137291 inhibited AR nuclear translocation and showed not only superior efficacy in prostate cancers with castration-resistance acquired from AR overexpression, mutation and outlaw mechanisms but also prolonged time to progression in hormone-sensitive prostate cancer compared with bicalutamide. CH5137291 is expected to contribute to novel therapeutic approaches against a broad spectrum of prostate cancers. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):A219.

Background: We hypothesized that the androgen receptor (AR) pure antagonists, which exhibit no agonist activities, would inhibit AR signaling completely and would be efficacious against castration-resistant prostate cancer (CRPC). Based on our understanding of estrogen receptor pure antagonists, we designed dihydrotestosterone (DHT) derivatives and nonsteroidal RU56187 derivatives that inhibit the folding of helix 12 of AR and screened AR pure antagonists. Although a non-steroidal derivative (CH4933468) showed improved metabolic stability compared with DHT derivatives, it metabolized into an extremely small amount of a dealkylated metabolite with strong agonist effect. Therefore, CH4933468 did not show antitumor activity against the CRPC xenograft model. Methods: For metabolic stability, we designed and synthesized sulfonamide-substituted aryl compounds without dealkylated agonist metabolites. The production of agonist metabolites was examined in rat, mouse, monkey and dog. The efficacy of our compounds was measured using an in vitro reporter assay, a cell growth assay and in vivo prostate cancer xenograft models. Results: As a result of our optimization, CH5137291 was discovered and found to be an orally-active androgen receptor pure antagonist without agonistic activities derived from dealkylated agonist metabolites in vivo in rat, mouse, monkey and dog. CH5137291 exhibited AR antagonistic activities of IC50 of 300 nM and no agonist activity even at 30,000 nM in the reporter gene assay. In LNCaP-BC2 cells (AR overexpression CRPC model), CH5137291 was more efficient than bicalutamide. CH5137291 completely inhibited cell growth, in contrast to the partial inhibition of bicalutamide. Furthermore, CH5137291 inhibited the tumor growth and PSA production in the LNCaP-BC2 xenograft model. CH5137291 also inhibited AR nuclear translocation in the presence of R1881. According to the binding models of CH5137291 and bicalutamide to wild type AR, the sulfonamide of CH5137291 directly collided with M895 and thus prevented the folding of helix 12 completely. On the other hand, bicalutamide did not collide with M895 from helix 12 and inhibited helix 12 folding indirectly through W741. Hence, CH5137291 might act as an AR pure antagonist, but bicalutamide apparently does not. Conclusions: CH5137291, a novel AR pure antagonist without agonistic activities derived from dealkylated agonist metabolites, has shown antitumor activities in both in vitro and in vivo CRPC models. Our experimental results for clinical candidate CH5137291 corroborate our hypothesis that pure antagonists inhibit the folding of helix 12 completely, leading to the complete inhibition of the transcription activity of AR. Based on the strong efficacy against CRPC xenograft model and the inhibition of AR nuclear translocation, CH5137291 may offer more benefit than bicalutamide. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):A220.