19F chemical library and 19F-NMR for a weakly bound complex structure.

Fragment-based drug discovery (FBDD), which involves small compounds <300 Da, has been recognized as one of the most powerful tools for drug discovery. In FBDD, the affinity of hit compounds tends to be low, and the analysis of protein-compound interactions becomes difficult. In an effort to overcome such difficulty, we developed a 19F-NMR screening method optimizing a 19F chemical library focusing on highly soluble monomeric molecules. Our method was successfully applied to four proteins, including protein kinases and a membrane protein. For FKBP12, hit compounds were carefully validated by protein thermal shift analysis, 1H-15N HSQC NMR spectroscopy, and isothermal titration calorimetry to determine dissociation constants and model complex structures. It should be noted that the 1H and 19F saturation transfer difference experiments were crucial to obtaining highly precise model structures. The combination of 19F-NMR analysis and the optimized 19F chemical library enables the modeling of the complex structure made up of a weak binder and its target protein.

Water molecule-mediated selective inhibition of bacterial zinc metalloproteinases by non-hydroxamate compounds: Ab initio molecular simulations.

UDP-3-O-acyl-N acetylglucosamine deacetylase (LpxC), Zn metalloenzyme for Gram-negative bacteria is an attractive target for developing novel therapeutic agents. Since LpxC has the similar binding pocket as the human matrix metalloproteinases (MMPs), LpxC inhibitors might also inhibit MMP functions producing side effects in human bodies. Here, we investigated specific interactions between LpxC/MMP and their inhibitors using ab initio molecular simulations to elucidate the reason of selective inhibition for LpxC by non-hydroxamate compounds. The evaluated binding properties between LpxC and the compounds are comparable to the trend of their observed inhibitory affinities. It was also elucidated that compound 22 binds most strongly to LpxC due to its specific interactions with Zn ion and Asp241 side chain of LpxC. In contrast, the interactions between the compounds and MMP are significantly weakened due to the water molecules, which are tightly coordinated with the Zn ion in MMP and interrupt the binding of the compounds to the Zn ion. Accordingly, the present molecular simulations revealed that these water molecules around the Zn ion in MMP are causally related to the selective inhibition of these compounds for LpxC rather than MMP.

Structure of macrophage migration inhibitory factor in complex with methotrexate.

Methotrexate (MTX) is an anticancer and anti-rheumatoid arthritis drug that is considered to block nucleotide synthesis and the cell cycle mainly by inhibiting the activity of dihydrofolate reductase (DHFR). Using affinity-matrix technology and X-ray analysis, the present study shows that MTX also interacts with macrophage migration inhibitory factor (MIF). Fragment molecular-orbital calculations quantified the interaction between MTX and MIF based on the structure of the complex and revealed the amino acids that are effective in the interaction of MTX and MIF. It should be possible to design new small-molecule compounds that have strong inhibitory activity towards both MIF and DHFR by structure-based drug discovery.

Proposal of novel potent inhibitors against androgen receptor based on ab initio molecular orbital calculations.

The androgen receptor (AR), a family of nuclear receptor proteins, stimulates the transcription of androgen-responsive genes. As its abnormal activation can cause the progression of prostate cancer, numerous types of ligands for AR have been developed as promising antagonists for the treatment of prostate cancer. We previously investigated the specific interactions between AR and nine types of existing non-steroidal ligands, using molecular simulations based on molecular mechanics and ab initio fragment molecular orbital methods. The results were confirmed to be comparable to the binding affinities of these ligands observed in experiments. We here propose novel ligands as potent inhibitors against AR and investigate their binding properties to AR, using the same molecular simulations. The results indicate that the most promising ligand binds stronger to AR than the existing non-steroidal ligands, and that our proposed ligand binds strongly to a mutant-type AR, which has drug resistance to the existing non-steroidal ligands.

Proposal of novel inhibitors for vitamin-D receptor: Molecular docking, molecular mechanics and ab initio molecular orbital simulations.

The specific binding of active vitamin-D to the vitamin-D receptor (VDR) is closely related to the onset of immunological diseases. To inhibit the binding, various compounds have been developed as potent inhibitors against VDR. Among them, a compound NS-54c, which was developed based on the first VDR antagonist TEI-9647 (25-dehydro-1α-hydroxyvitamin D3-26,23-lactone), was revealed to posse almost 1000-fold improved antagonistic activity over the original TEI-9647. However, the reason for this significant improvement has not been elucidated. In the present study, we investigated the specific interactions between VDR and these inhibitors, using molecular simulations based on molecular docking, molecular mechanics and ab initio fragment molecular orbital calculations. Based on the results simulated, we furthermore proposed novel inhibitors and investigated their binding properties to VDR. The results elucidate that the replacement of propyl group at the 24th site of NS-54c by a phenethyl group can enhance the binding affinity of the inhibitor to VDR. This finding provides useful information for developing novel potent inhibitors against VDR.

FMODB: The World's First Database of Quantum Mechanical Calculations for Biomacromolecules Based on the Fragment Molecular Orbital Method.

We developed the world's first web-based public database for the storage, management, and sharing of fragment molecular orbital (FMO) calculation data sets describing the complex interactions between biomacromolecules, named FMO Database (https://drugdesign.riken.jp/FMODB/). Each entry in the database contains relevant background information on how the data was compiled as well as the total energy of each molecular system and interfragment interaction energy (IFIE) and pair interaction energy decomposition analysis (PIEDA) values. Currently, the database contains more than 13 600 FMO calculation data sets, and a comprehensive search function implemented at the front-end. The procedure for selecting target proteins, preprocessing the experimental structures, construction of the database, and details of the database front-end were described. Then, we demonstrated a use of the FMODB by comparing IFIE value distributions of hydrogen bond, ion-pair, and XH/π interactions obtained by FMO method to those by molecular mechanics approach. From the comparison, the statistical analysis of the data provided standard reference values for the three types of interactions that will be useful for determining whether each interaction in a given system is relatively strong or weak compared to the interactions contained within the data in the FMODB. In the final part, we demonstrate the use of the database to examine the contribution of halogen atoms to the binding affinity between human cathepsin L and its inhibitors. We found that the electrostatic term derived by PIEDA greatly correlated with the binding affinities of the halogen containing cathepsin L inhibitors, indicating the importance of QM calculation for quantitative analysis of halogen interactions. Thus, the FMO calculation data in FMODB will be useful for conducting statistical analyses to drug discovery, for conducting molecular recognition studies in structural biology, and for other studies involving quantum mechanics-based interactions.

Structural change of retinoic-acid receptor-related orphan receptor induced by binding of inverse-agonist: Molecular dynamics and ab initio molecular orbital simulations.

To elucidate structural changes in the retinoic acid receptor-related orphan receptor gamma (RORγt) induced by the binding of an agonist or an inverse agonist, we conducted molecular dynamics (MD) simulations in explicit water. In addition, ab initio fragment molecular orbital calculations were carried out for certain characteristic structures obtained from the MD simulations to reveal important interactions between the amino acid residues of RORγt, and to distinguish the different effects in the binding of an agonist and an inverse agonist on the structure of RORγt. The results elucidate that the hydrogen bond between His479 of helix11 (H11) and Tyr502 of helix12 (H12) is important to keep the H12 conformation in the agonist-bound RORγt. In contrast, in the inverse-agonist-bound RORγt, the side chain of His479 rotates, significantly weakening the interaction between His479 and Tyr502, leading to a conformational change in H12. Therefore, the present molecular simulations clearly indicate that the conformational change in the side chain of His479 in the inverse-agonist-bound RORγt is the main reason for the H12 destabilization induced by the binding of the inverse agonist. Such a conformational change does not occur on the binding of the agonist in RORγt, owing to the strong hydrogen bond between His479 and Tyr502.

Ligand chirality can affect histidine protonation of vitamin-D receptor: ab initio molecular orbital calculations in water.

Vitamin D is recognized to play important roles in the onset of immunological diseases as well as the regulation of the amount of Ca in the blood. Since these physiological actions caused by active vitamin D are triggered by the specific interaction between the vitamin D receptor (VDR) and active vitamin D, many types of compounds have been developed as potent ligands against VDR. It was found that the binding affinity between VDR and its ligand depends significantly on the chirality of the ligand. However, the reason for the dependence has, thus far, not been elucidated. In the present study, we investigated the specific interactions between VDR and some ligands with different chirality, using ab initio fragment molecular orbital (FMO) calculations. The FMO results reveal that two histidine residues of VDR contribute significantly to the binding between VDR and ligand and that their protonation states can affect the specific interactions between VDR and ligand. We therefore considered other possible protonation states of these histidine residues and determined their most stable states, using the ab initio FMO calculations. The results illustrate the possibility that the difference in the chirality of a ligand can induce the change in protonation states of the histidine residues of VDR existing near the ligand. This finding provides an important warning that the protonation states of histidine residues existing near the ligand should be considered more precisely in the molecular simulations for investigating the specific interactions between protein and ligand.

Structural development of non-secosteroidal vitamin D receptor (VDR) ligands without any asymmetric carbon.

Non-secosteroidal VDR ligands without any assymmetric carbon were designed and synthesized based on the structure of the previously reported non-secosteroidal VDR agonist LG190178. The VDR-agonistic activity of all synthesized compounds was evaluated, and 7b emerged as a potent agonist activity with an EC50 value of 9.26 nM. Moreover, a docking simulation analysis was also performed to determine the binding mode of 7b with VDR-LBD.

Effects of 2-substitution on 14-epi-19-nortachysterol-mediated biological events: based on synthesis and X-ray co-crystallographic analysis with the human vitamin D receptor.

Both 2α- and 2ß-hydroxypropyl substituted 14-epi-1α,25-dihydroxy-19-nortachysterols were synthesized to study the human vitamin D receptor (hVDR) binding affinity, binding configurations, and interactions with amino acid residues in the ligand binding domain of hVDR by X-ray co-crystallographic analysis. In conjunction with our previous results on 14-epi-19-nortachysterol, 2-methylidene-, 2α-methyl-, 2ß-methyl, and 2α-hydroxypropoxy-14-epi-19-nortachysterol, we propose a variety of effects of substitution at the C2 position in the 14-epi-19-nortachysterol skeleton on biological activities.

Proposal of potent inhibitors for vitamin-D receptor based on ab initio fragment molecular orbital calculations.

Vitamin D plays an important role in the regulation of the calcium and phosphorus metabolism as well as in bone formation. These physiological actions caused by vitamin D are triggered by the specific binding of vitamin D to its receptor (VDR). Here we investigated the specific interactions and binding affinities between VDR and vitamin D derivatives, using ab initio fragment molecular orbital (FMO) calculations. The FMO results elucidate that relative position of the two hydroxyl groups of the derivatives is essential for the strong binding affinity between the derivative and Arg274 residue of VDR. It is therefore expected that novel potent ligands, which have a great binding affinity for VDR, are developed by adjusting the positions of the hydroxyl groups in the derivatives in such a way as these groups form strong hydrogen bonds with VDR residues. We proposed these novel derivatives and investigated their specific interactions with VDR at atomic and electronic levels to obtain a more potent ligand for VDR.

Specific interactions between androgen receptor and its ligand: ab initio molecular orbital calculations in water.

The Androgen Receptor (AR) is a family of nuclear receptor proteins and a ligand-activated transcription factor. Since its abnormal activation can cause the progression of prostate cancer, numerous types of antagonists against AR have been developed as promising agents for treating prostate cancers. We here investigated the specific interactions between AR and several types of non-steroid agents at an electronic level, using ab initio molecular simulations based on molecular mechanics and ab initio fragment molecular orbital (FMO) methods From the results obtained by FMO, we proposed novel agents as potent ligands against AR and investigated the binding properties between AR and these agents to confirm that some of them can bind more strongly with AR than the existing non-steroid agents and can be strongly effective ligands against AR.

Specific interactions between vitamin-D receptor and its ligands: Ab initio molecular orbital calculations in water.

Vitamin D is recognized to play important roles not only in the bone metabolism and the regulation of Ca amount in the blood but also in the onset of immunological diseases. These physiological actions caused by vitamin D are triggered by the specific interaction between vitamin D receptor (VDR) and vitamin D. In the present study, we investigated the interactions between VDR and vitamin D derivatives using ab initio molecular simulation, in order to elucidate the reason for the significant difference in their effects on VDR activity. Based on the results simulated, we elucidated which parts of the derivatives and which residues of VDR mainly contribute to the specific binding between VDR and the derivatives at an electronic level. This finding will be helpful for proposing new vitamin D derivatives as a potent modulator or inhibitor against VDR.

Synthesis and biological activities of vitamin D3 derivatives with cyanoalkyl side chain at C-2 position.

We synthesized and evaluated novel vitamin D3 derivatives with cyanoalkyl side chain at C-2 position on the basis of our previous research for 2α side chain which bears nitrogen atom-containing functional group. Through a study of X-ray co-crystal structures of human VDR and compound 3, we demonstrated that the 2α alkyl side chain in compound 3 shows a novel interaction in the complex of hVDR-LBD and ligand. This article is part of a Special Issue entitled '17th Vitamin D Workshop'.

Design and synthesis of 2α-(tetrazolylethyl)-1α,25-dihydroxyvitamin D3 as a high affinity ligand for vitamin D receptor.

X-ray cocrystallographic studies of the human vitamin D receptor (hVDR)-[2α-(3-hydroxypropyl)-1α,25-dihydroxyvitamin D3 (O1C3)] complex showed that the terminal hydroxy group of the 2α-functional group of O1C3 formed a hydrogen bond with Arg274 in the ligand binding domain (LBD) of hVDR to stabilize the complex; therefore, O1C3 showed 3-times greater binding affinity for VDR than the natural hormone. Here, the effects of a heteroaromatic ring on binding to hVDR instead of the terminal OH group of O1C3 and also on preliminary biological activities were studied. We synthesized 2α-[2-(tetrazol-2-yl)ethyl]-1α,25(OH)2D3 (1a) and its regioisomer 2α-[2-(tetrazol-1-yl)ethyl]-1α,25(OH)2D3 (1b), in which 1a showed much higher hVDR binding affinity and greater osteocalcin promoter transactivation activity in human osteosarcoma (HOS) cells than those of 1b. X-ray cocrystallographic analysis of the hVDR-1a complex showed new hydrogen bond formation between one of the nitrogen atoms of the tetrazole ring and Arg274. This article is part of a Special Issue entitled '16th Vitamin D Workshop'.

Structure of the ß-form of human MK2 in complex with the non-selective kinase inhibitor TEI-L03090.

Mitogen-activated protein kinase-activated protein kinase 2 (MK2 or MAPKAP-K2), a serine/threonine kinase from the p38 mitogen-activated protein kinase signalling pathway, plays an important role in the production of TNF-α and other cytokines. In a previous report, it was shown that MK2 in complex with the selective inhibitor TEI-I01800 adopts an α-helical glycine-rich loop that is induced by the stable nonplanar conformer of TEI-I01800. To understand the mechanism of the structural change, the structure of MK2 bound to TEI-L03090, which lacks the key substituent found in TEI-I01800, was determined. MK2-TEI-L03090 has a ß-sheet glycine-rich loop in common with other kinases, as predicted. This result suggests that a small compound can induce a drastic conformational change in the target protein structure and can be used to design potent and selective inhibitors.

Crystal structure of human cyclin-dependent kinase-2 complex with MK2 inhibitor TEI-I01800: insight into the selectivity.

Mitogen-activated protein kinase-activated protein kinase 2 (MK2 or MAPKAP-K2) is a Ser/Thr kinase from the p38 mitogen-activated protein kinase signalling pathway and plays an important role in inflammatory diseases. The crystal structure of the MK2-TEI-I01800 complex has been reported; its Gly-rich loop was found to form an α-helix, not a ß-sheet as has been observed for other Ser/Thr kinases. TEI-I01800 is 177-fold selective against MK2 compared with CDK2; in order to understand the inhibitory mechanism of TEI-I01800, the cyclin-dependent kinase 2 (CDK2) complex structure with TEI-I01800 was determined at 2.0 Å resolution. Interestingly, the Gly-rich loop of CDK2 formed a ß-sheet that was different from that of MK2. In MK2, TEI-I01800 changed the secondary structure of the Gly-rich loop from a ß-sheet to an α-helix by collision between Leu70 and a p-ethoxyphenyl group at the 7-position and bound to MK2. However, for CDK2, TEI-I01800 bound to CDK2 without this structural change and lost the interaction with the substituent at the 7-position. In summary, the results of this study suggest that the reason for the selectivity of TEI-I01800 is the favourable conformation of TEI-I01800 itself, making it suitable for binding to the α-form MK2.

Crystal structure of a complex of human chymase with its benzimidazole derived inhibitor.

The crystal structure of human chymase complexed with a novel benzimidazole inhibitor, TJK002, was determined at 2.8 Å resolution. The X-ray crystallographic study shows that the benzimidazole inhibitor forms a non-covalent interaction with the catalytic domain of human chymase. The hydrophobic fragment of the inhibitor occupies the S1 pocket. The carboxylic acid group of the inhibitor forms hydrogen bonds with the imidazole N(ℇ) atom of His57 and/or the O(γ) atom of Ser195 which are members of the catalytic triad. This imidazole ring of His57 induces π-π stacking to the benzene ring of the benzimidazole scaffold as P2 moiety. Fragment molecular orbital calculation of the atomic coordinates by X-ray crystallography shows that this imidazole ring of His57 could be protonated with the carboxyl group of Asp102 or hydroxyl group of Ser195 and the stacking interaction is stabilized. A new drug design strategy is proposed where the stacking to the protonated imidazole of the drug target protein with the benzimidazole scaffold inhibitor causes unpredicted potent inhibitory activity for some enzymes.

Structure basis 1/2SLPI and porcine pancreas trypsin interaction.

SLPI (secretory leukocyte protease inhibitor) is a 107-residue protease inhibitor which inhibits various serine proteases, including elastase, cathepsin G, chymotrypsin and trypsin. SLPI is obtained as a multiple inhibitor in lung defense and in chronic airway infection. X-ray crystal structures have so far reported that they are full-length SLPIs with bovine α-chymotrypsin and 1/2SLPI (recombinant C-terminal domain of SLPI; Arg58-Ala107) with HNE (human neutrophil elastase). To understand the role of this multiple inhibitory mechanism, the crystal structure of 1/2SLPI with porcine pancreas trypsin was solved and the binding modes of two other complexes compared. The Leu residue surprisingly interacts with the S1 site of trypsin, as with chymotrypsin and elastase. The inhibitory mechanism of 1/2SLPI using the wide primary binding site contacts (from P2' to P5) with various serine proteases is discussed. These inhibitory mechanisms have been acquired in the evolution of the protection system for acute inflammatory diseases.

Synthesis of novel C-2 substituted vitamin D derivatives having ringed side chains and their biological evaluation on bone.

Up to the present, numerous vitamin D derivatives have been synthesized, but most of them have straight side chains, and there are few publications described about in vitro and in vivo evaluations on bone by vitamin D derivatives. In our previous paper, we reported the synthesis of various C-2 substituted vitamin D derivatives (2b-2i) with a 2,2-dimethylcyclopentanone unit in the CD-ring side chains, and that the derivatives have strong activity for enhancing bone growth. On the basis of results, this time, we report the synthesis of 2α-substituted vitamin D3 derivatives with chiral cyclopentanone (3-6 and 12-16). These derivatives were obtained by Pd-coupling reaction with A-ring precursor and CD-rings precursor. We evaluated novel derivatives in vitro assays, for affinities for VDR and transactivation assays by human osteosarcoma (HOS) cells. In this research, we demonstrated that some novel vitamin D derivatives (12-MP, 13-MP, 15-MP and 16-LP) have strong transactivation activities in spite of lower affinity for VDR than 1. In addition, we also demonstrated that these derivatives have strong activities for enhancing bone growth using OVX therapeutic rats. This article is part of a Special Issue entitled 'Vitamin D Workshop'.