Drug discovery based on the structure of FKBP12: Design,synthesis and evaluation of <Emphasis Type="Italic">L</Emphasis>-1,4-thiazane-3-carboxylic acid derivatives as neuroimmunophilin ligands

By choosing neuroimmunophilin FKBP12 as a therapeutical target, we have attempted to discover a new structural drug for treating neurodegenerative disease. This drug should possess neurotrophic activity and not affect the immune system. Based on the crystal structure of FKBP12, FK506 and Calcineurin complex, a series of small organic molecules were designed. These molecules were to have the ability of binding to FKBP12 in a virtual screening. By using a solution parallel synthetic method, these compounds were synthesized. The neuroprotective and neuroregenerative activities of these compounds were evaluated by binding assays, PC12 cells survival and neurite outgrowth model, chick dorsal root ganglion cultures (DRG) and 6-OHDA lesioned mice sympathetic nerve endings model. The evaluation results of these compounds showed that compound N308 has great promise as a candidate for a neuroprotective and neuroregenerative agent. Supported by the National Basic Research Program of China (Grant No. G1998051107) and Hi-tech Research and Development Program of China (Grant No. 2002AA233051)     

In Silico Strategy for Targeting the mTOR Kinase at Rapamycin Binding Site by Small Molecules

Computer aided drug-design methods proved to be powerful tools for the identification of new therapeutic agents. We employed a structure-based workflow to identify new inhibitors targeting mTOR kinase at rapamycin binding site. By combining molecular dynamics (MD) simulation and pharmacophore modelling, a simplified structure-based pharmacophore hypothesis was built starting from the FKBP12-rapamycin-FRB ternary complex retrieved from RCSB Protein Data Bank (PDB code 1FAP). Then, the obtained model was used as filter to screen the ZINC biogenic compounds library, containing molecules derived from natural sources or natural-inspired compounds. The resulting hits were clustered according to their similarity; moreover, compounds showing the highest pharmacophore fit-score were chosen from each cluster. The selected molecules were subjected to docking studies to clarify their putative binding mode. The binding free energy of the obtained complexes was calculated by MM/GBSA method and the hits characterized by the lowest ΔG_bind values were identified as potential mTOR inhibitors. Furthermore, the stability of the resulting complexes was studied by means of MD simulation which revealed that the selected compounds were able to form a stable ternary complex with FKBP12 and FRB domain, thus underlining their potential ability to inhibit mTOR with a rapamycin-like mechanism.     

Selective Inhibitors of FKBP51 Employ Conformational Selection of Dynamic Invisible States

The recently discovered SAFit class of inhibitors against the Hsp90 co‐chaperone FKBP51 show greater than 10 000‐fold selectivity over its closely related paralogue FKBP52. However, the mechanism underlying this selectivity remained unknown. By combining NMR spectroscopy, biophysical and computational methods with mutational analysis, we show that the SAFit molecules bind to a transient pocket in FKBP51. This represents a weakly populated conformation resembling the inhibitor‐bound state of FKBP51, suggesting conformational selection rather than induced fit as the major binding mechanism. The inhibitor‐bound conformation of FKBP51 is stabilized by an allosteric network of residues located away from the inhibitor‐binding site. These residues stabilize the Phe67 side chain in a dynamic outward conformation and are distinct in FKBP52, thus rationalizing the basis for the selectivity of SAFit inhibitors. Our results represent a paradigm for the selective inhibition of transient binding pockets.     

Investigating Protein–Protein Allosteric Network using Current-Flow Scheme

Protein dynamic network analysis provides a powerful tool for investigating protein allosteric regulation. We recently developed a current-flow betweenness scheme for protein network analysis and demonstrated that this method, that is, using current-flow betweenness as edge weights, is more appropriate and more robust for investigating the signal transmission between two predefined protein residues or domains as compared with direct usage of correlation scores as edge weights. Here we seek to expand the current-flow scheme to study allosteric regulations involving protein–protein binding. Specifically, we investigated three gain-of-function mutations located at the binding interface of ALK2 (also known as ACVR1) kinase and its inhibitory protein FKBP12. We first searched for the optimal smoothing function for contact network construction and then calculated the subnetwork between FKBP12 protein and the kinase ATP binding site using current-flow betweenness. By comparing the networks between the wild-type and three mutants, we have identified statistically significant changes in the protein–protein networks that are common among all three mutants that allosterically shift the kinase toward a catalytically competent configuration. © 2019 Wiley Periodicals, Inc.     

The in silico identification of potent anti-cancer agents by targeting the ATP binding site of the N-domain of HSP90

To identify new HSP90 inhibitors, the ATP binding site of the N-domain of HSP90 was targeted by molecular docking of a library of 23,129,083 compounds (from the ZINC database) to the ATP binding site of the N-domain of HSP90. Structure-based virtual screen (SBVS) was performed using idock software on the istar web platform. Based on idock binding energies, 40 molecules were considered as HSP90 inhibitors. In the next step, the 40 molecules and the compound AT13387 (Onalespib) were docked to the XJX binding site using AutoDock Vina software. By comparing the binding energies of the 40 molecules selected with compound AT13387, 26 molecules were selected. By applying the rule of five, eight molecules were selected as hit compounds. The interactions of these eight compounds with the XJX binding site were obtained and investigated, and two-dimensional interaction maps were provided for the others. Finally, computing the toxicity of these compounds with the ProTox-II webserver shows that three compounds, namely ZINC89453765, ZINC23918431 and ZINC12414793, can be considered as good HSP90 inhibitors. These compounds are inactive for nuclear receptor signalling and stress response pathways including heat shock response, so do not have the limitations of common HSP90 inhibitors. They are also inactive for hepatotoxicity, carcinogenicity, immunotoxicity, mutagenicity and cytotoxicity.     

Direct calculation of the binding free energies of FKBP ligands

Direct calculations of the absolute free energies of binding for eight ligands to FKBP protein were performed using the Fujitsu BioServer massively parallel computer. Using the latest version of the general assisted model building with energy refinement (AMBER) force field for ligand model parameters and the Bennett acceptance ratio for computing free-energy differences, we obtained an excellent linear fit between the calculated and experimental binding free energies. The rms error from a linear fit is 0.4 kcal/mol for eight ligand complexes. In comparison with a previous study of the binding energies of these same eight ligand complexes, these results suggest that the use of improved model parameters can lead to more predictive binding estimates, and that these estimates can be obtained with significantly less computer time than previously thought. These findings make such direct methods more attractive for use in rational drug design.     

Standard free energy of releasing a localized water molecule from the binding pockets of proteins: double-decoupling method

Localized water molecules in the binding pockets of proteins play an important role in noncovalent association of proteins and small drug compounds. At times, the dominant contribution to the binding free energy comes from the release of localized water molecules in the binding pockets of biomolecules. Therefore, to quantify the energetic importance of these water molecules for drug design purposes, we have used the double-decoupling approach to calculate the standard free energy of tying up a water molecule in the binding pockets of two protein complexes. The double-decoupling approach is based on the underlying principle of statistical thermodynamics. We have calculated the standard free energies of tying up the water molecule in the binding pockets of these complexes to be favorable. These water molecules stabilize the protein-drug complexes by interacting with the ligands and binding pockets. Our results offer ideas that could be used in optimizing protein-drug interactions, by designing ligands that are capable of targeting localized water molecules in protein binding sites. The resulting free energy of ligand binding could benefit from the potential free energy gain accompanying the release of these water molecules. Furthermore, we have examined the theoretical background of the double-decoupling method and its connection to the molecular dynamics thermodynamic integration techniques.     

Chemical genetic control of protein levels: selective in vivo targeted degradation

Genetic loss of function analysis is a powerful method for the study of protein function. However, some cell biological questions are difficult to address using traditional genetic strategies often due to the lack of appropriate genetic model systems. Here, we present a general strategy for the design and syntheses of molecules capable of inducing the degradation of selected proteins in vivo via the ubiquitin-proteasome pathway. Western blot and fluorometric analyses indicated the loss of two different targets: green fluorescent protein (GFP) fused with FK506 binding protein (FKBP12) and GFP fused with the androgen receptor (AR), after treatment with PROteolysis TArgeting Chimeric moleculeS (PROTACS) incorporating a FKBP12 ligand and dihydrotestosterone, respectively. These are the first in vivo examples of direct small molecule-induced recruitment of target proteins to the proteasome for degradation upon addition to cultured cells. Moreover, PROTAC-mediated protein degradation offers a general strategy to create "chemical knockouts," thus opening new possibilities for the control of protein function.     

Structure activity relationship by NMR and by computer: a comparative study

There has recently been considerable interest in using NMR spectroscopy to identify ligand binding sites of macromolecules. In particular, a modular approach has been put forward by Fesik et al. (Shuker, S. B.; Hajduk, P. J.; Meadows, R. P.; Fesik, S. W. Science 1996, 274, 1531-1534) in which small ligands that bind to a particular target are identified in a first round of screening and subsequently linked together to form ligands of higher affinity. Similar strategies have also been proposed for in silico drug design, where the binding sites of small chemical groups are identified, and complete ligands are subsequently assembled from different groups that have favorable interactions with the macromolecular target. In this paper, we compare experimental and computational results on a selected target (FKBP12). The binding sites of three small ligands ((2S)1-acetylprolinemethylester, 1-formylpiperidine, 1-piperidinecarboxamide) in FKBP12 were identified independently by NMR and by computational methods. The subsequent comparison of the experimental and computational data showed that the computational method identified and ranked favorably ligand positions that satisfy the experimental NOE constraints.     

Examination of the role of intestinal fatty acid-binding protein in drug absorption using a parallel artificial membrane permeability assay

Transcellular diffusion across the absorptive epithelial cells (enterocytes) of the small intestine is the main route of absorption for most orally administered drugs. The process by which lipophilic compounds transverse the aqueous environment of the cytoplasm, however, remains poorly defined. In the present study, we have identified a structurally diverse group of lipophilic drugs that display low micromolar binding affinities for a cytosolic lipid-binding protein - intestinal fatty acid-binding protein (I-FABP). Binding to I-FABP significantly enhanced the transport of lipophilic drug molecules across a model membrane, and the degree of transport enhancement was related to both drug lipophilicity and I-FABP binding affinity. These data suggest that intracellular lipid-binding proteins such as I-FABP may enhance the membrane transport of lipophilic xenobiotics and facilitate drug access to the enterocyte cytoplasm and cytoplasmic organelles.     

TGF-β-signaling with small molecule FKBP12 antagonists that bind myristoylated FKBP12-TGF-β type I receptor fusion proteins

Background: Growth arrest in many cell types is triggered by transforming growth factor beta (TGF-β), which signals through two TGF-β receptors (type I, TGF-βRI, and type II, TGF-βRII). In the signaling pathway, TGF-β binds to the extracellular domain of TGF-βRII, which can then transphosphorylate TGF-βRI in its glycine/serine (GS)-rich box. Activated TGF-βRI phosphorylates two downstream effectors, Smad2 and Smad3, leading to their translocation into the nucleus. Cell growth is arrested and plasminogen activator inhibitor 1 (PAI-1) is upregulated. We investigated the role of the immunophilin FKBP12, which can bind to the GS box of TGF-βRI, in TGF-β signaling.Results: Overexpression of myristoylated TGF-βRI and TGF-βRII cytoplasmic tails caused constitutive nuclear translocation of a green-fluorescent-protein-Smad2 construct in COS-1 cells, and constitutive activation of a PAI-1 reporter plasmid in mink lung cells. Fusing FKBP12 to TGF-βRI resulted in repression of autosignaling that could be alleviated by FK506M or rapamycin (two small molecules that can bind to FKBP12). Mutation of the FKBP12-binding site in the FKBP12-TGF-βRI fusion protein restored constitutive signaling. An acidic mutation in the FKBP12-TGFβRI protein allowed FKBP12 antagonists to activate signaling in the absence of TGF-βRII. Further mutations in the TGF-βRI FKBP12-binding site resulted in TGF-β signaling that was independent of both TGF-βRII and FKBP12 antagonists.Conclusions: Fusing FKBP12 to TGF-βRI results in a novel receptor that is activated by small molecule FKBP12 antagonists. These results suggest that FKBP12 binding to TGF-βRI is inhibitory and that FKBP12 plays a role in inhibiting TGF-β superfamily signals.     

Inflammatory mimetic microfluidic chip by immobilization of cell adhesion molecules for T cell adhesion

Leukocyte adhesion to adhesion molecules on endothelial cells is important in immune function, cancer metastasis and inflammation. This cell-cell binding is mediated via cell adhesion molecules such as E-selectin, intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) found on endothelial cells. Because these adhesion molecules on endothelial cells vary significantly across several disease conditions such as autoimmune diseases, inflammation or cancer metastasis, investigations of therapeutic agents that down-regulate leukocyte-endothelial interactions have been based on in vitro models using endothelial cell lines. Here we report a new model, an inflammatory mimetic microfluidic chip, which emulates leukocyte binding to cell adhesion molecules (CAM) by controlling the types and ratio of adhesion molecules. In our model, E-selectin was essential for the synergic binding of Jurkat T cells. Immunosuppressive drugs, such as tacrolimus (FK506) and cyclosporine A (CsA), were used to inhibit T cell interactions under the physiologic model of T cell migration at a ratio of 5?:?4.3?:?3.9 (E-selectin?:?ICAM-1?:?VCAM-1). Our results support the potential usefulness of the inflammatory mimetic microfluidic chip as a T cell adhesion assay tool with modified adhesion molecules for applications such as immunosuppressive drug screening. The inflammatory mimetic microfluidic chip can also be used as a biosensor in clinical diagnostics, drug efficacy tests and high throughput drug screening due to the dynamic monitoring capability of the microfluidic chip.     

Native FKBP12 engineering by ligand-directed tosyl chemistry: labeling properties and application to photo-cross-linking of protein complexes in vitro and in living cells

The ability to modify target "native" (endogenous) proteins selectively in living cells with synthetic molecules should provide powerful tools for chemical biology. To this end, we recently developed a novel protein labeling technique termed ligand-directed tosyl (LDT) chemistry. This method uses labeling reagents in which a protein ligand and a synthetic probe are connected by a tosylate ester group. We previously demonstrated its applicability to the selective chemical labeling of several native proteins in living cells and mice. However, many fundamental features of this chemistry remain to be studied. In this work, we investigated the relationship between the LDT reagent structure and labeling properties by using native FK506-binding protein 12 (FKBP12) as a target protein. In vitro experiments revealed that the length and rigidity of the spacer structure linking the protein ligand and the tosylate group have significant effects on the overall labeling yield and labeling site. In addition to histidine, which we reported previously, tyrosine and glutamate residues were identified as amino acids that are modified by LDT-mediated labeling. Through the screening of various spacer structures, piperazine was found to be optimal for FKBP12 labeling in terms of labeling efficiency and site specificity. Using a piperazine-based LDT reagent containing a photoreactive probe, we successfully demonstrated the labeling and UV-induced covalent cross-linking of FKBP12 and its interacting proteins in vitro and in living cells. This study not only furthers our understanding of the basic reaction properties of LDT chemistry but also extends the applicability of this method to the investigation of biological processes in mammalian cells.     

Drug Discovery Based on the Structure of FKBPs： Design, Synthesis and Evaluation of L-1, 4-Thiazane-3-carboxylic Acid Derivatives as Neuroimmunophilin Ligands

Based on the structure of FK506, FKBP12 and calcineurin complex and the interactive characteristics of small molecular ligands with FKBPs, a series of L-1,4-thiazane-3-carboxylic acid derivatives as neuroimmunophilin ligands was designed and synthesized. The results of evaluation show that compound N308 has a great promise as a candidate of neuroprotective and neuroregenerative agent.     

Snooker: a structure-based pharmacophore generation tool applied to class A GPCRs

G-protein coupled receptors (GPCRs) are important drug targets for various diseases and of major interest to pharmaceutical companies. The function of individual members of this protein family can be modulated by the binding of small molecules at the extracellular side of the structurally conserved transmembrane (TM) domain. Here, we present Snooker, a structure-based approach to generate pharmacophore hypotheses for compounds binding to this extracellular side of the TM domain. Snooker does not require knowledge of ligands, is therefore suitable for apo-proteins, and can be applied to all receptors of the GPCR protein family. The method comprises the construction of a homology model of the TM domains and prioritization of residues on the probability of being ligand binding. Subsequently, protein properties are converted to ligand space, and pharmacophore features are generated at positions where protein ligand interactions are likely. Using this semiautomated knowledge-driven bioinformatics approach we have created pharmacophore hypotheses for 15 different GPCRs from several different subfamilies. For the beta-2-adrenergic receptor we show that ligand poses predicted by Snooker pharmacophore hypotheses reproduce literature supported binding modes for ～75% of compounds fulfilling pharmacophore constraints. All 15 pharmacophore hypotheses represent interactions with essential residues for ligand binding as observed in mutagenesis experiments and compound selections based on these hypotheses are shown to be target specific. For 8 out of 15 targets enrichment factors above 10-fold are observed in the top 0.5% ranked compounds in a virtual screen. Additionally, prospectively predicted ligand binding poses in the human dopamine D3 receptor based on Snooker pharmacophores were ranked among the best models in the community wide GPCR dock 2010.     

Design,synthesis and in silico study of pyridine based 1,3,4-oxadiazole embedded hydrazinecarbothioamide derivatives as potent anti-tubercular agent

Development of novel, safe and effective drug candidates combating the emerging drug resistance has remained a major focus in the mainstream of anti-tuberculosis research. Here, we inspired to design and synthesize series of new pyridin-4-yl-1,3,4-oxadiazol-2-yl-thio-ethylidene-hydrazinecarbothioamide derivatives as potential anti-tubercular agents. The anti-tubercular bioactive assay demonstrated that the synthesized compounds exhibit potent anti-tubercular activity (MIC = 3.9–7.81 μg/mL) in comparison with reference drugs Rifampicin and Isoniazid.We employed pharmacophore probing approach for the identification of CYP51 as a possible drug target for the synthesized compounds. To understand the preferable binding mode, the synthesized molecules were docked onto the active site of Sterol 14 α-demethylases (CYP51) target. From the binding free energy of the docking results it was revealed that the compounds were effective CYP51 inhibitors and acts as antitubercular agent.     

Proteome analysis of conditioned medium from cultured adult hippocampal progenitors

It is known that proliferation and survival of neural stem/progenitor cells in vitro not only depend on exogenous factors, but also on autocrine factors secreted into the conditioned medium. It is also well known that the identification of bioactive proteins secreted into the conditioned medium poses a substantial challenge. Recently, neural stem/progenitor cells were shown to secrete a survival factor, cystatin C, into the conditioned medium. Here, we demonstrate an approach to identify other low molecular weight proteins in conditioned medium from cultured adult rat hippocampal progenitor cells. A combination of preparative two-dimensional gel electrophoresis (2-DE) and mass spectrometry was utilized in the analysis. We were able to identify a number of proteins, which include Rho-guanine nucleotide dissociation inhibitor 1, phosphatidylethanolamine binding protein (PEBP), also termed Raf-1 kinase interacting protein, polyubiquitin, immunophilin FK506 binding protein 12 (FKBP12) and cystatin C. The presence of PEBP and FKBP12 in conditioned medium was confirmed immunologically. All nestin-positive progenitor cells showed immunoreactivity for antibodies against PEBP and FKBP12. To our knowledge we are the first to use this preparative proteomic approach to search for stem cell factors in conditioned medium. The method could be used to identify novel bioactive proteins secreted by stem/progenitor cells in vitro. Identification of bioactive proteins in vitro is of potential importance for the understanding of the regulatory mechanisms of the cells in vivo.