Tumor Necrosis Factor-α Converting Enzyme Inhibitors Research Articles

An alignment‐independent 3D‐QSAR study on series of hydroxamic acid‐based tumor necrosis factor‐α converting enzyme inhibitors

Grid‐independent descriptors are extensively used in 3D quantitative structure‐activity relationship (3D‐QSAR) studies. These kinds of descriptors represent the spatial arrangement of the atoms in a 3D fashion. In the current study, we have developed a 3D‐QSAR model for a set of hydroxamic acid‐based derivatives as tumor necrosis factor‐α converting enzyme (TACE) inhibitors. The generated model revealed the importance of some main moieties in the potency of these compounds. Quinolinyl and hydroxamate moieties have the ability to act as hydrogen bond acceptors and hot spot points. On the other hand, phenyl ring can participate in hydrophobic contacts with the receptor. 3D‐QSAR analyses resulted in the partial least squares model of 3 latent variables with internal and external validation yielding q2 values of 0.66 and 0.73, respectively. The result of the current study can be used in designing of potent TACE inhibitors where inhibition of TACE enzyme is required, such as inflammatory diseases, atherosclerosis, osteoporosis, autoimmune disorders, allograft rejection, and cancer.

Media effects in modulating the conformational equilibrium of a model compound for tumor necrosis factor converting enzyme inhibition

Small-molecule inhibitors of Tumor Necrosis Factor α Converting Enzyme (TACE) are a promising therapeutic tool for Rheumatoid Arthritis, Multiple Sclerosis and other autoimmune diseases. Here we report on an extensive chemical–physical analysis of the media effects in modulating the conformational landscape of MBET306, the common scaffold and a synthetic precursor of a family of recently discovered tartrate-based TACE inhibitors. The structural features of this molecule with potential pharmaceutical applications have been disclosed by interpreting extensive photophysical measurements in various solvents with the aid of enhanced sampling molecular dynamics simulations and time dependent density functional calculations. Using a combination of experimental and computational techniques, the paper provides a general protocol for studying the structure in solution of molecular systems characterized by the existence of conformational metastable states.

Pharmacophore modeling for the identification of small-molecule inhibitors of TACE

Tumor necrosis factor α-converting enzyme (TACE) plays a critical role in diverse physiological processes such as inflammation, hematopoiesis, and development. In this study, a pharmacophore model constructed from a training set of TACE inhibitors was used to screen an in-house database of organic compounds, from which compound 1 emerged as a top candidate. In a cell-free assay, compound 1 inhibited TACE enzymatic activity in a dose-dependent manner. Moreover, compound 1 inhibited the production of soluble TNF-α in human acute monocytic leukemia THP-1 cells without impacting nitric oxide production, and exhibited anti-proliferative activity against THP-1 cells. We envisage that compound 1 may be employed as a useful scaffold for the development of more potent TACE inhibitors. This study also validates the use of pharmacophore modeling to identify enzyme inhibitors.

Virtual screening-based identification of lead molecules as selective TACE inhibitors

Some novel selective inhibitors of tumor necrosis factor-α converting enzyme (TACE) have been identified from virtual screening of Asinex database containing 2,06,759 compounds using HTS docking with TACE and MMPs and application of Lipinski’s rule of five. Docking studies were carried out for known inhibitors along with lead compounds. Pyrrolidine-based tartrate diamides were docked into the active site of TACE along with MMPs to get insight regarding the binding interactions of active and inactive TACE inhibitors. The 3D orientations of the obtained hits from virtual screening were found to match with that of known inhibitors supporting the binding of the hit molecules into the active site of TACE.

Abstract 355: Autologous Cleavage of the GPIbalpha Receptor Promotes Platelet Dysfunction Following Trauma

Background: Trauma is the most common cause of death in individuals 0-50 years of age and is responsible for more year of life lost than cancer, heart disease, and stroke combined. One quarter of all trauma patients experience some degree of prolonged hemorrhage due to impaired blood clotting, or trauma-induced coagulopathy (TIC). Protein C activation and hyperfibrinolysis have been proposed as key drivers in developing TIC. While primary hemostasis is mediated by von Willebrand factor (vWF) binding to the GPIbα receptor on the platelet surface, tethering them to damaged endothelium. Whether this interaction is altered in TIC remains unexplored. Data from our laboratory has previously shown a significant reduction in platelet-vWF adhesion over time following injury as measured by platelet aggregation response to ristocetin. The aim of this study was to determine the mechanisms behind defective platelet-vWF adhesion in trauma patients. Methods and Results: We obtained samples from 25 transfused trauma patients on admission and at 3, 5, 12, 24, and 120 hours later. Expression of the GPIbα receptor on the platelet surface and circulating in the plasma was measured by flow cytometry and western blot, respectively. We observed a time-dependent decrease in platelet bound GPIbα receptor expression (p=0.01) which was accompanied by up to a 6-fold increase in GPIbα shed into the plasma. Due to its role in proteolytic regulation of platelet aggregation through cleavage of GPIbα, we then measured expression of tumor necrosis factor-α converting enzyme (TACE). Membrane-bound TACE was analyzed by flow cytometry on platelets and leukocytes. We found a significant increase in platelet-derived TACE compared to healthy controls (p<0.01) and over time in trauma patients (p<0.05). The highest expression of platelet TACE corresponded to the time at which we observed the lowest GPIbα expression and lowest platelet-vWF adhesion in these patients. Conclusions: These data suggest that upregulation of platelet-derived TACE results in increased proteolytic cleavage of the GPIbα receptor and reduced platelet-vWF adhesion. These results have important implications for the use of TACE inhibitors to promote effective primary hemostasis in severely injured patients.

Three-dimensional quantitative structure–activity relationship CoMFA/CoMSIA on pyrrolidine-based tartrate diamides as TACE inhibitors

A series of pyrrolidine-based tartrate diamides having selective tumor necrosis factor-α converting enzyme (TACE) inhibitory activity was selected for the three-dimensional quantitative structure–activity relationship (3D-QSAR) studies. Total 76 compounds were selected by considering a high deviation in the biological activity and structural variations. The quality and predictive power of 3D-QSAR, comparative molecular field analysis (CoMFA) and comparative molecular similarity indices analysis (CoMSIA) models for the atom-based, centroid/atom-based, data-based alignments were performed. Various models were developed with the help of these alignments. The best model was developed with data-based alignment. The optimal predictive CoMFA model was obtained with cross-validated r2 = 0.53 with six component, non-cross-validated r2 = 0.94, standard error of estimates 0.23, F-value = 121.98 and optimal CoMSIA model was obtained with cross-validated r2 = 0.53 with five components, non-cross-validated r2 = 0.93, standard error of estimates = 0.24 and F-value = 138.83. These models also showed the best test set prediction with predictive r2 value of 0.65 and 0.73, respectively. Thus, on the basis of predictive power COMSIA model appeared to be the best one. The statistical parameters from these models indicate that the data are being well fitted and also have high predictive ability. Moreover, the resulting 3D-CoMFA/CoMSIA contour maps provide useful guidance for designing of highly active TACE inhibitors.

Chemical–physical analysis of a tartrate model compound for TACE inhibition

We have synthesized and done an extensive chemical-physical analysis of the behavior of a new compound, named MBET306, a synthetic precursor of the recently discovered tartrate-based inhibitors of the protein Tumor Necrosis factor-α Converting Enzyme (TACE). Experimental and theoretical data have shown that in water solution MBET306 is overwhelmingly found as a monoanion at physiological pH, in a conformation that differs substantially from that detected in the known co-crystal structures of MBET306 derivatives bound to TACE. The body of collected experimental and theoretical data indicates that the monoanionic species binds Zn(ii) inducing a strong stabilization of the crystal-like arrangement of the central tartrate zinc-binding group, lending support for a two step TACE docking mechanism via a zinc-bound intermediate. The thorough chemical-physical characterization of the conformational behavior of free and zinc-bound MBET306 in water bulk solution opens new avenues for the rational drug design of tartrate-based highly specific TACE inhibitors.

Blockade of tumor necrosis factor-α converting enzyme (TACE) enhances IL-1β and IFN-γ via caspase-1 activation: A probable cause for loss of efficacy of TACE inhibitors in humans?

TNF-α converting enzyme (TACE) is a member of the ADAM (a disintegrin and metalloproteinase) family and is known as ADAM17, which processes precursor TNF-α in order to release soluble TNF-α (sTNF-α). Inhibition of TACE has been effective as a strategy to inhibit arthritis in animal models; however, it has not been translated in the clinic due to lack of efficacy or toxicity. We hypothesized that inhibition of TACE may activate a different pro-inflammatory pathway in human. To investigate this, we studied the effect of TACE inhibitor DPC-333 on cytokine levels in concanavalin A (Con A) activated human peripheral blood mononuclear cells (hPBMC). We have also studied the effects of DPC-333 on Con A induced cytokine levels in mice in vivo or in vitro in whole blood assay. DPC-333 treatment significantly up-regulated IL-1β and IFN-γ in Con A activated hPBMC. In contrast, pre-treatment with DPC-333 effectively suppressed IL-1β and IFN-γ in mice in vivo or in vitro. Interestingly, DPC-333 was found to up-regulate mRNA expression of caspase-1 in hPBMC in a dose dependent fashion and selective caspase-1 inhibitor completely restored DPC-333 induced IL-1β and IFN-γ. Furthermore, selective IL-1β receptor antagonist (anakinra) prevented DPC-333 induced IFN-γ. In conclusion, our data demonstrates that blockade of TACE enhances IL-1β in a caspase-1 dependent manner in vitro in hPBMC and the elevation of IFN-γ is secondarily mediated via IL-1β. This novel finding might explain the possible cause behind the loss of efficacy of TACE inhibitors in human.

Strategy for generation of new TACE inhibitors: pharmacophore and counter pharmacophore modeling to remove non-selective hits

Present study describes the ligand-based molecular modeling along with virtual screening (VS) approach for generation of new tumor necrosis factor-α converting enzyme (TACE) inhibitors. In ligand-based molecular modeling, two statistically reliable HipHop pharmacophore models Hip1 and counter pharmacophore (CP1) were generated using training set of 3 and 2 molecules, respectively. CP1 was generated using inhibitors of MMP-1 (matrix metalloproteinase-1), an important enzyme involved in musculoskeletal degradation. VS was performed with model Hip1 in in-house database of 1.2 million molecules. In addition, the retrieved molecules were screened with CP1. The combination of both models helped for generating new improved TACE inhibitor molecules.

Three Dimensional Molecular Field Analysis of Thiomorpholine Analogs of TACE Inhibitors Using Receptor Based Alignment

Thiomorpholine analogs (TML) have been identified as novel class of potent tumor necrosis factor-α converting enzyme (TACE) inhibitors. A computational strategy based on molecular docking studies, followed by MFA analysis has been performed to elucidate the atomic details of the TACE/TML interactions and to identify the most important features impacting TACE inhibitory activity of TMLs. The generated MFA model resulted to be well predictive, and gave r2 test 0.723, conventional r2 0.982 and r2 cv 0.811. The 3D-QSAR field contributions and the structural features of the TACE binding site showed a good correlation. These studies will be useful to design new TACE inhibitors with improved potency. Keywords: TACE inhibitors, Rheumatoid arthritis, 3D-QSAR, Molecular Field Analysis (MFA), Docking

Anthranilate derivatives as TACE inhibitors: Docking based CoMFA and CoMSIA analyses

Anthranilic acid based derivatives (ANTs) have been identified as a novel class of potent tumor necrosis factor-α converting enzyme (TACE) inhibitors. A computational strategy based on molecular docking studies, followed by CoMFA and CoMSIA analyses has been performed to elucidate the atomic details of the TACE/ANT interactions and also to identify the most important features impacting TACE inhibitory activity of ANTs. The CoMSIA model resulted to be slightly more predictive than CoMFA model, and gave conventional r² 0.991, r²(cv) 0.793, q² 0.777, SEE 0.050, F-value 655.610, and r²(test) 0.871. The 3D-QSAR field contributions and the structural features of the TACE binding site showed a good correlation. These studies will be useful to design new TACE inhibitors with improved potency.

Molecular modeling and biological effects of peptidomimetic inhibitors of TACE activity

We investigated the molecular basis of specificity for the interaction between tumor necrosis factor-α converting enzyme (TACE) and peptidomimetic inhibitors. Four novel peptidomimetic TACE inhibitors (8a–d) were designed and synthesized by introducing a substituted sulfur group and a hydrophobic group to a novel matrix metalloprotease (MMP) inhibitor. Inhibition was determined by in vitro lipopolysaccharide (LPS) cytotoxicity tests in HL-60 cell lines and by measuring the expression of mTNF-α using FCM techniques and immunohistochemistry in vivo. We simulated the interaction of the inhibitors with the 3D structure of the TACE active site in the Brookhaven Protein Database (PDB). The four inhibitors (8a–d) inhibited activity by 9.1%, 54.5%, 27.3%, and 54.5%, respectively. 8b and 8d showed significant in vitro inhibition in cytotoxicity tests, which corresponded to the molecular docking results. 8d also showed inhibitory activity in vivo. We explored the interface between enzyme and substrate by combining bioinformatics with experimental observations to further the development of specific TACE inhibitors to reduce inflammatory responses.

Generation of Selective TACE Inhibitors: Ligand and Structure Based Molecular Modeling, Virtual Screening, Counter Pharmacophore Screening to Get Selective Molecules

AbstractThis study describes the ligand based as well as structure based molecular modeling and virtual screening of selective tumor necrosis factor‐α converting enzyme (TACE) inhibitors. In ligand based molecular modeling, two statistically reliable pharmacophore models HypoA1 and HypoB1 were generated using a same training set of 22 molecules. HypoA1 consists of two hydrogen bond acceptor and three hydrophobic groups whereas HypoB1 consists of one hydrogen bond donor, one ring aromatic and three hydrophobic groups. Virtual screening was performed with both models in in‐house database of 1.2 million molecules. To remove non selective hits from screened molecules, a counter pharmacophore was generated using inhibitors of MMP‐1, an important enzyme involved in musculoskeletal degradation. In structure based molecular modeling, docking analysis was performed to explore the important interactions between ligands and protein. On comparison, HypoA1 and HypoB1 were found to be complementing with results of docking analysis suggesting high reliability of both models for their use in virtual screening/designing of new molecules.

Production of the Soluble Form of KIT, s-KIT, Abolishes Stem Cell Factor-Induced Melanogenesis in Human Melanocytes

The signaling of stem cell factor (SCF) and its receptor KIT (membrane-bound KIT; m-KIT) plays an important role in melanocyte development, survival, proliferation, and melanogenesis. It has been demonstrated in other systems that a soluble form of m-KIT released from the cell surface (s-KIT) regulates SCF signaling, although there have been no reports pertaining to the existence and the biological role of s-KIT in melanocytes. In this study, we therefore examined the involvement of s-KIT in melanogenesis. Western blotting analysis revealed that treatment with phorbol 12-myristate-13-acetate (PMA) or 4-aminophenylmercuric acetate (APMA) induced s-KIT production in cultured human melanocytes. Inhibitors of tumor necrosis factor-alpha-converting enzyme (TACE) and metalloproteinases (MMPs) muted this release of s-KIT into the media. Human recombinant s-KIT added to melanocytes inhibited SCF-induced phosphorylation of m-KIT, resulting in suppression of SCF-induced melanogenesis. Additionally, APMA-induced s-KIT production abolished SCF-induced melanogenesis as effectively as a KIT-neutralizing antibody. Concomitantly, APMA and TACE inhibitors significantly decreased and increased melanin synthesis, respectively, in an in vitro skin model. Taken together, these findings provided an insight into the elaborate mechanism of SCF/m-KIT signaling in human melanocytes and suggested that production of s-KIT contributes to the regulation of human skin pigmentation. Journal of Investigative Dermatology (2008) 128, 1763-1772; doi:10.1038/jid.2008.9; published online 31 January 2008.

Hydantoins, triazolones, and imidazolones as selective non-hydroxamate inhibitors of tumor necrosis factor-α converting enzyme (TACE)

We have discovered selective and potent inhibitors of TACE that replace the common hydroxamate zinc binding group with a hydantoin, triazolone, and imidazolone heterocycle. These novel heterocyclic inhibitors of a zinc metalloprotease were designed using a pharmacophore model that we previously described while developing hydantoin and pyrimidinetrione (barbiturate) inhibitors of TACE. The potency and binding orientation of these inhibitors is discussed and they are modeled into the X-ray crystal structure of TACE and compared to hydroxamate and earlier hydantoin TACE inhibitors which share the same 4-[(2-methyl-4-quinolinyl)methoxy]benzoyl P1′ group.

Non-hydroxamate 5-phenylpyrimidine-2,4,6-trione derivatives as selective inhibitors of tumor necrosis factor-α converting enzyme

New inhibitors of tumor necrosis factor-α converting enzyme (TACE) were discovered with a pyrimidine-2,4,6-trione in place of the commonly used hydroxamic acid. These non-hydroxamate TACE inhibitors were developed by incorporating a 4-(2-methyl-4-quinolinylmethoxy)phenyl group, an optimized TACE selective P1′ group. Several leads were identified with IC 50 values around 100 nM in a porcine TACE assay and selective over MMP-1, -2, -9, -13, and aggrecanase.

Design of selective and soluble inhibitors of tumor necrosis factor-alpha converting enzyme (TACE).

A program to improve upon the in vitro, in vivo, and physicochemical properties of N-hydroxyformamide TACE inhibitor GW 3333 (1) is described. Using the primary structure of pro-TNF-alpha, along with a homology model of the catalytic domain of TACE based on the X-ray diffraction coordinates of adamalysin, we synthesized N-hydroxyformamide TACE inhibitors containing a P2' arginine side chain. Introduction of nitro and sulfonyl electron-withdrawing groups covalently bound to the P2' guanidine moiety rendered the inhibitors electronically neutral at cellular pH and led to potent inhibition of TNF-alpha release from stimulated macrophages. Inhibitors containing these arginine mimetics were found to have increased solubility in simulated gastric fluid (SGF) relative to 1, allowing for the incorporation of lipophilic P1' side chains which had the effect of retaining potent TACE inhibition, but reducing potency against matrix metalloproteases (MMPs) thus increasing overall selectivity against MMP1, MMP3, and MMP9. Selected compounds showed good to excellent in vivo TNF inhibition when administered via subcutaneous injection. One inhibitor, 28a, with roughly 10x selectivity over MMP1 and MMP3 and high solubility in SGF, was evaluated in the rat zymosan-induced pleuisy model of inflammation and found to inhibit zymosan-stimulated pleural TNF-alpha elevation by 30%.