A small-molecule ligand of valosin-containing protein/p97 inhibits cancer cell-accelerated fibroblast migration.

Carcinoma-associated fibroblasts are fibroblasts activated by surrounding cancer cells. Carcinoma-associated fibroblasts exhibit enhanced cell migration, which plays an important role in cancer metastasis. Previously, we demonstrated enhanced migration of NIH3T3 fibroblasts when they were cultured in the presence of MCF7 breast cancer cells. Human fibroblasts displayed a similar phenomenon even when they were co-cultured with cancer cells other than MCF7 cells. In this study, we screened ∼16,000 compounds from the RIKEN Natural Products Depository chemical library for inhibitors of enhanced NIH3T3 cell migration in the presence of MCF7. We identified NPD8733 as an inhibitor of cancer cell-enhanced fibroblast migration. This inhibition was observed not only in a wound-healing co-culture assay but also in a Transwell migration assay. Using NPD8733 and a structurally similar but inactive derivative, NPD8126, on immobilized beads, we found that NPD8733, but not NPD8126, specifically binds to valosin-containing protein (VCP)/p97, a member of the ATPase-associated with diverse cellular activities (AAA+) protein family. Using VCP truncation variants, we found that NPD8733 binds to the D1 domain of VCP. Because VCP's D1 domain is important for its function, we concluded that NPD8733 may act on VCP by binding to this domain. siRNA-mediated silencing of VCP in NIH3T3 fibroblasts, but not in MCF7 cells, reduced the migration of the co-cultured NIH3T3 fibroblasts. These results indicate that MCF7 activates the migration of NIH3T3 cells through VCP and that NPD8733 binds VCP and thereby inhibits its activity.

Comparative chemical array screening for p38γ/δ MAPK inhibitors using a single gatekeeper residue difference between p38α/ß and p38γ/δ.

Mammalian p38 mitogen activated protein kinases (MAPKs) are responsive to a variety of cellular stresses. The development of specific pyridinyl imidazole inhibitors has permitted the characterization of the p38 MAPK isoform p38α, which is expressed in most cell types, whereas the physiological roles of p38γ and p38δ are poorly understood. In this study, we report an approach for identifying selective inhibitors against p38γ and p38δ by focusing on the difference in gatekeeper residues between p38α/ß and p38γ/δ. Using GST-fused p38α wild type and T106M mutant constructs, wherein the p38α gatekeeper residue (Thr-106) was substituted by the p38γ/δ-type (Met), we performed comparative chemical array screening to identify specific binders of the mutant and identified SU-002 bound to p38αT106M specifically. SU-002 was found to inhibit p38αT106M but not p38α kinase activity in in vitro kinase assays. SU-005, the analog of SU-002, had inhibitory effects against the kinase activity of p38γ and p38δ in vitro but not p38α. In addition, SU-005 inhibited both p38γ and p38δ auto-phosphorylation in HeLa and HEK293T cells. These results demonstrate that the comparative chemical array screening approach is a powerful technique to explore specific inhibitors for mutant proteins with even single amino-acid substitutions in a high-throughput manner.

Screening of a virtual mirror-image library of natural products.

We established a facile access to an unexplored mirror-image library of chiral natural product derivatives using d-protein technology. In this process, two chemical syntheses of mirror-image substances including a target protein and hit compound(s) allow the lead discovery from a virtual mirror-image library without the synthesis of numerous mirror-image compounds.

Toxic tau oligomer formation blocked by capping of cysteine residues with 1,2-dihydroxybenzene groups.

Neurofibrillary tangles, composed of hyperphosphorylated tau fibrils, are a pathological hallmark of Alzheimer's disease; the neurofibrillary tangle load correlates strongly with clinical progression of the disease. A growing body of evidence indicates that tau oligomer formation precedes the appearance of neurofibrillary tangles and contributes to neuronal loss. Here we show that tau oligomer formation can be inhibited by compounds whose chemical backbone includes 1,2-dihydroxybenzene. Specifically, we demonstrate that 1,2-dihydroxybenzene-containing compounds bind to and cap cysteine residues of tau and prevent its aggregation by hindering interactions between tau molecules. Further, we show that orally administered DL-isoproterenol, an adrenergic receptor agonist whose skeleton includes 1,2-dihydroxybenzene and which penetrates the brain, reduces the levels of detergent-insoluble tau, neuronal loss and reverses neurofibrillary tangle-associated brain dysfunction. Thus, compounds that target the cysteine residues of tau may prove useful in halting the progression of Alzheimer's disease and other tauopathies.

Construction and application of a photo-cross-linked chemical array.

Chemical array technology is a powerful tool for high-throughput screening of small-molecule ligand-protein interactions. A chemical array is a collection of small-molecule compounds spotted and immobilized on a glass slide surface, providing a multiplex platform to identify small-molecule compounds binding to a protein of interest in high-throughput screening. Several research groups have developed a variety of methods for the immobilization of small molecules onto a solid matrix. We have developed a unique photo-cross-linked chemical array for immobilizing small molecules in a functional-group-independent manner. In this chapter, we describe in detail a protocol for the construction of a photo-cross-linked chemical array and its application for ligand screening by using a tag-fused protein.

Identification and characterization of an inhibitor of trichothecene 3-O-acetyltransferase, TRI101, by the chemical array approach.

Trichothecene 3-O-acetyltransferase (TRI101) is an indispensable enzyme for the biosynthesis of trichothecenes, a group of mycotoxins produced by Fusarium graminearum. In this study, an inhibitor of TRI101 was identified by chemical array analysis using compounds from the RIKEN Natural Products Depository (NPDepo) library. Although the addition of the identified enzyme inhibitor to the fungal culture did not inhibit trichothecene production, it can serve as a candidate lead compound in the development of a mycotoxin inhibitor that inactivates fungal defense mechanisms.

Boron-based inhibitors of acyl protein thioesterases 1 and 2.

Ras proteins are of importance in cell proliferation, and hence their mutated forms play causative roles in many kinds of cancer in different tissues. Inhibition of the Ras-depalmitoylating enzyme acyl protein thioesterases APT1 and -2 is a new approach to modulating the Ras cycle. Here we present boronic and borinic acid derivatives as a new class of potent and nontoxic APT inhibitors. These compounds were detected by extensive library screening using chemical arrays and turned out to inhibit human APT1 and -2 in a competitive mode. Furthermore, one of the molecules was demonstrated to inhibit Erk1/2 phosphorylation significantly.

Discovery of novel antiviral agents directed against the influenza A virus nucleoprotein using photo-cross-linked chemical arrays.

The nucleoprotein (NP) of the influenza virus is expressed in the early stage of infection and plays important roles in numerous steps of viral replication. NP is relatively well conserved compared with viral surface spike proteins. This study experimentally demonstrates that NP is a novel target for the development of new antiviral drugs against the influenza virus. First, artificial analogs of mycalamide A in a chemical array bound specifically with high affinity to NP. Second, the compounds inhibited multiplication of the influenza virus. Furthermore, surface plasmon resonance imaging experiments demonstrated that the binding activity of each compound to NP correlated with its antiviral activity. Finally, it was shown that these compounds bound NP within the N-terminal 110-amino acid region but their binding abilities were dramatically reduced when the N-terminal 13-amino acid tail was deleted, suggesting that the compounds might bind to this region, which mediates the nuclear transport of NP and its binding to viral RNA. These data suggest that compound binding to the N-terminal 13-amino acid tail region may inhibit viral replication by inhibiting the functions of NP. Collectively, these results strongly suggest that chemical arrays are convenient tools for the screening of viral product inhibitors.