Synthesis and evaluation of 3'- and 4'-substituted cyclohexyl noviomimetics that modulate mitochondrial respiration.

KU-32 (2) and KU-596 (3), are first and second generation cytoprotective novologues that are derivatives of novobiocin (1), a heat shock protein 90 (Hsp90) C-terminal inhibitor. Although 2 and 3 improve mitochondrial bioenergetics and have demonstrated considerable cytoprotective activity, they contain a synthetically demanding noviose sugar. This issue was initially addressed by creating noviomimetics, such as KU-1202 (4), which replaced the noviose sugar with ether-linked cyclohexyl derivatives that retained some cytoprotective potential due to their ability to increase mitochondrial bioenergetics. Based on structure-activity relationship (SAR) studies of KU-1202 (4), the current study investigated 3'- and 4'-substituted cyclohexyl scaffolds as noviomimetics and determined their efficacy at increasing mitochondrial bioenergetic as a marker for cytoprotective potential.

Synthesis and Evaluation of Small Molecule Disruptors of the Aha1/Hsp90 Complex for the Reduction of Tau Aggregation.

KU-177 was recently shown to disrupt interactions between Hsp90 and Aha1 in vitro. Subsequent studies in recombinant thioflavin T (ThT) assays demonstrated that KU-177 ablates Aha1-driven enhancement of Hsp90-dependent tau aggregation, which was confirmed by TEM. Using KU-177 as a lead compound, derivatives of KU-177 were synthesized and evaluated for their ability to disrupt Aha1/Hsp90 interactions and inhibit P301L tau aggregation. Preliminary structure-activity relationships were revealed, which led to the identification of a new lead compound that contains a cis-like amide bond. The new lead compounds retain the ability to disrupt Aha1/Hsp90 interactions in SH-SY5Y and SK-BR-3 cells without direct inhibition of Hsp90, providing a new scaffold for subsequent drug discovery efforts.

Rational Design and Identification of Harmine-Inspired, N-Heterocyclic DYRK1A Inhibitors Employing a Functional Genomic In Vivo Drosophila Model System.

Deregulation of dual-specificity tyrosine phosphorylation-regulated kinase 1A (DYRK1A) plays a significant role in developmental brain defects, early-onset neurodegeneration, neuronal cell loss, dementia, and several types of cancer. Herein, we report the discovery of three new classes of N-heterocyclic DYRK1A inhibitors based on the potent, yet toxic kinase inhibitors, harmine and harmol. An initial in vitro evaluation of the small molecule library assembled revealed that the core heterocyclic motifs benzofuranones, oxindoles, and pyrrolones, showed statistically significant DYRK1A inhibition. Further, the utilization of a low cost, high-throughput functional genomic in vivo model system to identify small molecule inhibitors that normalize DYRK1A overexpression phenotypes is described. This in vivo assay substantiated the in vitro results, and the resulting correspondence validates generated classes as architectural motifs that serve as potential DYRK1A inhibitors. Further expansion and analysis of these core compound structures will allow discovery of safe, more effective chemical inhibitors of DYRK1A to ameliorate phenotypes caused by DYRK1A overexpression.

Structure-Based Design, Synthesis, and Biological Evaluation of Hsp90ß-Selective Inhibitors.

The 90 kDa heat shock proteins (Hsp90) are molecular chaperones that are responsible for the folding and/or trafficking of ∼400 client proteins, many of which are directly associated with cancer progression. Consequently, inhibition of Hsp90 can exhibit similar activity as combination therapy as multiple signaling nodes can be targeted simultaneously. In fact, seventeen small-molecule inhibitors that bind the Hsp90 N-terminus entered clinical trials for the treatment of cancer, all of which exhibited pan-inhibitory activity against all four Hsp90 isoforms. Unfortunately, most demonstrated undesired effects alongside induction of the pro-survival heat shock response. As a result, isoform-selective inhibitors have been sought to overcome these detriments. Described herein is a structure-based approach to design Hsp90ß-selective inhibitors along with preliminary SAR. In the end, compound 5 was shown to manifest ∼370-fold selectivity for Hsp90ß versus Hsp90α, and induced the degradation of select Hsp90ß-dependent clients. These data support the development of Hsp90ß-selective inhibitors as a new paradigm to overcome the detriments associated with pan-inhibition of Hsp90.

Biological Evaluation of 5'-(N-Ethylcarboxamido)adenosine Analogues as Grp94-Selective Inhibitors.

The heat shock protein 90 kDa (Hsp90) family of chaperones is highly sought-after for the treatment of cancer and neurodegenerative diseases. Glucose regulated protein 94 (Grp94) is the endoplasmic reticulum localized isoform that is responsible for the maturation of proteins involved in cell adhesion and the immune response, including Toll-like receptors, immunoglobulins, and integrins. Consequently, Grp94 has been implicated in many different diseases including cancer metastasis, glaucoma, and viral infection. 5'-(N-Ethylcarboxamido)adenosine (NECA) was identified from a high-throughput screen as one of the first molecules to exhibit isoform selectivity toward Grp94, with the ethyl group projecting into a unique pocket within the ATP binding site of Grp94. This pocket has since been exploited by several groups to develop Grp94 selective inhibitors. Despite success in the development of other classes of inhibitors, relatively little work has been done to further develop inhibitors with the NECA scaffold. Unfortunately, NECA is also a potent adenosine receptor agonist, which is likely to confound any biological activity. Therefore, structure-activity relationship studies were performed on the NECA scaffold leading to the discovery of several molecules that displayed similar selectivity and affinity as the parent compound.

Selective Inhibition of the Hsp90α Isoform.

The 90 kDa heat shock protein (Hsp90) is a molecular chaperone that processes nascent polypeptides into their biologically active conformations. Many of these proteins contribute to the progression of cancer, and consequently, inhibition of the Hsp90 protein folding machinery represents an innovative approach toward cancer chemotherapy. However, clinical trials with Hsp90 N-terminal inhibitors have encountered deleterious side effects and toxicities, which appear to result from the pan-inhibition of all four Hsp90 isoforms. Therefore, the development of isoform-selective Hsp90 inhibitors is sought to delineate the pathological role played by each isoform. Herein, we describe a structure-based approach that was used to design the first Hsp90α-selective inhibitors, which exhibit >50-fold selectivity versus other Hsp90 isoforms.

The Development of Hsp90ß-Selective Inhibitors to Overcome Detriments Associated with pan-Hsp90 Inhibition.

The 90 kD heat shock proteins (Hsp90) are molecular chaperones that are responsible for the folding of select proteins, many of which are directly associated with cancer progression. Consequently, inhibition of the Hsp90 protein folding machinery results in a combinatorial attack on numerous oncogenic pathways. Seventeen small-molecule inhibitors of Hsp90 have entered clinical trials for the treatment of cancer, all of which bind the Hsp90 N-terminus and exhibit pan-inhibitory activity against all four Hsp90 isoforms, which may lead to adverse effects. The development of Hsp90 isoform-selective inhibitors represents an alternative approach toward the treatment of cancer and may limit some of these detriments. Described herein, is a structure-based approach to develop isoform-selective inhibitors of Hsp90ß, which induces the degradation of select Hsp90 clients without concomitant induction of Hsp90 levels. Together, these initial studies support the development of Hsp90ß-selective inhibitors as a method for overcoming the detriments associated with pan-inhibition.

Assay design and development strategies for finding Hsp90 inhibitors and their role in human diseases.

Heat shock protein 90 (Hsp90) is a molecular chaperone that facilitates the maturation of its client proteins including protein kinases, transcription factors, and steroid hormone receptors which are structurally and functionally diverse. These client proteins are involved in various cellular signaling pathways, and Hsp90 is implicated in various human diseases including cancer, inflammation, and diseases associated with protein misfolding; thus making Hsp90 a promising target for drug discovery. Some of its client proteins are well-known cancer targets. Instead of targeting these client proteins individually, however, targeting Hsp90 is more practical for cancer drug development. Efforts have been invested in recognizing potential drugs for clinical use that inhibit Hsp90 activity and result in the prevention of Hsp90 client maturation and dampening of subsequent signaling cascades. Here, we discuss current assays and technologies used to find and characterize Hsp90 inhibitors that include biophysical, biochemical, cell-based assays and computational modeling. This review highlights recent discoveries that N-terminal isoform-selective compounds and inhibitors that target the Hsp90 C-terminus that may offer the potential to overcome some of the detriments observed with pan Hsp90 inhibitors. The tools and assays summarized in this review should be used to develop Hsp90-targeting drugs with high specificity, potency, and drug-like properties that may prove immensely useful in the clinic.

Mitochondrial-targeted Hsp90 C-terminal inhibitors manifest anti-proliferative activity.

The development of C-terminal heat shock protein 90 kDa (Hsp90) inhibitors has emerged as a potential treatment for cancer. Similarly, small molecules that target the mitochondria have proven to be efficacious towards cancer, as the reprogramming of mitochondrial function is often associated with oncogenic transformation. Herein, we report the development of triphenylphosphonium (TPP)-conjugated Hsp90 C-terminal inhibitors, their anti-proliferative activity, and accumulation in the mitochondria. In general, TPP-conjugated Hsp90 C-terminal inhibitors were found to manifest increased activity against various cancer cell lines when compared to the parent compounds.

Tryptophan 299 is a conserved residue of human pregnane X receptor critical for the functional consequence of ligand binding.

PXR is a xenobiotic receptor that regulates drug metabolism by regulating the expression of drug-metabolizing enzymes including CYP3A4. It can be modulated by chemicals with different structures, functional groups and sizes. X-ray crystal structures of the ligand binding domain of human PXR (hPXR) alone or bound with agonists reveal a highly hydrophobic ligand binding pocket where the aromatic amino acid residue W299 appears to play a critical role in ligand binding. Here, we have investigated the role of W299 on the functional consequence of hPXR ligand binding. We first found that substitution of W299 with a hydrophobic residue retained its response to rifampicin, but substitution with a charged residue altered such agonist response in activating the transcription of CYP3A4. The activity of hPXR mutants on CYP3A4 expression correlates with the ability of hPXR mutants to interact with co-activator SRC-1. We further demonstrated that the effect of replacing W299 by residues with different side chains on hPXR's function varied depending on the specific agonist used. Finally we interpreted the cellular activity of the hPXR mutants by analyzing reported crystallographic data and proposing a model. Our findings reveal the essential role of W299 in the transactivation of hPXR in response to agonist binding, and provide useful information for designing modulators of hPXR.

PDZ Domain Recognition: Insight from Human Tax-Interacting Protein 1 (TIP-1) Interaction with Target Proteins.

Cellular signaling is primarily directed via protein-protein interactions. PDZ (PSD-95/Discs large/ZO-1 homologous) domains are well known protein-protein interaction modules involved in various key signaling pathways. Human Tax-interacting protein 1 (TIP-1), also known as glutaminase interaction protein (GIP), is a Class I PDZ domain protein that recognizes the consensus binding motif X-S/T-X-V/I/L-COOH of the C-terminus of its target proteins. We recently reported that TIP-1 not only interacts via the C-terminus of its target partner proteins but also recognizes an internal motif defined by the consensus sequence S/T-X-V/L-D in the target protein. Identification of new target partners containing either a C-terminal or internal recognition motif has rapidly expanded the TIP-1 protein interaction network. TIP-1 being composed solely of a single PDZ domain is unique among PDZ containing proteins. Since it is involved in many important signaling pathways, it is a possible target for drug design. In this mini review, we have discussed human TIP-1, its structure, mechanism of function, its interactions with target proteins containing different recognition motifs, and its involvement in human diseases. Understanding the molecular mechanisms of TIP-1 interactions with distinct target partners and their role in human diseases will be useful for designing novel therapeutics.

Targeting xenobiotic receptors PXR and CAR in human diseases.

Nuclear receptors such as the pregnane X receptor (PXR) and constitutive androstane receptor (CAR) are xenobiotic receptors regulating not only drug metabolism and disposition but also various human diseases such as cancer, diabetes, inflammatory disease, metabolic disease and liver diseases, suggesting that PXR and CAR are promising targets for drug discovery. Consequently, there is an urgent need to discover and develop small molecules that target these PXR- and/or CAR-mediated human-disease-related pathways for relevant therapeutic applications. This review proposes approaches to target PXR and CAR, either individually or simultaneously, in the context of various human diseases, taking into consideration the structural differences between PXR and CAR.

Thiazide-like diuretic drug metolazone activates human pregnane X receptor to induce cytochrome 3A4 and multidrug-resistance protein 1.

Human pregnane X receptor (hPXR) regulates the expression of drug-metabolizing enzyme cytochrome P450 3A4 (CYP3A4) and drug transporters such as multidrug-resistance protein 1 (MDR1). PXR can be modulated by small molecules, including Federal Drug Administration (FDA)-approved drugs, thus altering drug metabolism and causing drug-drug interactions. To determine the role of FDA-approved drugs in PXR-mediated regulation of drug metabolism and clearance, we screened 1481 FDA-approved small-molecule drugs by using a luciferase reporter assay in HEK293T cells and identified the diuretic drug metolazone as an activator of hPXR. Our data showed that metolazone activated hPXR-mediated expression of CYP3A4 and MDR1 in human hepatocytes and intestine cells and increased CYP3A4 promoter activity in various cell lines. Mammalian two-hybrid assays showed that hPXR recruits its co-activator SRC-1 upon metolazone binding in HepG2 cells, explaining the mechanism of hPXR activation. To understand the role of other commonly-used diuretics in hPXR activation and the structure-activity relationship of metolazone, thiazide and non-thiazide diuretics drugs were also tested but only metolazone activates hPXR. To understand the molecular mechanism, docking studies and mutational analysis were carried out and showed that metolazone binds in the ligand-binding pocket and interacts with mostly hydrophobic amino acid residues. This is the first report showing that metolazone activates hPXR. Because activation of hPXR might cause drug-drug interactions, metolazone should be used with caution for drug treatment in patients undergoing combination therapy.

Differential regulation of CYP3A4 promoter activity by a new class of natural product derivatives binding to pregnane X receptor.

The pregnane X receptor (PXR) regulates drug metabolism by regulating the expression of drug-metabolizing enzymes such as cytochrome P450 3A4 (CYP3A4), which is involved in the metabolism of >50% of clinically prescribed drugs. The activity of PXR can be controlled by the binding of small molecule agonists or antagonists. Because of its unique ligand binding pocket, PXR binds promiscuously to structurally diverse chemicals. To study the structure-activity relationship, novel modulators for PXR are needed. Here we report the virtual screening of ∼25,000 natural product derivatives from the ZINC database using the Molecular Operating Environment docking software tool against the PXR-rifampicin complex X-ray crystal structure. Our screening resulted in identification of compounds based on the lowest S score, which measures Gibbs free energy. Interestingly, we found that the compounds that bind directly to PXR, as revealed in an intrinsic tryptophan fluorescence assay, modulate CYP3A4 promoter activity differentially in HepG2 cells. Mutational analysis and docking studies showed that these compounds bind broadly in the ligand binding pocket but interact with different amino acid residues. We further investigated the mechanism of binding by analyzing the functional groups that are important for distinguishing agonists from antagonists. The approach we used to identify novel modulators that bind to PXR can be useful for finding novel modulators of PXR.

Modulation of xenobiotic receptors by steroids.

Nuclear receptors (NRs) are ligand-activated transcription factors that regulate the expression of their target genes. NRs play important roles in many human diseases, including metabolic diseases and cancer, and are therefore a key class of therapeutic targets. Steroids play important roles in regulating nuclear receptors; in addition to being ligands of steroid receptors, steroids (and their metabolites) also regulate other NRs, such as the pregnane X receptor and constitutive androstane receptor (termed xenobiotic receptors), which participate in steroid metabolism. Xenobiotic receptors have promiscuous ligand-binding properties, and their structurally diverse ligands include steroids and their metabolites. Therefore, steroids, their metabolism and metabolites, xenobiotic receptors, steroid receptors, and the respective signaling pathways they regulate have functional interactions. This review discusses these functional interactions and their implications for activities mediated by steroid receptors and xenobiotic receptors, focusing on steroids that modulate pathways involving the pregnane X receptor and constitutive androstane receptor. The emphasis of the review is on structure-function studies of xenobiotic receptors bound to steroid ligands.

New partner proteins containing novel internal recognition motif for human glutaminase interacting protein (hGIP).

Regulation of gene expression in cells is mediated by protein-protein, DNA-protein and receptor-ligand interactions. PDZ (PSD-95/Discs-large/ZO-1) domains are protein-protein interaction modules. PDZ-containing proteins function in the organization of multi-protein complexes controlling spatial and temporal fidelity of intracellular signaling pathways. In general, PDZ proteins possess multiple domains facilitating distinct interactions. The human glutaminase interacting protein (hGIP) is an unusual PDZ protein comprising entirely of a single PDZ domain and plays pivotal roles in many cellular processes through its interaction with the C-terminus of partner proteins. Here, we report the identification by yeast two-hybrid screening of two new hGIP-interacting partners, DTX1 and STAU1. Both proteins lack the typical C-terminal PDZ recognition motif but contain a novel internal hGIP recognition motif recently identified in a phage display library screen. Fluorescence resonance energy transfer and confocal microscopy analysis confirmed the in vivo association of hGIP with DTX1 and STAU1 in mammalian cells validating the previous discovery of S/T-X-V/L-D as a consensus internal motif for hGIP recognition. Similar to hGIP, DTX1 and STAU1 have been implicated in neuronal function. Identification of these new interacting partners furthers our understanding of GIP-regulated signaling cascades and these interactions may represent potential new drug targets in humans.

Specificity and promiscuity in human glutaminase interacting protein recognition: insight from the binding of the internal and C-terminal motif.

A large number of cellular processes are mediated by protein-protein interactions, often specified by particular protein binding modules. PDZ domains make up an important class of protein-protein interaction modules that typically bind to the C-terminus of target proteins. These domains act as a scaffold where signaling molecules are linked to a multiprotein complex. Human glutaminase interacting protein (GIP), also known as tax interacting protein 1, is unique among PDZ domain-containing proteins because it is composed almost exclusively of a single PDZ domain rather than one of many domains as part of a larger protein. GIP plays pivotal roles in cellular signaling, protein scaffolding, and cancer pathways via its interaction with the C-terminus of a growing list of partner proteins. We have identified novel internal motifs that are recognized by GIP through combinatorial phage library screening. Leu and Asp residues in the consensus sequence were identified to be critical for binding to GIP through site-directed mutagenesis studies. Structure-based models of GIP bound to two different surrogate peptides determined from nuclear magnetic resonance constraints revealed that the binding pocket is flexible enough to accommodate either the smaller carboxylate (COO(-)) group of a C-terminal recognition motif or the bulkier aspartate side chain (CH(2)COO(-)) of an internal motif. The noncanonical ILGF loop in GIP moves in for the C-terminal motif but moves out for the internal recognition motifs, allowing binding to different partner proteins. One of the peptides colocalizes with GIP within human glioma cells, indicating that GIP might be a potential target for anticancer therapeutics.

Identification of brain-specific angiogenesis inhibitor 2 as an interaction partner of glutaminase interacting protein.

The vast majority of physiological processes in living cells are mediated by protein-protein interactions often specified by particular protein sequence motifs. PDZ domains, composed of 80-100 amino acid residues, are an important class of interaction motif. Among the PDZ-containing proteins, glutaminase interacting protein (GIP), also known as Tax Interacting Protein TIP-1, is unique in being composed almost exclusively of a single PDZ domain. GIP has important roles in cellular signaling, protein scaffolding and modulation of tumor growth and interacts with a number of physiological partner proteins, including Glutaminase L, ß-Catenin, FAS, HTLV-1 Tax, HPV16 E6, Rhotekin and Kir 2.3. To identify the network of proteins that interact with GIP, a human fetal brain cDNA library was screened using a yeast two-hybrid assay with GIP as bait. We identified brain-specific angiogenesis inhibitor 2 (BAI2), a member of the adhesion-G protein-coupled receptors (GPCRs), as a new partner of GIP. BAI2 is expressed primarily in neurons, further expanding GIP cellular functions. The interaction between GIP and the carboxy-terminus of BAI2 was characterized using fluorescence, circular dichroism (CD) and nuclear magnetic resonance (NMR) spectroscopy assays. These biophysical analyses support the interaction identified in the yeast two-hybrid assay. This is the first study reporting BAI2 as an interaction partner of GIP.

Promiscuous binding at the crossroads of numerous cancer pathways: insight from the binding of glutaminase interacting protein with glutaminase L.

The glutaminase interacting protein (GIP) is composed of a single PDZ domain that interacts with a growing list of partner proteins, including glutaminase L, that are involved in a number of cell signaling and cancer pathways. Therefore, GIP makes a good target for structure-based drug design. Here, we report the solution structures of both free GIP and GIP bound to the C-terminal peptide analogue of glutaminase L. This is the first reported nuclear magnetic resonance structure of GIP in a complex with one of its binding partners. Our analysis of both free GIP and GIP in a complex with the glutaminase L peptide provides important insights into how a promiscuous binding domain can have affinity for multiple binding partners. Through a detailed chemical shift perturbation analysis and backbone dynamics studies, we demonstrate here that the binding of the glutaminase L peptide to GIP is an allosteric event. Taken together, the insights reported here lay the groundwork for the future development of a specific inhibitor for GIP.

A novel method of production and biophysical characterization of the catalytic domain of yeast oligosaccharyl transferase.

Oligosaccharyl transferase (OT) is a multisubunit enzyme that catalyzes N-linked glycosylation of nascent polypeptides in the lumen of the endoplasmic reticulum. In the case of Saccharomyces cerevisiae, OT is composed of nine integral membrane protein subunits. Defects in N-linked glycosylation cause a series of disorders known as congenital disorders of glycosylation (CDG). The C-terminal domain of the Stt3p subunit has been reported to contain the acceptor protein recognition site and/or catalytic site. We report here the subcloning, overexpression, and a robust but novel method of production of the pure C-terminal domain of Stt3p at 60-70 mg/L in Escherichia coli. CD spectra indicate that the C-terminal Stt3p is highly helical and has a stable tertiary structure in SDS micelles. The well-dispersed two-dimensional (1)H-(15)N HSQC spectrum in SDS micelles indicates that it is feasible to determine the atomic structure by NMR. The effect of the conserved D518E mutation on the conformation of the C-terminal Stt3p is particularly interesting. The replacement of a key residue, Asp(518), located within the WWDYG signature motif (residues 516-520), led to a distinct tertiary structure, even though both proteins have similar overall secondary structures, as demonstrated by CD, fluorescence and NMR spectroscopies. This observation strongly suggests that Asp(518) plays a critical structural role, in addition to the previously proposed catalytic role. Moreover, the activity of the protein was confirmed by saturation transfer difference and nuclear magnetic resonance titration studies.