Crystal structures of the DExH-box RNA helicase DHX9.

DHX9 is a DExH-box RNA helicase with versatile functions in transcription, translation, RNA processing and regulation of DNA replication. DHX9 has recently emerged as a promising target for oncology, but to date no mammalian structures have been published. Here, crystal structures of human, dog and cat DHX9 bound to ADP are reported. The three mammalian DHX9 structures share identical structural folds. Additionally, the overall architecture and the individual domain structures of DHX9 are highly conserved with those of MLE, the Drosophila orthologue of DHX9 previously solved in complex with RNA and a transition-state analogue of ATP. Due to differences in the bound substrates and global domain orientations, the localized loop conformations and occupancy of dsRNA-binding domain 2 (dsRBD2) differ between the mammalian DHX9 and MLE structures. The combined effects of the structural changes considerably alter the RNA-binding channel, providing an opportunity to compare active and inactive states of the helicase. Finally, the mammalian DHX9 structures provide a potential tool for structure-based drug-design efforts.

Development of assays to support identification and characterization of modulators of DExH-box helicase DHX9.

DHX9 is a DExH-box RNA helicase that utilizes hydrolysis of all four nucleotide triphosphates (NTPs) to power cycles of 3' to 5' directional movement to resolve and/or unwind double stranded RNA, DNA, and RNA/DNA hybrids, R-loops, triplex-DNA and G-quadraplexes. DHX9 activity is important for both viral amplification and maintaining genomic stability in cancer cells; therefore, it is a therapeutic target of interest for drug discovery efforts. Biochemical assays measuring ATP hydrolysis and oligonucleotide unwinding for DHX9 have been developed and characterized, and these assays can support high-throughput compound screening efforts under balanced conditions. Assay development efforts revealed DHX9 can use double stranded RNA with 18-mer poly(U) 3' overhangs and as well as significantly shorter overhangs at the 5' or 3' end as substrates. The enzymatic assays are augmented by a robust SPR assay for compound validation. A mechanism-derived inhibitor, GTPγS, was characterized as part of the validation of these assays and a crystal structure of GDP bound to cat DHX9 has been solved. In addition to enabling drug discovery efforts for DHX9, these assays may be extrapolated to other RNA helicases providing a valuable toolkit for this important target class.

Correction: Crystal structures of human procathepsin H.

[This corrects the article DOI: 10.1371/journal.pone.0200374.].

Crystal structures of human procathepsin H.

Cathepsin H is a member of the papain superfamily of lysosomal cysteine proteases. It is the only known aminopeptidase in the family and is reported to be involved in cancer and other major diseases. Like many other proteases, it is synthesized as an inactive proenzyme. Although the crystal structure of mature porcine cathepsin H revealed the binding of the mini-chain and provided structural basis for the aminopeptidase activity, detailed structural and functional information on the inhibition and activation of procathepsin H has been elusive. Here we present the crystal structures of human procathepsin H at 2.00 Å and 1.66 Å resolution. These structures allow us to explore in detail the molecular basis for the inhibition of the mature domain by the prodomain. Comparison with cathepsin H structure reveals how mini-chain reorients upon activation. We further demonstrate that procathepsin H is not auto-activated but can be trans-activated by cathepsin L.

Structural Basis for Apelin Control of the Human Apelin Receptor.

Apelin receptor (APJR) is a key regulator of human cardiovascular function and is activated by two different endogenous peptide ligands, apelin and Elabela, each with different isoforms diversified by length and amino acid sequence. Here we report the 2.6-Å resolution crystal structure of human APJR in complex with a designed 17-amino-acid apelin mimetic peptide agonist. The structure reveals that the peptide agonist adopts a lactam constrained curved two-site ligand binding mode. Combined with mutation analysis and molecular dynamics simulations with apelin-13 binding to the wild-type APJR, this structure provides a mechanistic understanding of apelin recognition and binding specificity. Comparison of this structure with that of other peptide receptors suggests that endogenous peptide ligands with a high degree of conformational flexibility may bind and modulate the receptors via a similar two-site binding mechanism.

The Panitumumab EGFR Complex Reveals a Binding Mechanism That Overcomes Cetuximab Induced Resistance.

Panitumumab and cetuximab target the epidermal growth factor receptor for the treatment of metastatic colorectal cancer. These therapies provide a significant survival benefit to patients with metastatic colorectal cancer with wild-type RAS. A single point mutation in the ectodomain of EGFR (S468R) confers acquired or secondary resistance in cetuximab treated patients, which is not observed in panitumumab-treated patients. Structural and biophysical studies presented here show this mutation directly blocks cetuximab binding to EGFR domain III and describes a unique mechanism by which panitumumab uses a central cavity to accommodate this mutation.

Facile Modulation of Antibody Fucosylation with Small Molecule Fucostatin Inhibitors and Cocrystal Structure with GDP-Mannose 4,6-Dehydratase.

The efficacy of therapeutic antibodies that induce antibody-dependent cellular cytotoxicity can be improved by reduced fucosylation. Consequently, fucosylation is a critical product attribute of monoclonal antibodies produced as protein therapeutics. Small molecule fucosylation inhibitors have also shown promise as potential therapeutics in animal models of tumors, arthritis, and sickle cell disease. Potent small molecule metabolic inhibitors of cellular protein fucosylation, 6,6,6-trifluorofucose per-O-acetate and 6,6,6-trifluorofucose (fucostatin I), were identified that reduces the fucosylation of recombinantly expressed antibodies in cell culture in a concentration-dependent fashion enabling the controlled modulation of protein fucosylation levels. 6,6,6-Trifluorofucose binds at an allosteric site of GDP-mannose 4,6-dehydratase (GMD) as revealed for the first time by the X-ray cocrystal structure of a bound allosteric GMD inhibitor. 6,6,6-Trifluorofucose was found to be incorporated in place of fucose at low levels (<1%) in the glycans of recombinantly expressed antibodies. A fucose-1-phosphonate analog, fucostatin II, was designed that inhibits fucosylation with no incorporation into antibody glycans, allowing the production of afucosylated antibodies in which the incorporation of non-native sugar is completely absent-a key advantage in the production of therapeutic antibodies, especially biosimilar antibodies. Inhibitor structure-activity relationships, identification of cellular and inhibitor metabolites in inhibitor-treated cells, fucose competition studies, and the production of recombinant antibodies with varying levels of fucosylation are described.

Fragment-Linking Approach Using (19)F NMR Spectroscopy To Obtain Highly Potent and Selective Inhibitors of ß-Secretase.

Fragment-based drug discovery (FBDD) has become a widely used tool in small-molecule drug discovery efforts. One of the most commonly used biophysical methods in detecting weak binding of fragments is nuclear magnetic resonance (NMR) spectroscopy. In particular, FBDD performed with (19)F NMR-based methods has been shown to provide several advantages over (1)H NMR using traditional magnetization-transfer and/or two-dimensional methods. Here, we demonstrate the utility and power of (19)F-based fragment screening by detailing the identification of a second-site fragment through (19)F NMR screening that binds to a specific pocket of the aspartic acid protease, ß-secretase (BACE-1). The identification of this second-site fragment allowed the undertaking of a fragment-linking approach, which ultimately yielded a molecule exhibiting a more than 360-fold increase in potency while maintaining reasonable ligand efficiency and gaining much improved selectivity over cathepsin-D (CatD). X-ray crystallographic studies of the molecules demonstrated that the linked fragments exhibited binding modes consistent with those predicted from the targeted screening approach, through-space NMR data, and molecular modeling.

Pharmaceutical Optimization of Peptide Toxins for Ion Channel Targets: Potent, Selective, and Long-Lived Antagonists of Kv1.3.

To realize the medicinal potential of peptide toxins, naturally occurring disulfide-rich peptides, as ion channel antagonists, more efficient pharmaceutical optimization technologies must be developed. Here, we show that the therapeutic properties of multiple cysteine toxin peptides can be rapidly and substantially improved by combining direct chemical strategies with high-throughput electrophysiology. We applied whole-molecule, brute-force, structure-activity analoging to ShK, a peptide toxin from the sea anemone Stichodactyla helianthus that inhibits the voltage-gated potassium ion channel Kv1.3, to effectively discover critical structural changes for 15× selectivity against the closely related neuronal ion channel Kv1.1. Subsequent site-specific polymer conjugation resulted in an exquisitely selective Kv1.3 antagonist (>1000× over Kv1.1) with picomolar functional activity in whole blood and a pharmacokinetic profile suitable for weekly administration in primates. The pharmacological potential of the optimized toxin peptide was demonstrated by potent and sustained inhibition of cytokine secretion from T cells, a therapeutic target for autoimmune diseases, in cynomolgus monkeys.

Design and preparation of a potent series of hydroxyethylamine containing ß-secretase inhibitors that demonstrate robust reduction of central ß-amyloid.

A series of potent hydroxyethyl amine (HEA) derived inhibitors of ß-site APP cleaving enzyme (BACE1) was optimized to address suboptimal pharmacokinetics and poor CNS partitioning. This work identified a series of benzodioxolane analogues that possessed improved metabolic stability and increased oral bioavailability. Subsequent efforts focused on improving CNS exposure by limiting susceptibility to Pgp-mediated efflux and identified an inhibitor which demonstrated robust and sustained reduction of CNS ß-amyloid (Aß) in Sprague-Dawley rats following oral administration.

Design and synthesis of potent, orally efficacious hydroxyethylamine derived ß-site amyloid precursor protein cleaving enzyme (BACE1) inhibitors.

We have previously shown that hydroxyethylamines can be potent inhibitors of the BACE1 enzyme and that the generation of BACE1 inhibitors with CYP 3A4 inhibitory activities in this scaffold affords compounds (e.g., 1) with sufficient bioavailability and pharmacokinetic profiles to reduce central amyloid-ß peptide (Aß) levels in wild-type rats following oral dosing. In this article, we describe further modifications of the P1-phenyl ring of the hydroxyethylamine series to afford potent, dual BACE1/CYP 3A4 inhibitors which demonstrate improved penetration into the CNS. Several of these compounds caused robust reduction of Aß levels in rat CSF and brain following oral dosing, and compound 37 exhibited an improved cardiovascular safety profile relative to 1.

A Potent and Orally Efficacious, Hydroxyethylamine-Based Inhibitor of ß-Secretase.

ß-Secretase inhibitors are potentially disease-modifying treatments for Alzheimer's disease. Previous efforts in our laboratory have resulted in hydroxyethylamine-derived inhibitors such as 1 with low nanomolar potency against ß-site amyloid precursor protein cleaving enzyme (BACE). When dosed intravenously, compound 1 was also shown to significantly reduce Aß40 levels in plasma, brain, and cerebral spinal fluid. Herein, we report further optimizations that led to the discovery of inhibitor 16 as a novel, potent, and orally efficacious BACE inhibitor.

From fragment screening to in vivo efficacy: optimization of a series of 2-aminoquinolines as potent inhibitors of beta-site amyloid precursor protein cleaving enzyme 1 (BACE1).

Using fragment-based screening of a focused fragment library, 2-aminoquinoline 1 was identified as an initial hit for BACE1. Further SAR development was supported by X-ray structures of BACE1 cocrystallized with various ligands and molecular modeling studies to expedite the discovery of potent compounds. These strategies enabled us to integrate the C-3 side chain on 2-aminoquinoline 1 extending deep into the P2' binding pocket of BACE1 and enhancing the ligand's potency. We were able to improve the BACE1 potency to subnanomolar range, over 10(6)-fold more potent than the initial hit (900 µM). Further elaboration of the physical properties of the lead compounds to those more consistent with good blood-brain barrier permeability led to inhibitors with greatly improved cellular activity and permeability. Compound 59 showed an IC(50) value of 11 nM on BACE1 and cellular activity of 80 nM. This compound was advanced into rat pharmacokinetic and pharmacodynamic studies and demonstrated significant reduction of Aß levels in cerebrospinal fluid (CSF).

Alternative conformations at the RNA-binding surface of the N-terminal U2AF(65) RNA recognition motif.

The essential pre-mRNA splicing factor, U2 auxiliary factor 65KD (U2AF(65)) recognizes the polypyrimidine tract (Py-tract) consensus sequence of the pre-mRNA using two RNA recognition motifs (RRMs), the most prevalent class of eukaryotic RNA-binding domain. The Py-tracts of higher eukaryotic pre-mRNAs are often interrupted with purines, yet U2AF(65) must identify these degenerate Py-tracts for accurate pre-mRNA splicing. Previously, the structure of a U2AF(65) variant in complex with poly(U) RNA suggested that rearrangement of flexible side-chains or bound water molecules may contribute to degenerate Py-tract recognition by U2AF(65). Here, the X-ray structure of the N-terminal RRM domain of U2AF(65) (RRM1) is described at 1.47 A resolution in the absence of RNA. Notably, RNA-binding by U2AF(65) selectively stabilizes pre-existing alternative conformations of three side-chains located at the RNA interface (Arg150, Lys225, and Arg227). Additionally, a flexible loop connecting the beta2/beta3 strands undergoes a conformational change to interact with the RNA. These pre-existing alternative conformations may contribute to the ability of U2AF(65) to recognize a variety of Py-tract sequences. This rare, high-resolution view of an important member of the RRM class of RNA-binding domains highlights the role of alternative side-chain conformations in RNA recognition.

Structural basis for polypyrimidine tract recognition by the essential pre-mRNA splicing factor U2AF65.

The essential pre-mRNA splicing factor, U2AF(65), guides the early stages of splice site choice by recognizing a polypyrimidine (Py) tract consensus sequence near the 3' splice site. Since Py tracts are relatively poorly conserved in higher eukaryotes, U2AF(65) is faced with the problem of specifying uridine-rich sequences, yet tolerating a variety of nucleotide substitutions found in natural Py tracts. To better understand these apparently contradictory RNA binding characteristics, the X-ray structure of the U2AF(65) RNA binding domain bound to a Py tract composed of seven uridines has been determined at 2.5 A resolution. Specific hydrogen bonds between U2AF(65) and the uracil bases provide an explanation for polyuridine recognition. Flexible side chains and bound water molecules form the majority of the base contacts and potentially could rearrange when the U2AF(65) structure adapts to different Py tract sequences. The energetic importance of conserved residues for Py tract binding is established by analysis of site-directed mutant U2AF(65) proteins using surface plasmon resonance.

Crystallization and preliminary X-ray analysis of a U2AF65 variant in complex with a polypyrimidine-tract analogue by use of protein engineering.

The large subunit of the essential pre-mRNA splicing factor U2 auxiliary factor (U2AF65) binds the polypyrimidine tract near the 3' splice site of pre-mRNA introns and directs the association of the U2 small nuclear ribonucleoprotein particle (U2 snRNP) of the spliceosome with the pre-mRNA. Protein engineering, in which the flexible linker region connecting tandem RNA-recognition motifs (RRMs) within the U2AF65 RNA-binding domain was partially deleted, allowed successful crystallization of the protein-nucleic acid complex. Cocrystals of a U2AF65 variant with a deoxyuridine dodecamer diffract X-rays to 2.9 angstroms resolution and contain one complex per asymmetric unit.

X-ray structure of a Rex-family repressor/NADH complex insights into the mechanism of redox sensing.

The redox-sensing repressor Rex regulates transcription of respiratory genes in response to the intra cellular NADH/NAD(+) redox poise. As a step toward elucidating the molecular mechanism of NADH/NAD(+) sensing, the X-ray structure of Thermus aquaticus Rex (T-Rex) bound to effector NADH has been determined at 2.9 A resolution. The fold of the C-terminal domain of T-Rex is characteristic of NAD(H)-dependent enzymes, whereas the N-terminal domain is similar to a winged helix DNA binding motif. T-Rex dimerization is primarily mediated by "domain-swapped" alpha helices. Each NADH molecule binds to the C-terminal domain near the dimer interface. In contrast to NAD(H)-dependent enzymes, the nicotinamide is deeply buried within a hydrophobic pocket that appears to preclude substrate entry. We show that T-Rex binds to the Rex operator, and NADH but not NAD(+) inhibits T-Rex/DNA binding activity. A mechanism for redox sensing by Rex family members is proposed by analogy with domain closure of NAD(H)-dependent enzymes.

The crystal structure of the UvsW helicase from bacteriophage T4.

In bacteriophage T4, the WXY system repairs DNA damage by a process that involves homologous recombination. This system comprises three proteins, the RecA-like recombination protein UvsX, a recombination mediator protein UvsY, and a helicase UvsW. Here we report the 2.0 A resolution crystal structure of the N-terminal two domains of the UvsW helicase (UvsWNF; residues 1-282). The structure reveals a typical helicase RecA-like domain linked to a small N-terminal alpha/beta domain that likely binds the nucleic acid substrate. The missing C-terminal portion of UvsW almost certainly corresponds to the second RecA-like domain typically found in monomeric helicases. The putative substrate binding domain is unique within the known helicase structures, and it resembles the novel "double-wing" DNA binding domain from the phage T4 MotA transcription factor that mediates the expression of T4 middle genes. The functional implications of this homology for the role of UvsW in T4 DNA metabolism are discussed.

The MotA transcription factor from bacteriophage T4 contains a novel DNA-binding domain: the 'double wing' motif.

MotA is a transcription factor from bacteriophage T4 that helps adapt the host Escherichia coli transcription apparatus to T4 middle promoters. We have determined the crystal structure of the C-terminal DNA-binding domain of MotA (MotCF) to 1.6 A resolution using multiwavelength, anomalous diffraction methods. The structure reveals a novel DNA-binding alpha/beta motif that contains an exposed beta-sheet surface that mediates interactions with the DNA. Independent biochemical experiments have shown that MotCF binds to one surface of a single turn of DNA through interactions in adjacent major and minor grooves. We present a model of the interaction in which beta-ribbons at opposite corners of the six-stranded beta-sheet penetrate the DNA grooves, and call the motif a 'double wing' to emphasize similarities to the 'winged-helix' motif. The model is consistent with data on how MotA functions at middle promoters, and provides an explanation for why MotA can form non-specific multimers on DNA.