Identification of multidentate tyrosyl-DNA phosphodiesterase 1 (TDP1) inhibitors that simultaneously access the DNA, protein and catalytic-binding sites by oxime diversification.

Tyrosyl-DNA phosphodiesterase 1 (TDP1) is a member of the phospholipase D family that can downregulate the anticancer effects of the type I topoisomerase (TOP1) inhibitors by hydrolyzing the 3'-phosphodiester bond between DNA and the TOP1 residue Y723 in the critical stalled intermediate that is the foundation of TOP1 inhibitor mechanism of action. Thus, TDP1 antagonists are attractive as potential enhancers of TOP1 inhibitors. However, the open and extended nature of the TOP1-DNA substrate-binding region has made the development of TDP1 inhibitors extremely challenging. In this study, starting from our recently identified small molecule microarray (SMM)-derived TDP1-inhibitory imidazopyridine motif, we employed a click-based oxime protocol to extend the parent platform into the DNA and TOP1 peptide substrate-binding channels. We applied one-pot Groebke-Blackburn-Bienayme multicomponent reactions (GBBRs) to prepare the needed aminooxy-containing substrates. By reacting these precursors with approximately 250 aldehydes in microtiter format, we screened a library of nearly 500 oximes for their TDP1 inhibitory potencies using an in vitro florescence-based catalytic assay. Select hits were structurally explored as their triazole- and ether-based isosteres. We obtained crystal structures of two of the resulting inhibitors bound to the TDP1 catalytic domain. The structures reveal that the inhibitors form hydrogen bonds with the catalytic His-Lys-Asn triads ("HKN" motifs: H263, K265, N283 and H493, K495, N516), while simultaneously extending into both the substrate DNA and TOP1 peptide-binding grooves. This work provides a structural model for developing multivalent TDP1 inhibitors capable of binding in a tridentate fashion with a central component situated within the catalytic pocket and extensions that project into both the DNA and TOP1 peptide substrate-binding regions.

Self-inhibited State of Venezuelan Equine Encephalitis Virus (VEEV) nsP2 Cysteine Protease: A Crystallographic and Molecular Dynamics Analysis.

The Venezuelan equine encephalitis virus (VEEV) belongs to the Togaviridae family and is pathogenic to both humans and equines. The VEEV non-structural protein 2 (nsP2) is a cysteine protease (nsP2pro) that processes the polyprotein and thus it is a drug target for inhibitor discovery. The atomic structure of the VEEV nsP2 catalytic domain was previously characterized by both X-ray crystallography and computational studies. A modified nsP2pro harboring a N475A mutation in the N terminus was observed to exhibit an unexpected conformation: the N-terminal residues bind to the active site, mimicking binding of a substrate. The large conformational change of the N terminus was assumed to be induced by the N475A mutation, as N475 has an important role in stabilization of the N terminus and the active site. This conformation was first observed in the N475A mutant, but we also found it while determining a crystal structure of the catalytically active nsP2pro containing the wild-type N475 active site residue and K741A/K767A surface entropy reduction mutations. This suggests that the N475A mutation is not a prerequisite for self-inhibition. Here, we describe a high resolution (1.46 Å) crystal structure of a truncated nsP2pro (residues 463-785, K741A/K767A) and analyze the structure further by molecular dynamics to study the active and self-inhibited conformations of nsP2pro and its N475A mutant. A comparison of the different conformations of the N-terminal residues sheds a light on the interactions that play an important role in the stabilization of the enzyme.

Phosphonic acid-containing inhibitors of tyrosyl-DNA phosphodiesterase 1.

Tyrosyl-DNA phosphodiesterase 1 (TDP1) repairs stalled type I topoisomerase (TOP1)-DNA complexes by hydrolyzing the phosphodiester bond between the TOP1 Y723 residue and the 3'-phosphate of its DNA substrate. Although TDP1 antagonists could potentially reduce the dose of TOP1 inhibitors needed to achieve effective anticancer effects, the development of validated TDP1 inhibitors has proven to be challenging. This may, in part, be due to the open and extended nature of the TOP1 substrate binding region. We have previously reported imidazopyrazines and imidazopyridines that can inhibit TDP1 catalytic function in vitro. We solved the TDP1 crystal structures with bound inhibitors of this class and found that the dicarboxylic acid functionality within the N-(3,4-dicarboxyphenyl)-2-diphenylimidazo [1,2-a]pyridin-3-amine platform overlaps with aspects of phosphoryl substrate recognition. Yet phosphonic acids could potentially better-replicate cognate TOP1-DNA substrate binding interactions than carboxylic acids. As reported herein, we designed phosphonic acid-containing variants of our previously reported carboxylic acid-containing imidazopyrazine and imidazopyridine inhibitors and effected their synthesis using one-pot Groebke-Blackburn-Bienayme multicomponent reactions. We obtained crystal structures of TDP1 complexed with a subset of inhibitors. We discuss binding interactions of these inhibitors within the context of phosphate-containing substrate and carboxylic acid-based inhibitors. These compounds represent a new structural class of small molecule ligands that mimic aspects of the 3'-processed substrate that results from TDP1 catalysis.

Small molecule microarray identifies inhibitors of tyrosyl-DNA phosphodiesterase 1 that simultaneously access the catalytic pocket and two substrate binding sites.

Tyrosyl-DNA phosphodiesterase 1 (TDP1) is a member of the phospholipase D family of enzymes, which catalyzes the removal of both 3'- and 5'-DNA phosphodiester adducts. Importantly, it is capable of reducing the anticancer effects of type I topoisomerase (TOP1) inhibitors by repairing the stalled covalent complexes of TOP1 with DNA. It achieves this by promoting the hydrolysis of the phosphodiester bond between the Y723 residue of human TOP1 and the 3'-phosphate of its DNA substrate. Blocking TDP1 function is an attractive means of enhancing the efficacy of TOP1 inhibitors and overcoming drug resistance. Previously, we reported the use of an X-ray crystallographic screen of more than 600 fragments to identify small molecule variations on phthalic acid and hydroxyquinoline motifs that bind within the TDP1 catalytic pocket. Yet, the majority of these compounds showed limited (millimolar) TDP1 inhibitory potencies. We now report examining a 21 000-member library of drug-like Small Molecules in Microarray (SMM) format for their ability to bind Alexa Fluor 647 (AF647)-labeled TDP1. The screen identified structurally similar N,2-diphenylimidazo[1,2-a]pyrazin-3-amines as TDP1 binders and catalytic inhibitors. We then explored the core heterocycle skeleton using one-pot Groebke-Blackburn-Bienayme multicomponent reactions and arrived at analogs having higher inhibitory potencies. Solving TDP1 co-crystal structures of a subset of compounds showed their binding at the TDP1 catalytic site, while mimicking substrate interactions. Although our original fragment screen differed significantly from the current microarray protocol, both methods identified ligand-protein interactions containing highly similar elements. Importantly inhibitors identified through the SMM approach show competitive inhibition against TDP1 and access the catalytic phosphate-binding pocket, while simultaneously providing extensions into both the substrate DNA and peptide-binding channels. As such, they represent a platform for further elaboration of trivalent ligands, that could serve as a new genre of potent TDP1 inhibitors.

Characterization of a broadly specific cadaverine N-hydroxylase involved in desferrioxamine B biosynthesis in Streptomyces sviceus.

N-hydroxylating flavin-dependent monooxygenases (FMOs) are involved in the biosynthesis of hydroxamate siderophores, playing a key role in microbial virulence. Herein, we report the first structural and kinetic characterization of a novel alkyl diamine N-hydroxylase DesB from Streptomyces sviceus (SsDesB). This enzyme catalyzes the first committed step in the biosynthesis of desferrioxamine B, a clinical drug used to treat iron overload disorders. X-ray crystal structures of the SsDesB holoenzyme with FAD and the ternary complex with bound NADP+ were solved at 2.86 Å and 2.37 Å resolution, respectively, providing a structural view of the active site environment. SsDesB crystallized as a tetramer and the structure of the individual protomers closely resembles the structures of homologous N-hydroxylating FMOs from Erwinia amylovora (DfoA), Pseudomonas aeruginosa (PvdA), and Aspergillus fumigatus (SidA). Using NADPH oxidation, oxygen consumption, and product formation assays, kinetic parameters were determined for various substrates with SsDesB. SsDesB exhibited typical saturation kinetics with substrate inhibition at high concentrations of NAD(P)H as well as cadaverine. The apparent kcat values for NADPH in steady-state NADPH oxidation and oxygen consumption assays were 0.28 ± 0.01 s-1 and 0.24 ± 0.01 s-1, respectively. However, in product formation assays used to measure the rate of N-hydroxylation, the apparent kcat for NADPH (0.034 ± 0.008 s-1) was almost 10-fold lower under saturating FAD and cadaverine concentrations, reflecting an uncoupled reaction, and the apparent NADPH KM was 33 ± 24 µM. Under saturating FAD and NADPH concentrations, the apparent kcat and KM for cadaverine in Csaky assays were 0.048 ± 0.004 s-1 and 19 ± 9 µM, respectively. SsDesB also N-hydroxylated putrescine, spermidine, and L-lysine substrates but not alkyl (di)amines that were branched or had fewer than four methylene units in an alkyl chain. These data demonstrate that SsDesB has wider substrate scope compared to other well-studied ornithine and lysine N-hydroxylases, making it an amenable biocatalyst for the production of desferrioxamine B, derivatives, and other N-substituted products.

Identification of a ligand binding hot spot and structural motifs replicating aspects of tyrosyl-DNA phosphodiesterase I (TDP1) phosphoryl recognition by crystallographic fragment cocktail screening.

Tyrosyl DNA-phosphodiesterase I (TDP1) repairs type IB topoisomerase (TOP1) cleavage complexes generated by TOP1 inhibitors commonly used as anticancer agents. TDP1 also removes DNA 3' end blocking lesions generated by chain-terminating nucleosides and alkylating agents, and base oxidation both in the nuclear and mitochondrial genomes. Combination therapy with TDP1 inhibitors is proposed to synergize with topoisomerase targeting drugs to enhance selectivity against cancer cells exhibiting deficiencies in parallel DNA repair pathways. A crystallographic fragment screening campaign against the catalytic domain of TDP1 was conducted to identify new lead compounds. Crystal structures revealed two fragments that bind to the TDP1 active site and exhibit inhibitory activity against TDP1. These fragments occupy a similar position in the TDP1 active site as seen in prior crystal structures of TDP1 with bound vanadate, a transition state mimic. Using structural insights into fragment binding, several fragment derivatives have been prepared and evaluated in biochemical assays. These results demonstrate that fragment-based methods can be a highly feasible approach toward the discovery of small-molecule chemical scaffolds to target TDP1, and for the first time, we provide co-crystal structures of small molecule inhibitors bound to TDP1, which could serve for the rational development of medicinal TDP1 inhibitors.

The molecular mechanism of dsRNA processing by a bacterial Dicer.

Members of the ribonuclease (RNase) III family regulate gene expression by processing dsRNAs. It was previously shown that Escherichia coli (Ec) RNase III recognizes dsRNA with little sequence specificity and the cleavage products are mainly 11 nucleotides (nt) long. It was also shown that the mutation of a glutamate (EcE38) to an alanine promotes generation of siRNA-like products typically 22 nt long. To fully characterize substrate specificity and product size of RNase IIIs, we performed in vitro cleavage of dsRNAs by Ec and Aquifex aeolicus (Aa) enzymes and delineated their products by next-generation sequencing. Surprisingly, we found that both enzymes cleave dsRNA at preferred sites, among which a guanine nucleotide was enriched at a specific position (+3G). Based on sequence and structure analyses, we conclude that RNase IIIs recognize +3G via a conserved glutamine (EcQ165/AaQ161) side chain. Abolishing this interaction by mutating the glutamine to an alanine eliminates the observed +3G preference. Furthermore, we identified a second glutamate (EcE65/AaE64), which, when mutated to alanine, also enhances the production of siRNA-like products. Based on these findings, we created a bacterial Dicer that is ideally suited for producing heterogeneous siRNA cocktails to be used in gene silencing studies.

A Small-Molecule Microarray Approach for the Identification of E2 Enzyme Inhibitors in Ubiquitin-Like Conjugation Pathways.

E2 enzymes in ubiquitin-like conjugation pathways are important, highly challenging pharmacological targets, and despite significant efforts, few noncovalent modulators have been discovered. Small-molecule microarray (SMM)-based screening was employed to identify an inhibitor of the "undruggable" small ubiquitin-like modifier (SUMO) E2 enzyme Ubc9. The inhibitor, a degradation product from a commercial screening collection, was chemically synthesized and evaluated in biochemical, mechanistic, and structure-activity relationship studies. Binding to Ubc9 was confirmed through the use of ligand-detected nuclear magnetic resonance, and inhibition of sumoylation in a reconstituted enzymatic cascade was found to occur with an IC50 of 75 µM. This work establishes the utility of the SMM approach for identifying inhibitors of E2 enzymes, targets with few known small-molecule modulators.

Insights Into the Allosteric Inhibition of the SUMO E2 Enzyme Ubc9.

Conjugation of the small ubiquitin-like modifier (SUMO) to protein substrates is an important disease-associated posttranslational modification, although few inhibitors of this process are known. Herein, we report the discovery of an allosteric small-molecule binding site on Ubc9, the sole SUMO E2 enzyme. An X-ray crystallographic screen was used to identify two distinct small-molecule fragments that bind to Ubc9 at a site distal to its catalytic cysteine. These fragments and related compounds inhibit SUMO conjugation in biochemical assays with potencies of 1.9-5.8 mm. Mechanistic and biophysical analyses, coupled with molecular dynamics simulations, point toward ligand-induced rigidification of Ubc9 as a mechanism of inhibition.

Structures of the Middle East respiratory syndrome coronavirus 3C-like protease reveal insights into substrate specificity.

Middle East respiratory syndrome coronavirus (MERS-CoV) is a highly pathogenic virus that causes severe respiratory illness accompanied by multi-organ dysfunction, resulting in a case fatality rate of approximately 40%. As found in other coronaviruses, the majority of the positive-stranded RNA MERS-CoV genome is translated into two polyproteins, one created by a ribosomal frameshift, that are cleaved at three sites by a papain-like protease and at 11 sites by a 3C-like protease (3 CL(pro)). Since 3 CL(pro) is essential for viral replication, it is a leading candidate for therapeutic intervention. To accelerate the development of 3 CL(pro) inhibitors, three crystal structures of a catalytically inactive variant (C148A) of the MERS-CoV 3 CL(pro) enzyme were determined. The aim was to co-crystallize the inactive enzyme with a peptide substrate. Fortuitously, however, in two of the structures the C-terminus of one protomer is bound in the active site of a neighboring molecule, providing a snapshot of an enzyme-product complex. In the third structure, two of the three protomers in the asymmetric unit form a homodimer similar to that of SARS-CoV 3 CL(pro); however, the third protomer adopts a radically different conformation that is likely to correspond to a crystallographic monomer, indicative of substantial structural plasticity in the enzyme. The results presented here provide a foundation for the structure-based design of small-molecule inhibitors of the MERS-CoV 3 CL(pro) enzyme.

Structural enzymology and inhibition of the bi-functional folate pathway enzyme HPPK-DHPS from the biowarfare agent Francisella tularensis.

UNLABELLED: Two valid targets for antibiotic development, 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase (HPPK) and dihydropteroate synthase (DHPS), catalyze consecutive reactions in folate biosynthesis. In Francisella tularensis (Ft), these two activities are contained in a single protein, FtHPPK-DHPS. Although Pemble et al. (PLoS One 5, e14165) determined the structure of FtHPPK-DHPS, they were unable to measure the kinetic parameters of the enzyme. In this study, we elucidated the binding and inhibitory activities of two HPPK inhibitors (HP-18 and HP-26) against FtHPPK-DHPS, determined the structure of FtHPPK-DHPS in complex with HP-26, and measured the kinetic parameters for the dual enzymatic activities of FtHPPK-DHPS. The biochemical analyses showed that HP-18 and HP-26 have significant isozyme selectivity, and that FtHPPK-DHPS is unique in that the catalytic efficiency of its DHPS activity is only 1/260,000 of that of Escherichia coli DHPS. Sequence and structural analyses suggest that HP-26 is an excellent lead for developing therapeutic agents for tularemia, and that the very low DHPS activity is due, at least in part, to the lack of a key residue that interacts with the substrate p-aminobenzoic acid (pABA). A BLAST search of the genomes of ten F. tularensis strains indicated that the bacterium contains a single FtHPPK-DHPS. The marginal DHPS activity and the single copy existence of FtHPPK-DHPS in F. tularensis make this bacterium more vulnerable to DHPS inhibitors. Current sulfa drugs are ineffective against tularemia; new inhibitors targeting the unique pABA-binding pocket may be effective and less subject to resistance because any mutations introducing resistance may make the marginal DHPS activity unable to support the growth of F. tularensis. DATABASE: The coordinates and structure factors have been deposited in the Protein Data Bank under accession code 4PZV.

Rescuing aggregation-prone proteins in Escherichia coli with a dual His6-MBP tag.

Insolubility of recombinant proteins in Escherichia coli is a major impediment to their production for structural and functional studies. One way around this problem is to fuse an aggregation-prone protein to a highly soluble partner. E. coli maltose-binding protein (MBP) is widely recognized as a premier solubilizing agent. In this chapter, we describe how to construct dual His6-MBP-tagged fusion proteins by Gateway(®) recombinational cloning and how to predict their yield and solubility. We also describe a simple and rapid procedure to test the ability of a His6-MBP fusion protein to bind to Ni-NTA resin and to be digested by tobacco etch virus (TEV) protease, along with a method to assess the solubility of the target protein after it has been separated from His6-MBP.

Affibody-DyLight conjugates for in vivo assessment of HER2 expression by near-infrared optical imaging.

PURPOSE: Amplification of the HER2/neu gene and/or overexpression of the corresponding protein have been identified in approximately 20% of invasive breast carcinomas. Assessment of HER2 expression in vivo would advance development of new HER2-targeted therapeutic agents and, potentially, facilitate choice of the proper treatment strategy offered to the individual patient. We present novel HER2-specific probes for in vivo evaluation of the receptor status by near-infrared (NIR) optical imaging. EXPERIMENTAL DESIGN: Affibody molecules were expressed, purified, and labeled with NIR-fluorescent dyes. The binding affinity and specificity of the obtained probe were tested in vitro. For in vivo validation, the relationship of the measured NIR signal and HER2 expression was characterized in four breast cancer xenograft models, expressing different levels of HER2. Accumulation of Affibody molecules in tumor tissue was further confirmed by ex vivo analysis. RESULTS: Affibody-DyLight conjugates showed high affinity to HER2 (K(D) = 3.66±0.26). No acute toxicity resulted from injection of the probes (up to 0.5 mg/kg) into mice. Pharmacokinetic studies revealed a relatively short (37.53±2.8 min) half-life of the tracer in blood. Fluorescence accumulation in HER2-positive BT-474 xenografts was evident as soon as a few minutes post injection and reached its maximum at 90 minutes. On the other hand, no signal retention was observed in HER2-negative MDA-MB-468 xenografts. Immunostaining of extracted tumor tissue confirmed penetration of the tracer into tumor tissue. CONCLUSIONS: The results of our studies suggest that Affibody-DyLight-750 conjugate is a powerful tool to monitor HER2 status in a preclinical setting. Following clinical validation, it might provide complementary means for assessment of HER2 expression in breast cancer patients (assuming availability of proper NIR scanners) and/or be used to facilitate detection of HER2-positive metastatic lesions during NIR-assisted surgery.

Hyperthermia-triggered intracellular delivery of anticancer agent to HER2(+) cells by HER2-specific affibody (ZHER2-GS-Cys)-conjugated thermosensitive liposomes (HER2(+) affisomes).

We previously reported the formulation and physical properties of HER2 (human epidermal growth factor receptor 2)-specific affibody (ZHER2:342-Cys) conjugated thermosensitive liposomes (HER2(+)affisomes). Here we examined localized delivery potential of these affisomes by monitoring cellular interactions, intracellular uptake, and hyperthermia-induced effects on drug delivery. We modified ZHER2:342-Cys by introducing a glycine-serine spacer before the C-terminus cysteine (called ZHER2-GS-Cys) to achieve accessibility to cell surface expressed HER2. This modification did not affect HER2-specific binding and ZHER2-GS-Cys retained its ability to conjugate to the liposomes containing dipalmitoyl phosphatidyl choline: DSPE-PEG2000-Malemide, 96:04 mole ratios (HER2(+)affisomes). HER2(+)affisomes were either (i) fluorescently labeled with rhodamine-PE and calcein or (ii) loaded with an anticancer drug doxorubicin (DOX). Fluorescently labeled HER2(+) affisomes showed at least 10-fold increase in binding to HER2(+) cells (SK-BR-3) when compared to HER2(-) cells (MDA-MB-468) at 37°C. A competition experiment using free ZHER2-GS-Cys blocked HER2(+) affisome-SK-BR-3 cell associations. Imaging with confocal microscopy showed that HER2(+) affisomes accumulated in the cytosol of SK-BR-3 cells at 37°C. Hyperthermia-induced intracellular release experiments showed that the treatment of HER2(+) affisome/SK-BR-3 cell complexes with a 45°C (±1°C) pre-equilibrated buffer resulted in cytosolic delivery of calcein. Substantial calcein release was observed within 20min at 45°C, with no effect on cell viability under these conditions. Similarly, DOX-loaded HER2(+)affisomes showed at least 2- to 3-fold higher accumulation of DOX in SK-BR-3 cells as compared to control liposomes. DOX-mediated cytotoxicity was more pronounced in SK-BR-3 cells especially at lower doses of HER2(+)affisomes. Brief exposure of liposome-cell complexes at 45°C prior to the onset of incubations for cell killing assays resulted in enhanced cytotoxicity for affisomes and control liposomes. However, Doxil (a commercially available liposome formulation) showed significantly lower toxicity under identical conditions. Therefore, our data demonstrate that HER2(+)affisomes encompass both targeting and triggering potential and hence may prove to be viable nanodrug delivery carriers for breast cancer treatment.

Sequence of the Escherichia coli O121 O-antigen gene cluster and detection of enterohemorrhagic E. coli O121 by PCR amplification of the wzx and wzy genes.

The DNA sequence of the 15,155-bp O-antigen gene cluster of Escherichia coli O121 was determined, and 14 open reading frames were identified (all had the same transcriptional direction). Analyses of results indicated that the wzx (O-antigen flippase) and wzy (O-antigen polymerase) genes were E. coli O121 specific, so regions in these two genes were chosen for development of PCR assays. The PCR assays using DNA from 99 E. coli O121 strains, strains representative of non-O121 E. coli serogroups, and strains of other bacterial genera and PCR assays using DNA from seven enrichments of swine fecal samples naturally contaminated with E. coli O121 showed specificity for E. coli O121. Thus, the PCR assay can be employed to reliably identify E. coli O121 and to potentially detect the organism in food, fecal, and environmental samples.