SPOP-mutant prostate cancer: Translating fundamental biology into patient care.

Comprehensive cancer genome studies have revealed genetically-defined subtypes of prostate cancer with distinct truncal driver mutations. Because prostate cancer has been largely seen as a rather uniform disease, the clinical significance of this discovery remained largely obscure. However, recent findings imply distinct biological features and therapeutic vulnerabilities linked to specific truncal mutations. Here we review our current understanding of prostate cancers harboring recurrent point mutations in the ubiquitin ligase adaptor protein SPOP and discuss opportunities for future clinical translation. More specifically, activation of the androgen receptor (AR) signaling emerges as the key oncogenic pathway. SPOP-mutant prostate cancer patients respond to AR inhibition in various clinical settings. Molecular insights on how mutant SPOP promotes tumorigenesis may open more specific therapeutic avenues which, in combination with conventional AR-targeting agents, could improve the outcome of patients with SPOP-mutant prostate cancer.

The Tumor Suppressor SASH1 Interacts With the Signal Adaptor CRKL to Inhibit Epithelial-Mesenchymal Transition and Metastasis in Colorectal Cancer.

Background & Aims: The tumor-suppressor sterile α motif- and Src-homology 3-domain containing 1 (SASH1) has clinical relevance in colorectal carcinoma and is associated specifically with metachronous metastasis. We sought to identify the molecular mechanisms linking decreased SASH1 expression with distant metastasis formation. Methods: SASH1-deficient, SASH1-depleted, or SASH1-overexpressing HCT116 colon cancer cells were generated by the Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated 9-method, RNA interference, and transient plasmid transfection, respectively. Epithelial-mesenchymal transition (EMT) was analyzed by quantitative reverse-transcription polymerase chain reaction, immunoblotting, immunofluorescence microscopy, migration/invasion assays, and 3-dimensional cell culture. Yeast 2-hybrid assays and co-immunoprecipitation/mass-spectrometry showed V-Crk avian sarcoma virus CT10 oncogene homolog-like (CRKL) as a novel interaction partner of SASH1, further confirmed by domain mapping, site-directed mutagenesis, co-immunoprecipitation, and dynamic mass redistribution assays. CRKL-deficient cells were generated in parental or SASH1-deficient cells. Metastatic capacity was analyzed with an orthotopic mouse model. Expression and significance of SASH1 and CRKL for survival and response to chemotherapy was assessed in patient samples from our department and The Cancer Genome Atlas data set. Results: SASH1 expression is down-regulated during cytokine-induced EMT in cell lines from colorectal, pancreatic, or hepatocellular cancer, mediated by the putative SASH1 promoter. Deficiency or knock-down of SASH1 induces EMT, leading to an aggressive, invasive phenotype with increased chemoresistance. SASH1 counteracts EMT through interaction with the oncoprotein CRKL, inhibiting CRKL-mediated activation of SRC kinase, which is crucially required for EMT. SASH1-deficient cells form significantly more metastases in vivo, depending entirely on CRKL. Patient tumor samples show significantly decreased SASH1 and increased CRKL expression, associated with significantly decreased overall survival. Patients with increased CRKL expression show significantly worse response to adjuvant chemotherapy. Conclusions: We propose SASH1 as an inhibitor of CRKL-mediated SRC signaling, introducing a potentially druggable mechanism counteracting chemoresistance and metastasis formation.

Homology Model and Docking-Based Virtual Screening for Ligands of Human Dyskerin as New Inhibitors of Telomerase for Cancer Treatment.

Immortality is one of the main features of cancer cells. Tumor cells have an unlimited replicative potential, principally due to the holoenzyme telomerase. Telomerase is composed mainly by dyskerin (DKC1), a catalytic retrotranscriptase (hTERT) and an RNA template (hTR). The aim of this work is to develop new inhibitors of telomerase, selecting the interaction between hTR⁻DKC1 as a target. We designed two models of the human protein DKC1: homology and ab initio. These models were evaluated by different procedures, revealing that the homology model parameters were the most accurate. We selected two hydrophobic pockets contained in the PUA (pseudouridine synthase and archaeosine transglycosylase) domain, using structural and stability analysis. We carried out a docking-based virtual screen on these pockets, using the reported mutation K314 as the center of the docking. The hDKC1 model was tested against a library of 450,000 drug-like molecules. We selected the first 10 molecules that showed the highest affinity values to test their inhibitory activity on the cell line MDA MB 231 (Monroe Dunaway Anderson Metastasis Breast cancer 231), obtaining three compounds that showed inhibitory effect. These results allowed us to validate our design and set the basis to continue with the study of telomerase inhibitors for cancer treatment.

Identification of potential inhibitors against nuclear Dam1 complex subunit Ask1 of Candida albicans using virtual screening and MD simulations.

Identification of hit compounds against specific target form the starting point for a drug discovery program. A consistent decline of new chemical entities (NCEs) in recent years prompted a challenge to explore newer approaches to discover potential hit compounds that in turn can be converted into leads, and ultimately drug with desired therapeutic efficacy. The vast amount of omics and activity data available in public databases offers an opportunity to identify novel targets and their potential inhibitors. State of the art in silico methods viz., clustering of compounds, virtual screening, molecular docking, MD simulations and MMPBSA calculations were employed in a pipeline to identify potential 'hits' against those targets as well whose structures, as of now, could only predict through threading approaches. In the present work, we have started from scratch, amino acid sequence of target and compounds retrieved from PubChem compound database, modeled it in such a way that led to the identification of possible inhibitors of Dam1 complex subunit Ask1 of Candida albicans. We also propose a ligand based binding site determination approach. We have identified potential inhibitors of Ask1 subunit of a Dam1 complex of C. albicans, which is required to prevent precocious spindle elongation in pre-mitotic phases. The proposed scheme may aid to find virtually potential inhibitors of other unique targets against candida.

Impact of Target Warhead and Linkage Vector on Inducing Protein Degradation: Comparison of Bromodomain and Extra-Terminal (BET) Degraders Derived from Triazolodiazepine (JQ1) and Tetrahydroquinoline (I-BET726) BET Inhibitor Scaffolds.

The design of proteolysis-targeting chimeras (PROTACs) is a powerful small-molecule approach for inducing protein degradation. PROTACs conjugate a target warhead to an E3 ubiquitin ligase ligand via a linker. Here we examined the impact of derivatizing two different BET bromodomain inhibitors, triazolodiazepine JQ1 and the more potent tetrahydroquinoline I-BET726, via distinct exit vectors, using different polyethylene glycol linkers to VHL ligand VH032. Triazolodiazepine PROTACs exhibited positive cooperativities of ternary complex formation and were more potent degraders than tetrahydroquinoline compounds, which showed negative cooperativities instead. Marked dependency on linker length was observed for BET-degrading and cMyc-driven antiproliferative activities in acute myeloid leukemia cell lines. This work exemplifies as a cautionary tale how a more potent inhibitor does not necessarily generate more potent PROTACs and underscores the key roles played by the conjugation. The provided insights and framework for structure-activity relationships of bivalent degraders are anticipated to have wide future applicability.

Conformational stabilization as a strategy to prevent nucleophosmin mislocalization in leukemia.

Nucleophosmin (NPM) is a nucleolar protein involved in ribosome assembly and cell homeostasis. Mutations in the C-terminal domain of NPM that impair native folding and localization are associated with acute myeloid leukemia (AML). We have performed a high-throughput screening searching for compounds that stabilize the C-terminal domain. We identified three hit compounds which show the ability to increase the thermal stability of both the C-terminal domain as well as full-length NPM. The best hit also seemed to favor folding of an AML-like mutant. Computational pocket identification and molecular docking support a stabilization mechanism based on binding of the phenyl/benzene group of the compounds to a particular hydrophobic pocket and additional polar interactions with solvent-accessible residues. Since these results indicate a chaperoning potential of our candidate hits, we tested their effect on the subcellular localization of AML-like mutants. Two compounds partially alleviated the aggregation and restored nucleolar localization of misfolded mutants. The identified hits appear promising as pharmacological chaperones aimed at therapies for AML based on conformational stabilization of NPM.

Machine-Learning-Assisted Approach for Discovering Novel Inhibitors Targeting Bromodomain-Containing Protein 4.

Bromodomain-containing protein 4 (BRD4) is implicated in the pathogenesis of a number of different cancers, inflammatory diseases and heart failure. Much effort has been dedicated toward discovering novel scaffold BRD4 inhibitors (BRD4is) with different selectivity profiles and potential antiresistance properties. Structure-based drug design (SBDD) and virtual screening (VS) are the most frequently used approaches. Here, we demonstrate a novel, structure-based VS approach that uses machine-learning algorithms trained on the priori structure and activity knowledge to predict the likelihood that a compound is a BRD4i based on its binding pattern with BRD4. In addition to positive experimental data, such as X-ray structures of BRD4-ligand complexes and BRD4 inhibitory potencies, negative data such as false positives (FPs) identified from our earlier ligand screening results were incorporated into our knowledge base. We used the resulting data to train a machine-learning model named BRD4LGR to predict the BRD4i-likeness of a compound. BRD4LGR achieved a 20-30% higher AUC-ROC than that of Glide using the same test set. When conducting in vitro experiments against a library of previously untested, commercially available organic compounds, the second round of VS using BRD4LGR generated 15 new BRD4is. Moreover, inverting the machine-learning model provided easy access to structure-activity relationship (SAR) interpretation for hit-to-lead optimization.

Beneficial Effects of Lemon Balm Leaf Extract on In Vitro Glycation of Proteins, Arterial Stiffness, and Skin Elasticity in Healthy Adults.

Glycation, a non-enzymatic glycosylation of proteins, induces tissue damage in association with various diseases and aging phenomena. Pentosidine, an advanced glycation end product, is involved in aging phenomena such as tissue stiffness. In this study, we aimed to find a potent anti-glycation food material and to verify its health benefits by clinical trial. From among 681 hot water plant extracts, lemon balm (Melissa officinalis; LB) leaf extract was selected and revealed to have more potent inhibitory activity for pentosidine formation than a representative anti-glycation agent, aminoguanidine. Rosmarinic acid (RA), a typical polyphenol in Lamiaceae plants, was identified as a major active component in LB extract (LBE). Furthermore, LBE or RA dose-dependently suppressed glycation-associated reactions such as increased fluorescence, yellowing of collagen fiber sheets, and degeneration of the fibrous structure of elastin fiber sheets. An open-label, parallel-group comparative trial was conducted in 28 healthy Japanese subjects aged 31-65 y who consumed LB tea (LB group) or barley tea (Control group) for 6 wk. The LB group showed significant reductions in brachial-ankle pulse wave velocity, reflecting arterial stiffness, and b* (yellow) color values in forearm skin compared with the Control group. A gender-stratified analysis revealed that cheek skin elasticity was significantly improved in the LB group compared with the Control group only in female subjects. It is concluded that the hot water extract of LB leaf has the potential to provide health benefits with regard to glycation-associated tissue damage in blood vessels and skin of healthy adults.

Plant and Human MORC Proteins Have DNA-Modifying Activities Similar to Type II Topoisomerases, but Require One or More Additional Factors for Full Activity.

To elucidate one or more mechanisms through which microrchidia (MORC) proteins impact immunity, epigenetic gene silencing, and DNA modifications, the enzymatic activities of plant MORCs were characterized. Previously, we showed that plant MORC1s have ATPase and DNA endonuclease activities. Here, we demonstrate that plant MORCs have topoisomerase type II (topo II)-like activities, as they i) covalently bind DNA, ii) exhibit DNA-stimulated ATPase activity, iii) relax or nick supercoiled DNA, iv) catenate DNA, and v) decatenante kinetoplast DNA. Mutational analysis of tomato SlMORC1 suggests that a K loop-like sequence is required to couple DNA binding to ATPase stimulation as well as for efficient SlMORC1's DNA relaxation and catenation activities and in planta suppression of INF1-induced cell death, which is related to immunity. Human MORCs were found to exhibit the same topo II-like DNA modification activities as their plant counterparts. In contrast to typical topo IIs, SlMORC1 appears to require one or more accessory factors to complete some of its enzymatic activities, since addition of tomato extracts were needed for ATP-dependent, efficient conversion of supercoiled DNA to nicked/relaxed DNA and catenanes and for formation of topoisomer intermediates. Both plant and human MORCs bind salicylic acid; this suppresses their decatenation but not relaxation activity.

Development of 4,5-dihydro-benzodiazepinone derivatives as a new chemical series of BRD4 inhibitors.

Bromodomains (BRDs) are protein interaction modules that selectively recognize ε -N-lysine residues, serving as key epigenetic readers and play a key role in epigenetic regulation of gene transcription. Bromodomain-containing protein 4 (BRD4), a protein containing two BRDs termed BD1 and BD2, has emerged as an attractive candidate for the development of inhibitors targeting gene transcription in several types of cancers. In this study, we made structural modifications of previously reported BRD4 inhibitors, to develop new chemical scaffold 3,4-dihydroquinoxalin-2(1H)-one. Four series of compounds (compounds 7-10) were synthesized, and the BRD4-inhibitory activity and anti-proliferative effect of these compounds were evaluated. We found compound 10d has remarkable anti-proliferative activities toward leukemia cells and could induce apoptosis by mitochondrial pathways. Notably, the analysis of molecular docking suggested that hydrophobic interaction was essential for compound 10d to bind to BD1. In conclusion, these results demonstrate the potential of compound 10d to be utilized as a BRD4 inhibitor with apoptosis inducing effect in future leukemia therapy.

Identification of novel potent human testis-specific and bromodomain-containing protein (BRDT) inhibitors using crystal structure-based virtual screening.

Human testis-specific and bromodomain-containing protein (hBRDT) is essential for chromatin remodeling during spermatogenesis and is therefore an attractive target for the discovery of male contraceptive drugs. In this study, pharmacophore modeling was carried out based on the crystal structure of hBRDT in complex with the inhibitor, JQ1. The established pharmacophore model was used as a 3D search query to identify potent hBRDT inhibitors from an in-house chemical database. A molecular docking analysis was carried out to filter the obtained hit compounds. A total of 125 compounds was finally selected based on the ranking order and visual examination. These compounds were further evaluated by a protein-based in vitro assay. Four compounds with new chemical scaffolds were identified to be hBRDT inhibitors. The most active of these compounds, T480, had a half maximal inhibitory concentration (IC50) of 9.02 µM. The detailed analysis of the binding mode of compound T480 provides important information for the further development of novel BRDT inhibitors.

Evaluating p97 Inhibitor Analogues for Potency against p97-p37 and p97-Npl4-Ufd1 Complexes.

We previously found that the p97 cofactor, p47, significantly decreased the potency of some ATP-competitive p97 inhibitors such as ML240 [2-(2-amino-1H-benzo[d]imidazol-1-yl)-N-benzyl-8-methoxyquinazolin-4-amine] and ML241 [2-(2H-benzo[b][1,4]oxazin-4(3H)-yl)-N-benzyl-5,6,7,8 tetrahydroquinazolin-4-amine]. In this study, we aimed to evaluate inhibitor potencies against two additional p97 cofactor complexes, p97-p37 and p97-Npl4-Ufd1. We focused on these two cofactor complexes, because the protein sequence of p37 is 50 % identical to that of p47, and the Npl4-Ufd1 heterodimer (NU) is the most-studied p97 cofactor complex. We screened 200 p97 inhibitor analogues for their ability to inhibit the ATPase activity of p97 alone and of p97-p37 and p97-NU complexes. In contrast to the effect of p47, p37 and NU did not significantly change the potencies of most of the compounds. These results highlight differences among p97 cofactors in influencing p97 conformation and effects of inhibitors on p97 complexes, as compared to p97 alone. Continued efforts are needed to advance the development of complex-specific p97 inhibitors.

Crystal structures and mutagenesis of PPP-family ser/thr protein phosphatases elucidate the selectivity of cantharidin and novel norcantharidin-based inhibitors of PP5C.

Cantharidin is a natural toxin and an active constituent in a traditional Chinese medicine used to treat tumors. Cantharidin acts as a semi-selective inhibitor of PPP-family ser/thr protein phosphatases. Despite sharing a common catalytic mechanism and marked structural similarity with PP1C, PP2AC and PP5C, human PP4C was found to be insensitive to the inhibitory activity of cantharidin. To explore the molecular basis for this selectivity, we synthesized and tested novel C5/C6-derivatives designed from quantum-based modeling of the interactions revealed in the co-crystal structures of PP5C in complex with cantharidin. Structure-activity relationship studies and analysis of high-resolution (1.25Å) PP5C-inhibitor co-crystal structures reveal close contacts between the inhibitor bridgehead oxygen and both a catalytic metal ion and a non-catalytic phenylalanine residue, the latter of which is substituted by tryptophan in PP4C. Quantum chemistry calculations predicted that steric clashes with the bulkier tryptophan side chain in PP4C would force all cantharidin-based inhibitors into an unfavorable binding mode, disrupting the strong coordination of active site metal ions observed in the PP5C co-crystal structures, thereby rendering PP4C insensitive to the inhibitors. This prediction was confirmed by inhibition studies employing native human PP4C. Mutation of PP5C (F446W) and PP1C (F257W), to mimic the PP4C active site, resulted in markedly suppressed sensitivity to cantharidin. These observations provide insight into the structural basis for the natural selectivity of cantharidin and provide an avenue for PP4C deselection. The novel crystal structures also provide insight into interactions that provide increased selectivity of the C5/C6 modifications for PP5C versus other PPP-family phosphatases.

Crystal Structure of the Herpesvirus Nuclear Egress Complex Provides Insights into Inner Nuclear Membrane Remodeling.

Although nucleo-cytoplasmic transport is typically mediated through nuclear pore complexes, herpesvirus capsids exit the nucleus via a unique vesicular pathway. Together, the conserved herpesvirus proteins pUL31 and pUL34 form the heterodimeric nuclear egress complex (NEC), which, in turn, mediates the formation of tight-fitting membrane vesicles around capsids at the inner nuclear membrane. Here, we present the crystal structure of the pseudorabies virus NEC. The structure revealed that a zinc finger motif in pUL31 and an extensive interaction network between the two proteins stabilize the complex. Comprehensive mutational analyses, characterized both in situ and in vitro, indicated that the interaction network is not redundant but rather complementary. Fitting of the NEC crystal structure into the recently determined cryoEM-derived hexagonal lattice, formed in situ by pUL31 and pUL34, provided details on the molecular basis of NEC coat formation and inner nuclear membrane remodeling.

Identification of nuclear target proteins for S-nitrosylation in pathogen-treated Arabidopsis thaliana cell cultures.

Nitric oxide (NO) is a significant signalling molecule involved in the regulation of many different physiological processes in plants. One of the most imperative regulatory modes of action of NO is protein S-nitrosylation--the covalent attachment of an NO group to the sulfur atom of cysteine residues. In this study, we focus on S-nitrosylation of Arabidopsis nuclear proteins after pathogen infection. After treatment of Arabidopsis suspension cell cultures with pathogens, nuclear proteins were extracted and treated with the S-nitrosylating agent S-nitrosoglutathione (GSNO). A biotin switch assay was performed and biotin-labelled proteins were purified by neutravidin affinity chromatography and identified by mass spectrometry. A total of 135 proteins were identified, whereas nuclear localization has been described for 122 proteins of them. 117 of these proteins contain at least one cysteine residue. Most of the S-nitrosylated candidates were involved in protein and RNA metabolism, stress response, and cell organization and division. Interestingly, two plant-specific histone deacetylases were identified suggesting that nitric oxide regulated epigenetic processes in plants. In sum, this work provides a new collection of targets for protein S-nitrosylation in Arabidopsis and gives insight into the regulatory function of NO in the nucleus during plant defense response. Moreover, our data extend the knowledge on the regulatory function of NO in events located in the nucleus.

Selective activators of protein phosphatase 5 target the auto-inhibitory mechanism.

Protein phosphatase 5 (PP5) is an evolutionary conserved serine/threonine phosphatase. Its dephosphorylation activity modulates a diverse set of cellular factors including protein kinases and the microtubule-associated tau protein involved in neurodegenerative disorders. It is auto-regulated by its heat-shock protein (Hsp90)-interacting tetratricopeptide repeat (TPR) domain and its C-terminal α-helix. In the present study, we report the identification of five specific PP5 activators [PP5 small-molecule activators (P5SAs)] that enhance the phosphatase activity up to 8-fold. The compounds are allosteric modulators accelerating efficiently the turnover rate of PP5, but do barely affect substrate binding or the interaction between PP5 and the chaperone Hsp90. Enzymatic studies imply that the compounds bind to the phosphatase domain of PP5. For the most promising compound crystallographic comparisons of the apo PP5 and the PP5-P5SA-2 complex indicate a relaxation of the auto-inhibited state of PP5. Residual electron density and mutation analyses in PP5 suggest activator binding to a pocket in the phosphatase/TPR domain interface, which may exert regulatory functions. These compounds thus may expose regulatory mechanisms in the PP5 enzyme and serve to develop optimized activators based on these scaffolds.

Dual Screening of BPTF and Brd4 Using Protein-Observed Fluorine NMR Uncovers New Bromodomain Probe Molecules.

Bromodomain-containing protein dysregulation is linked to cancer, diabetes, and inflammation. Selective inhibition of bromodomain function is a newly proposed therapeutic strategy. We describe a (19)F NMR dual screening method for small molecule discovery using fluorinated tryptophan resonances on two bromodomain-containing proteins. The chemical shift dispersion of (19)F resonances within fluorine-labeled proteins enables the simultaneous analysis of two fluorinated bromodomains by NMR. A library of 229 small molecules was screened against the first bromodomain of Brd4 and the BPTF bromodomain. We report the first small molecule selective for BPTF over Brd4, termed AU1. The Kd = 2.8 µM for AU1, which is active in a cell-based reporter assay. No binding is detected with Brd4. Three new Brd4 inhibitors with submicromolar affinity were also discovered. Brd4 hits were validated in a thermal stability assay and potency determined via fluorescence anisotropy. The speed, ease of interpretation, and low protein concentration needed for protein-observed (19)F NMR experiments in a multiprotein format offers a new method to discover and characterize selective ligands for bromodomain-containing proteins.

Non-canonical Bromodomain within DNA-PKcs Promotes DNA Damage Response and Radioresistance through Recognizing an IR-Induced Acetyl-Lysine on H2AX.

Regulatory mechanisms underlying γH2AX induction and the associated cell fate decision during DNA damage response (DDR) remain obscure. Here, we discover a bromodomain (BRD)-like module in DNA-PKcs (DNA-PKcs-BRD) that specifically recognizes H2AX acetyl-lysine 5 (K5ac) for sequential induction of γH2AX and concurrent cell fate decision(s). First, top-down mass spectrometry of radiation-phenotypic, full-length H2AX revealed a radiation-inducible, K5ac-dependent induction of γH2AX. Combined approaches of sequence-structure modeling/docking, site-directed mutagenesis, and biochemical experiments illustrated that through docking on H2AX K5ac, this non-canonical BRD determines not only the H2AX-targeting activity of DNA-PKcs but also the over-activation of DNA-PKcs in radioresistant tumor cells, whereas a Kac antagonist, JQ1, was able to bind to DNA-PKcs-BRD, leading to re-sensitization of tumor cells to radiation. This study elucidates the mechanism underlying the H2AX-dependent regulation of DNA-PKcs in ionizing radiation-induced, differential DDR, and derives an unconventional, non-catalytic domain target in DNA-PKs for overcoming resistance during cancer radiotherapy.

Dual-probe electrochemical DNA biosensor based on the "Y" junction structure and restriction endonuclease assisted cyclic enzymatic amplification for detection of double-strand DNA of PML/RARα related fusion gene.

Taking advantage of "Y" junction structure and restriction endonuclease assisted cyclic enzymatic amplification, a dual-probe electrochemical DNA (DE-DNA) biosensor was designed to detect double-stranded DNA (dsDNA) of acute promyelocytic leukemia (APL) related gene. Two groups of detection probes were designed, and each group was composed of a biotinylated capture probe and an assisted probe. They were separately complementary with two strands of target dsDNA in order to prevent the reannealing of the two separate strands from target dsDNA. First, thiol functionalized capture probes (C1 and C2) were severally assembled onto two different gold electrodes, followed by hybridizing with target dsDNA (S1a-S1b) and assistant probes to form two Y-junction-structure ternary complexes. Subsequently, restriction sites on the ternary complexes were digested by Rsa I, which can release S1a, S1b and biotins from the electrode surfaces. Meanwhile, the released S1a and S1b can further hybridize with the unhybridized corresponding detection probes and then initiate another new hybridization-cleavage-separation cycle. Finally, the current signals were produced by the enzyme-catalyzed reaction of streptavidin-horse reddish peroxidase (streptavidin-HRP). The distinct difference in current signals between different sequences allowed detection of target dsDNA down to a low detection limit of 47 fM and presented excellent specificity with discriminating only a single-base mismatched dsDNA sequence. Moreover, this biosensor was also used for assay of polymerase chain reaction (PCR) samples with satisfactory results. According to the results, the power of the DE-DNA biosensor as a promising tool for the detection of APL and other diseases.

A Fragment-Based Ligand Screen Against Part of a Large Protein Machine: The ND1 Domains of the AAA+ ATPase p97/VCP.

The ubiquitous AAA+ ATPase p97 functions as a dynamic molecular machine driving several cellular processes. It is essential in regulating protein homeostasis, and it represents a potential drug target for cancer, particularly when there is a greater reliance on the endoplasmic reticulum-associated protein degradation pathway and ubiquitin-proteasome pathway to degrade an overabundance of secreted proteins. Here, we report a case study for using fragment-based ligand design approaches against this large and dynamic hexamer, which has multiple potential binding sites for small molecules. A screen of a fragment library was conducted by surface plasmon resonance (SPR) and followed up by nuclear magnetic resonance (NMR), two complementary biophysical techniques. Virtual screening was also carried out to examine possible binding sites for the experimental hits and evaluate the potential utility of fragment docking for this target. Out of this effort, 13 fragments were discovered that showed reversible binding with affinities between 140 µM and 1 mM, binding stoichiometries of 1:1 or 2:1, and good ligand efficiencies. Structural data for fragment-protein interactions were obtained with residue-specific [U-(2)H] (13)CH3-methyl-labeling NMR strategies, and these data were compared to poses from docking. The combination of virtual screening, SPR, and NMR enabled us to find and validate a number of interesting fragment hits and allowed us to gain an understanding of the structural nature of fragment binding.