Genetic association between the Pfk13 gene mutation and artemisinin resistance phenotype in Plasmodium falciparum isolates from Yunnan Province, China.

BACKGROUND: The problem of anti-malarial drug resistance is a long-term challenge faced by malaria control in Yunnan Province. Recently, the detection rates of chloroquine-resistant molecular markers (Plasmodium falciparum chloroquine resistant transporter, Pfcrt) and artemisinin-resistant molecular markers (P. falciparum kelch13 gene, ork13) were 85% and 35%, respectively. To understand the association of k13 gene mutation with artemisinin resistance in falciparum malaria cases, the difference in k13 gene differentiation between two populations and artemisinin resistance phenotype on falciparum malaria cases in Myanmar were analysed in this study. METHODS: This research involved all of falciparum malaria cases diagnosed continuously in Yunnan Province from 2013 to 2015 and some of falciparum malaria cases found in Lazar, Myanmar. Blood samples were taken from the former group for molecular epidemiological analysis of k13 gene mutations, and artemisinin resistance phenotypes of P. falciparum were observed in the latter group using the in vivo testing method recommended by the World Health Organization. Nested PCR was used to amplify the propeller domain of the k13 gene in P. falciparum, followed by sequencing. RESULTS: A total of 202 blood samples were collected from Yunnan Province and 382 blood samples were collected from falciparum malaria cases in Myanmar. 49 of 382 Myanmar cases were in vivo tested for artesunate resistance phenotype through full treatment course observation. At the same time, all the blood samples were screened for k13 gene mutation of P. falciparum. The genetic diversity of k13 was higher in the Plasmodium isolates from Yunnan Province than those from Myanmar cases. The genetic differentiation index of the two populations was 0.0410, where the intra- and inter-group variations were 95.9% and 4.1%, respectively. The odds ratio of artemisinin resistance phenotype and mutation at the locus 446 in k13 gene in Myanmar cases was 1.640, while the value was 1.840 based on the estimations of the mutations in the 12 loci. CONCLUSION: Although the Plasmodium isolates from Yunnan Province and those from Myanmar were collected from different sites, they still belong to the same geographical population. It is, therefore, reasonable to contrast the artemisinin resistance status of the Plasmodium population from Myanmar with the Plasmodium population from Yunnan Province. As a result, based on the molecular epidemiological investigation on k13 mutations of Plasmodium isolates in Yunnan Province and the determination of the artemisinin resistance on falciparum malaria cases in Myanmar, the positively genetic correlated was found between the k13 locus mutations with artemisinin resistance phenotype. This provides a basis for further monitoring the artemisinin resistance by detection some molecular markers in k13 gene of Plasmodium in Yunnan Province.

Hairpin loop-enhanced fluorescent copper nanoclusters and application in S1 nuclease detection.

Novel highly fluorescent copper nanoclusters (CuNCs) were prepared by using 24 adenine-thymine pair dsDNA (AT24) with six-base (X6) loops (AT24-X6-hairpin DNA) as an effective template. The AT24 double strand stem serves as a template for CuNC formation, and the six-base sequence loop acts as specific regions to enhance the fluorescence intensity of CuNCs. Relative to the AT24-CuNCs, AT24-X6-hairpin CuNCs have greater fluorescence (5 times enhancement). What's more, the influence of the hairpin loop with different base types and base numbers on the fluorescence of CuNCs was first proposed and investigated. By choosing an AT24 double strand stem, any types of base loops can enhance the fluorescence of CuNCs. However, the fluorescence enhancement would be reduced with an increasing number of hairpin loop sequences. Besides this, the successful detection of S1 nuclease demonstrates its potential to be a new and robust fluorescent probe for sensing applications.

[Polymorphism analysis of the block 5 region in merozoite surface protein-1 gene of imported and local Plasmodium vivax in Yunnan Province].

Objective: To analyze the sequence of Plasmodium vivax merozoite surface protein-1(PvMSP-1) and allele polymorphism in imported and local vivax malaria parasites in Yunnan Province. Methods: Blood samples on filter paper were collected from imported and local vivax malaria cases in Yunnan Province during August 2012 and September 2015 and information of epidemiological history was recorded. Plasmodium DNA was extracted by a DNA extraction kit, and the block 5 region in PvMSP-1 gene was amplified by PCR. The PCR products were sequenced and blasted with reference sequences M75674, AAN86237, M60807, ABJ53045, AAN86238 and BAA18977. The sequence polymorphism in block 5 region of PvMSP-1 was analyzed with MEGA 5.04 and Arlequin3.5.1 softwares. The conserved sites, genetic distances among sequences and Shannon-wiener index among alleles were calculated. The clustering tree was drawn according to the genetic distances between the amino-acid sequences. Results: A total of 847 blood samples were collected from the malaria cases, comprising of 61 samples from local cases, 66 from imported cases from Africa, and 720 from Myanmar. The block 5 region in PvMSP-1 was successfully amplified in 278 samples, and sequencing was successfully made in 206 of them. The peptide coded by the block 5 region had a length of 193 to 222 aa. The amino acid sequence alignment showed that in 206 samples the proportion of genotypes of Sal-1, Belem and Recombine was 59.2%(122/206),23.3%(48/206) and 17.5%(36/206), respectively. The proportion of Sal-1 genotype in imported cases from Myanmar and Africa and in local cases was 58.8%(104/177),73.3%(11/15) and 50%(7/14), respectively. The genotypes Sal-1, Belem and Recombine had 51, 9 and 6 different alleles. The 66 alleles had a Shannon Wiener index (H') of 0.955 and an expected heterozygosis (He) of 0.567. The 206 DNA sequences had a 665-bp homologous locus, comprising of 75 conserved sites (11.3%,75/665) and 590 variable sites (88.7%, 590/665). The genetic distances between sequences were all less than 0.4. The clustering analysis showed that the 206 sequences were clustered into two categories with three branches. The homology of Recombine with Belem genotype was 91%-92%, higher than with Sal-1 genotype (82%-83%). Conclusion: The block 5 region in PvMSP-1 gene from local and imported Plasmodium vivax in Yunnan Province has varied forms of alleles, and the Sal-1 genotype is predominant among the three genotypes.

[Polymorphism Analysis of Plasmodium falciparum K13 Gene Kelch Domain Associated with Resistance to Artemisinin in Yunnan Province].

Objective: To investigate the polymorphism of Plasmodium falciparum K13 gene kelch domain region and provide basis for understanding the artemisinin resistance of falciparum malaria in Yunnan Province. Methods: The filter blood samples and relative information of falciparum malaria cases were collected in 16 prefectures of Yunnan Province from January 2013 to December 2015. The source of infection was determined by epidemiological investigation and the place of case discovery was confirmed according to the China Information System for Disease Control and Prevention Epidemic Registration. The K13 gene kelch domain region was amplified by nested PCR, sequenced, and blasted against the reference strain 3D7（PF3D7_1343700）. The K13 gene kelch domain region polymorphism was analyzed with Mega 5.04. The variable sites and genetic distance between sequences were analyzed. The constituent ratio of amino acid mutation sites was calculated and analyzed with χ2 test. Results: A total of 202 blood samples were collected from 2013 to 2015, comprising 190 from imported cases, 12 from local cases in Yunnan Province. The constitutent ratio of infection cases were 30.7% （62/202）, 34.2% （69/202） and 35.1% （71/202） respectively, increased year by year. The K13 gene kelch domain was successfully amplified from 192 samples and 190 were successfully sequenced, detecting missense mutation of K13 gene in 66 samples, the mutation rate was 34.7% （66/190）. The detection rate of K13 gene mutation was 40.9% （27/66）, 37.9% （25/66） and 21.2% （14/66） respectively, decreased year by year. In this study, ten types of mutations were detected, which were F446I, A578S, N458Y, P574L, A676D, G449A, C469Y, V494I, E556D and S16L. The highest mutation rate occurred in F446I which was 72.7% （48/66）. The proportion of F446I mutation type was 58.3% （28/48） in an age-range of 18-56 years, 70.8% （34/48） in farmers, and 91.7% （44/48） in patients with infection source in Southeast Asia, all significantly higher than that of other groups with the same characteristics （41.7%, 20/48; 29.2%, 14/48; and 8.3%, 4/48, respectively）（χ2=4.633, 5.556 and 5.152, both P＜0.05）. There was a 248 bp homologous sequence in the 190 sequences, composed of 235 conservative sites （94.8%）, 13 variable sites （5.2%）, 5 parsim-info sites （2.0%）, and 8 singleton sites （3.2%）. The genetic distance among the 190 sequences ranged 0.000-0.036, with an average of 0.001±0.001. Conclusion: There are 10 types of mutations in the K13 kelch domain in Yunnan Province, the predominant mutation type was F446I.

High-throughput assays for superoxide and hydrogen peroxide: design of a screening workflow to identify inhibitors of NADPH oxidases.

Recent progress characterizing the reaction mechanism(s) of fluorescent probes with reactive oxygen species has made it possible to rigorously analyze these reactive species in biological systems. We have developed rapid high throughput-compatible assays for monitoring cellular production of superoxide radical anion and hydrogen peroxide using hydropropidine and coumarin boronic acid probes, respectively. Coupling plate reader-based fluorescence measurements with HPLC-based simultaneous monitoring of superoxide radical anion and hydrogen peroxide provides the basis for the screening protocol for NADPH oxidase (Nox) inhibitors. Using this newly developed approach along with the medium-throughput plate reader-based oximetry and EPR spin trapping as confirmatory assays, it is now eminently feasible to rapidly and reliably identify Nox enzyme inhibitors with a markedly lower rate of false positives. These methodological advances provide an opportunity to discover selective inhibitors of Nox isozymes, through enhanced conceptual understanding of their basic mechanisms of action.

A sensitive assay for trypsin using poly(thymine)-templated copper nanoparticles as fluorescent probes.

A new, simple and sensitive fluorescence strategy was developed for the trypsin assay based on copper nanoparticles (CuNPs) and its different fluorescence response toward trypsin-catalyzed hydrolysis of cytochrome c (Cyt c). Polythymine (poly T)-templated CuNPs served as effective fluorescent probes. Cyt c is well-known to act as a quencher. However, herein, a low concentration of Cyt c was designed specially to act as the substrate of trypsin to avoid the quenching effects by electron transfer from Cyt c to CuNPs. In the presence of trypsin, Cyt c hydrolyzes to small peptides, releasing free cysteine residues. Nonfluorescent coordination complexes were formed upon exposure to free cysteine residues by a metal-ligand bond between Cu atoms and sulfur atoms, leading to a decreased fluorescence response to CuNPs. This novel method for the quantitative determination of trypsin has a linear detection range from 0.25 µg mL(-1) to 1000 µg mL(-1) and a relatively low detection limit of 42 ng mL(-1). To the best of our knowledge, this is the first application of the trypsin-catalyzed hydrolysis reaction of Cyt c to produce quenching effect in bioanalysis, which provided a novel approach for the biochemical sensing strategy.

Dual myxovirus screen identifies a small-molecule agonist of the host antiviral response.

As we are confronted with an increasing number of emerging and reemerging viral pathogens, the identification of novel pathogen-specific and broad-spectrum antivirals has become a major developmental objective. Targeting of host factors required for virus replication presents a tangible approach toward obtaining novel hits with a broadened indication range. However, the identification of developable host-directed antiviral candidates remains challenging. We describe a novel screening protocol that interrogates the myxovirus host-pathogen interactome for broad-spectrum drug candidates and simultaneously probes for conventional, pathogen-directed hits. With resource efficiency and pan-myxovirus activity as the central developmental parameters, we explored coscreening against two distinct, independently traceable myxoviruses in a single-well setting. Having identified a pair of unrelated pathogenic myxoviruses (influenza A virus and measles virus) with comparable replication kinetics, we observed unimpaired coreplication of both viruses, generated suitable firefly and Renilla luciferase reporter constructs, respectively, and validated the protocol for up to a 384-well plate format. Combined with an independent counterscreen using a recombinant respiratory syncytial virus luciferase reporter, implementation of the protocol identified candidates with a broadened antimyxovirus profile, in addition to pathogen-specific hits. Mechanistic characterization revealed a newly discovered broad-spectrum lead that does not block viral entry but stimulates effector pathways of the innate cellular antiviral response. In summary, we provide proof of concept for the efficient discovery of broad-spectrum myxovirus inhibitors in parallel to para- and orthomyxovirus-specific hit candidates in a single screening campaign. The newly identified compound provides a basis for the development of a novel broad-spectrum small-molecule antiviral class.

Antiviral atropisomers: conformational energy surfaces by NMR for host-directed myxovirus blockers.

Biologically active organic molecules characterized by a high single bond torsional barrier generate isolable isomers (atropisomers) and offer a unique stereochemical component to the design of selective therapeutic agents. The present work presents a nanomolar active inhibitor of myxoviruses, which most likely acts by blocking one or more cellular host proteins but also, serendipitously, exhibits axial chirality with an energy barrier of ΔG((++)) ≥30 kcal/mol. The latter has been probed by variable temperature NMR and microwave irradiation and by high level DFT transition state analysis and force field calculations. Full conformational profiles of the corresponding (aR,S) and (aS,S) atropisomers at ambient temperature were derived by conformer deconvolution with NAMFIS (NMR Analysis by Molecular Flexibility In Solution) methodology to generate seven and eight individual conformations, each assigned a % population. An accurate evaluation of a key torsion angle at the center of the molecules associated with a (3)JC-S-C-H coupling constant was obtained by mapping the S-C bond rotation with the MPW1PW91/6-31G-d,p DFT method followed by fitting the resulting dihedral angles and J-values to a Karplus expression. Accordingly, we have developed a complete conformational profile of diastereomeric atropisomers consistent with both high and low rotational barriers. We expect this assessment to assist the rationalization of the selectivity of the two (aR,S) and (aS,S) forms against host proteins, while offering insights into their divergent toxicity behavior.

A model that predicts a real-time tumour surface using intra-treatment skin surface and end-of-expiration and end-of-inhalation planning CT images.

OBJECTIVES: To develop a mapping model between skin surface motion and internal tumour motion and deformation using end-of-exhalation (EOE) and end-of-inhalation (EOI) 3D CT images for tracking lung tumours during respiration. METHODS: Before treatment, skin and tumour surfaces were segmented and reconstructed from the EOE and the EOI 3D CT images. A non-rigid registration algorithm was used to register the EOE skin and tumour surfaces to the EOI, resulting in a displacement vector field that was then used to construct a mapping model. During treatment, the EOE skin surface was registered to the real-time, yielding a real-time skin surface displacement vector field. Using the mapping model generated, the input of a real-time skin surface can be used to calculate the real-time tumour surface. The proposed method was validated with and without simulated noise on 4D CT images from 15 patients at Léon Bérard Cancer Center and the 4D-lung dataset. RESULTS: The average centre position error, dice similarity coefficient (DSC), 95%-Hausdorff distance and mean distance to agreement of the tumour surfaces were 1.29 mm, 0.924, 2.76 mm, and 1.13 mm without simulated noise, respectively. With simulated noise, these values were 1.33 mm, 0.920, 2.79 mm, and 1.15 mm, respectively. CONCLUSIONS: A patient-specific model was proposed and validated that was constructed using only EOE and EOI 3D CT images and real-time skin surface images to predict internal tumour motion and deformation during respiratory motion. ADVANCES IN KNOWLEDGE: The proposed method achieves comparable accuracy to state-of-the-art methods with fewer pre-treatment planning CT images, which holds potential for application in precise image-guided radiation therapy.

Neuroprotection by selective allosteric potentiators of the EP2 prostaglandin receptor.

Activation of the Galphas-coupled EP2 receptor for prostaglandin E2 (PGE(2)) promotes cell survival in several models of tissue damage. To advance understanding of EP2 functions, we designed experiments to develop allosteric potentiators of this key prostaglandin receptor. Screens of 292,000 compounds identified 93 that at 20 microM (i) potentiated the cAMP response to a low concentration of PGE(2) by > 50%; (ii) had no effect on EP4 or beta2 adrenergic receptors, the cAMP assay itself, or the parent cell line; and (iii) increased the potency of PGE(2) on EP2 receptors at least 3-fold. In aqueous solution, the active compounds are largely present as nanoparticles that appear to serve as active reservoirs for bioactive monomer. From 94 compounds synthesized or purchased, based on the modification of one hit compound, the most active increased the potency of PGE(2) on EP2 receptors 4- to 5-fold at 10 to 20 microM and showed substantial neuroprotection in an excitotoxicity model. These small molecules represent previously undescribed allosteric modulators of a PGE(2) receptor. Our results strongly reinforce the notion that activation of EP2 receptors by endogenous PGE(2) released in a cell-injury setting is neuroprotective.

Asymmetric synthesis of host-directed inhibitors of myxoviruses.

High-throughput screening (HTS) previously identified benzimidazole 1 (JMN3-003) as a compound with broad antiviral activity against different influenza viruses and paramyxovirus strains. In pursuit of a lead compound from this series for development, we sought to increase both the potency and the aqueous solubility of 1. Lead optimization has achieved compounds with potent antiviral activity against a panel of myxovirus family members (EC(50) values in the low nanomolar range) and much improved aqueous solubilities relative to that of 1. Additionally, we have devised a robust synthetic strategy for preparing 1 and congeners in an enantio-enriched fashion, which has allowed us to demonstrate that the (S)-enantiomers are generally 7- to 110-fold more potent than the corresponding (R)-isomers.

Activation of AKT is associated with metastasis of nasopharyngeal carcinoma.

Although radiotherapy results of nasopharyngeal carcinoma (NPC) at an early stage are better than other tumors, there is still a portion of patients with NPC who die before 5 years after the treatment; the underlying mechanism remains to be further understood. This study aims to investigate the mechanism by which NPC cells escape from irradiation. Patients with NPC at stage I was included in this study. All the patients were treated with irradiation. NPC biopsies were obtained from each patient before and 1 week after the start of radiotherapy. Expression of AKT in NPC tissue was assessed by Western blotting. NPC cell line, SUNE-1 cells, was treated with irradiation. The levels of AKT in SUNE-1 cells were also assessed. The frequency of apoptotic SUNE-1 cells was evaluated by flow cytometry. The levels of AKT were markedly increased in NPC tissue after treatment with irradiation, which was significantly correlated with NPC metastasis and mortality. After irradiation, NPC cell line, SUNE-1 cells, expressed higher levels of AKT than control cells. The knockdown of AKT in SUNE-1 cells markedly increased apoptotic cell rate. Radiotherapy can increase the levels of AKT in NPC cells that are associated with NPC metastasis and increase in mortality.

Monoketone analogs of curcumin, a new class of Fanconi anemia pathway inhibitors.

BACKGROUND: The Fanconi anemia (FA) pathway is a multigene DNA damage response network implicated in the repair of DNA lesions that arise during replication or after exogenous DNA damage. The FA pathway displays synthetic lethal relationship with certain DNA repair genes such as ATM (Ataxia Telangectasia Mutated) that are frequently mutated in tumors. Thus, inhibition of FANCD2 monoubiquitylation (FANCD2-Ub), a key step in the FA pathway, might target tumor cells defective in ATM through synthetic lethal interaction. Curcumin was previously identified as a weak inhibitor of FANCD2-Ub. The aim of this study is to identify derivatives of curcumin with better activity and specificity. RESULTS: Using a replication-free assay in Xenopus extracts, we screened monoketone analogs of curcumin for inhibition of FANCD2-Ub and identified analog EF24 as a strong inhibitor. Mechanistic studies suggest that EF24 targets the FA pathway through inhibition of the NF-kB pathway kinase IKK. In HeLa cells, nanomolar concentrations of EF24 inhibited hydroxyurea (HU)-induced FANCD2-Ub and foci in a cell-cycle independent manner. Survival assays revealed that EF24 specifically sensitizes FA-competent cells to the DNA crosslinking agent mitomycin C (MMC). In addition, in contrast with curcumin, ATM-deficient cells are twofold more sensitive to EF24 than matched wild-type cells, consistent with a synthetic lethal effect between FA pathway inhibition and ATM deficiency. An independent screen identified 4H-TTD, a compound structurally related to EF24 that displays similar activity in egg extracts and in cells. CONCLUSIONS: These results suggest that monoketone analogs of curcumin are potent inhibitors of the FA pathway and constitute a promising new class of targeted anticancer compounds.

Target analysis of the experimental measles therapeutic AS-136A.

No effective therapeutic is currently in place for improved case management of severe measles or the rapid control of outbreaks. Through high-throughput screening, we recently identified a novel small-molecule class that potently blocks activity of the measles virus (MeV) RNA-dependent RNA polymerase (RdRp) complex in transient replicon assays. However, the nature of the block in RdRp activity and the physical target of the compound remained elusive. Through real-time reverse transcription-PCR analysis, we demonstrate that the lead compound AS-136A blocks viral RNA synthesis in the context of an infection. Adaptation of different MeV strains to growth in the presence of the compound identified three candidate hot spots for resistance that are located in conserved domains of the viral polymerase (L protein) subunit of the RdRp complex. Rebuilding of individual mutations in RdRp-driven reporter assays and recombinant MeV traced the molecular basis for resistance to specific mutations in L. Mutations responsible for resistance cluster in the immediate vicinity of the proposed catalytic center for phosphodiester bond formation and neighboring conserved domains of L, providing support for effective inhibition of a paramyxovirus RdRp complex through interaction of a nonnucleoside small-molecule inhibitor with the L protein. Resistance mutations are located in regions of L that are fully conserved among viral isolates, and recombinant MeV harboring individual resistance mutations show some delay in the onset of viral growth in vitro. Taken together, these data support the hypothesis that acquiring mutations in these L domains may reduce virus fitness.

Curcumin analog cytotoxicity against breast cancer cells: exploitation of a redox-dependent mechanism.

A series of novel curcumin analogs, symmetrical dienones, were previously shown to possess cytotoxic, anti-angiogenic and anti-tumor activities. Analogs 1 (EF24) and 2 (EF31) share the dienone scaffold and serve as Michael acceptors. We propose that the anti-cancer effects of 1 and 2 are mediated in part by redox-mediated induction of apoptosis. In order to support this concept, 1 and 2 were treated with L-glutathione (GSH) and cysteine-containing dipeptides under mild conditions to form colorless water-soluble adducts, which were identified by LC/MS. Comparison of the cytotoxic action of 1, 2 and the corresponding conjugates, 1-(GSH)(2) and 2-(GSH)(2), illustrated that the two classes of compounds exhibit essentially identical cell killing capabilities. Compared with the yellow, somewhat light sensitive and nearly water insoluble compounds 1 and 2, the glutathione conjugates represent a promising new series of stable and soluble anti-tumor pro-drugs.

Inhibition of IkappaB kinase-nuclear factor-kappaB signaling pathway by 3,5-bis(2-flurobenzylidene)piperidin-4-one (EF24), a novel monoketone analog of curcumin.

The nuclear factor-kappaB (NF-kappaB) signaling pathway has been targeted for therapeutic applications in a variety of human diseases, includuing cancer. Many naturally occurring substances, including curcumin, have been investigated for their actions on the NF-kappaB pathway because of their significant therapeutic potential and safety profile. A synthetic monoketone compound termed 3,5-bis(2-flurobenzylidene)piperidin-4-one (EF24) was developed from curcumin and exhibited potent anticancer activity. Here, we report a mechanism by which EF24 potently suppresses the NF-kappaB signaling pathway through direct action on IkappaB kinase (IKK). We demonstrate that 1) EF24 induces death of lung, breast, ovarian, and cervical cancer cells, with a potency about 10 times higher than that of curcumin; 2) EF24 rapidly blocks the nuclear translocation of NF-kappaB, with an IC(50) value of 1.3 microM compared with curcumin, with an IC(50) value of 13 microM; 3) EF24 effectively inhibits tumor necrosis factor (TNF)-alpha-induced IkappaB phosphorylation and degradation, suggesting a role of this compound in targeting IKK; and 4) EF24 indeed directly inhibits the catalytic activity of IKK in an in vitro-reconstituted system. Our study identifies IKK as an effective target for EF24 and provides a molecular explanation for a superior activity of EF24 over curcumin. The effective inhibition of TNF-alpha-induced NF-kappaB signaling by EF24 extends the therapeutic application of EF24 to other NF-kappaB-dependent diseases, including inflammatory diseases such as rheumatoid arthritis.

High-throughput screening-based identification of paramyxovirus inhibitors.

Several members of the paramyxovirus family constitute major human pathogens that, collectively, are responsible for major morbidity and mortality worldwide. In an effort to develop novel therapeutics against measles virus (MV), a prominent member of the paramyxovirus family, the authors report a high-throughput screening protocol that uses a nonrecombinant primary MV strain as targets. Implementation of the assay has yielded 60 hit candidates from a 137,500-entry library. Counterscreening and generation of dose-response curves narrows this pool to 35 compounds with active concentrations < or =15.3 microM against the MV-Alaska strain and specificity indices ranging from 36 to >500. Library mining for structural analogs of several confirmed hits combined with retesting of identified candidates reveals a high accuracy of primary hit identification. Eleven of the confirmed hits interfere with viral entry, whereas the remaining 24 compounds target postentry steps of the viral life cycle. Activity testing against selected members of the paramyxovirus family reveals 3 patterns of activity: 1) exclusively MV-specific blockers, 2) inhibitors of MV and related viruses of the same genus, and 3) broader range inhibitors with activity against a different Paramyxovirinae genus. Representatives of the last class may open avenues for the development of broad-range paramyxovirus inhibitors through hit-to-lead chemistry.

Synthesis and SAR study of N-(4-hydroxy-3-(2-hydroxynaphthalene-1-yl)phenyl)-arylsulfonamides: heat shock protein 90 (Hsp90) inhibitors with submicromolar activity in an in vitro assay.

Heat shock protein 90 is emerging as an important target in cancer chemotherapy. In a program directed toward identifying novel chemical probes for Hsp90, we found N-(4-hydroxy-3-(2-hydroxynaphthalene-1-yl)phenyl)benzene sulfonamide as an Hsp90 inhibitor with very weak activity. In this report, we present a new and general method for the synthesis of a variety of analogs around this scaffold, and discuss their structure-activity relationships.

Discovery of aminoquinolines as a new class of potent inhibitors of heat shock protein 90 (Hsp90): Synthesis, biology, and molecular modeling.

The molecular chaperone Hsp90 plays important roles in maintaining malignant phenotypes. Recent studies suggest that Hsp90 exerts high-affinity interactions with multiple oncoproteins, which are essential for the growth of tumor cells. As a result, research has focused on finding Hsp90 probes as potential and selective anticancer agents. In a high-throughput screening exercise, we identified quinoline 7 as a moderate inhibitor of Hsp90. Further hit identification, SAR studies, and biological investigation revealed several synthetic analogs in this series with micromolar activities in both fluorescent polarization (FP) assay and a cell-based Western blot (WB) assay. These compounds represent a new class of Hsp90 inhibitors with simple chemical structures.

Targeting tissue factor-expressing tumor angiogenesis and tumors with EF24 conjugated to factor VIIa.

Tissue factor (TF) is aberrantly expressed on tumor vascular endothelial cells (VECs) and on cancer cells in many malignant tumors, but not on normal VECs, making it a promising target for cancer therapy. As a transmembrane receptor for coagulation factor VIIa (fVIIa), TF forms a high-affinity complex with its cognate ligand, which is subsequently internalized through receptor-mediated endocytosis. Accordingly, we developed a method for selectively delivering EF24, a potent synthetic curcumin analog, to TF-expressing tumor vasculature and tumors using fVIIa as a drug carrier. EF24 was chemically conjugated to fVIIa through a tripeptide-chloromethyl ketone. After binding to TF-expressing targets by fVIIa, EF24 will be endocytosed along with the drug carrier and will exert its cytotoxicity. Our results showed that the conjugate inhibits vascular endothelial growth factor-induced angiogenesis in a rabbit cornea model and in a Matrigel model in athymic nude mice. The conjugate-induced apoptosis in tumor cells and significantly reduced tumor size in human breast cancer xenografts in athymic nude mice as compared with the unconjugated EF24. By conjugating potent drugs to fVIIa, this targeted drug delivery system has the potential to enhance therapeutic efficacy, while reducing toxic side effects. It may also prove to be useful for treating drug-resistant tumors and micro-metastases in addition to primary tumors.