Discovery of a macrocyclic o-aminobenzamide Hsp90 inhibitor with heterocyclic tether that shows extended biomarker activity and in vivo efficacy in a mouse xenograft model

A novel series of macrocyclic ortho-aminobenzamide Hsp90 inhibitors is reported. In continuation of our research, heterocycle-containing tethers were explored with the intent to further improve potency and minimize hERG liabilities. This effort culminated in the discovery of compound 10, which efficiently suppressed proliferation of HCT116 and U87 cells. This compound showed prolonged Hsp90-inhibitory activity at least 24 h post-administration consistent with elevated and prolonged exposure in the tumor. When studied in a xenograft model, the compound demonstrated significant suppression of tumor growth.     

Characterization of a homolog of DEAD-box RNA helicases in Toxoplasma gondii as a marker of cytoplasmic mRNP stress granules

Toxoplasma gondii is an obligate intracellular protozoan which infects one-third of the human population. Due to its high infection prevalence, Toxoplasma offers an ideal system for the study of host–parasite interaction. Similar to other eukaryotes, Toxoplasma maintains levels and localization of cytoplasmic mRNAs throughout its life cycle as part of a gene regulation network to meet all cellular and biochemical requirements. More recently, it was reported that the presence of cytoplasmic mRNA granules could contribute to the parasite pathogenesis and viability. Here we identified a novel Toxoplasma DEAD-box RNA helicase, referred to as Toxoplasma gondiiHomolog of DOZI (TgHoDI), because of its high homology (81%) to Plasmodium DOZI. TgHoDI is the functional ortholog of yeast DHH1, and its function was authenticated by complementation studies in Δdhh1 yeast strain. We demonstrated that TgHoDI is a marker of cytoplasmic RNA stress granules, which assemble when the parasites experience cellular stresses and translational arrest.     

Design and SAR of macrocyclic Hsp90 inhibitors with increased metabolic stability and potent cell-proliferation activity

A novel series of macrocyclic ortho-aminobenzamide Hsp90 inhibitors is reported. A basic nitrogen within the tether linking the aniline nitrogen atom to a tetrahydroindolone moiety allowed access to compounds with good physical properties. Important structure-activity relationship information was obtained from this series which led to the discovery of a soluble and stable compound which is potent in an Hsp90 binding and cell-proliferation assay.     

Upregulated miR-106a plays an oncogenic role in pancreatic cancer

Carcinogenesis is a complex process during which cells undergo genetic and epigenetic alterations. MicroRNAs control gene expression by negatively regulating protein-coding mRNAs. Several reports demonstrated that miR-106a is up-regulated in gastric and colorectal cancers and promotes tumor progression. In contrast, in glioma miR-106a plays the role of a tumor suppressor gene rather than an oncogene. Here we demonstrate that a high level of miR-106a expression is present in pancreatic cancer. Furthermore, our investigation shows that miR-106a has an oncogenic role in pancreatic tumorigenesis by promoting cancer cell proliferation, epithelial–mesenchymal transition and invasion by targeting tissue inhibitors of metalloproteinase 2 (TIMP-2). MiR-106a could be a critical therapeutic target in pancreatic cancer.     

Specific activity of a Bacillus thuringiensis strain against Locusta migratoria manilensis

Bacillus thuringiensis (Bt) has played an important role in biocontrol of pests. However, insecticidal activity of B. thuringiensis against locusts has been rarely reported. Bt strain BTH-13 exhibiting specific activity to locusts was isolated from a soil sample in China and characterized. Its bipyramidal parasporal crystal is mainly composed of a protein of 129 kDa, and produces a mature toxin of 64 kDa after activation. The pattern of total DNA from BTH-13 showed a large and three small plasmid bands. Known δ-endotoxin genes, cry1Aa, cry1Ab, cry1Ac, cry1C, cry3, cry4 and cry7Aa were not found from strain BTH-13 by PCR amplification. The sequence analysis of a DNA fragment produced by PCR amplification with degenerate cry-selective primers revealed that the fragment encoded a δ-endotoxin segment, which exhibited some similarity to several Cry proteins (41% of the highest similarity to Cry7Ba1). Toxicity tests were performed against Locusta migratoria manilensis, and the results demonstrated that trypsin-treated sporulated cultures and crystal proteins had high toxicity to larval and adult locusts. Cry toxin of BTH-13 was detected on the midguts of treated locusts using immunofluorescent technology, which confirmed the site of action of the crystal proteins in their toxicity for locusts.     

Divergence of cofactor recognition across evolution: coenzyme A binding in a prokaryotic arylamine N-acetyltransferase

Arylamine N-acetyltransferase (NAT) enzymes are widespread in nature. They serve to acetylate xenobiotics and/or endogenous substrates using acetyl coenzyme A (CoA) as a cofactor. Conservation of the architecture of the NAT enzyme family from mammals to bacteria has been demonstrated by a series of prokaryotic NAT structures, together with the recently reported structure of human NAT1. We report here the cloning, purification, kinetic characterisation and crystallographic structure determination of NAT from Mycobacterium marinum, a close relative of the pathogenic Mycobacterium tuberculosis. We have also determined the structure of M. marinum NAT in complex with CoA, shedding the first light on cofactor recognition in prokaryotic NATs. Surprisingly, the principal CoA recognition site in M. marinum NAT is located some 30 Å from the site of CoA recognition in the recently deposited structure of human NAT2 bound to CoA. The structure explains the Ping-Pong Bi-Bi reaction mechanism of NAT enzymes and suggests mechanisms by which the acetylated enzyme intermediate may be protected. Recognition of CoA in a much wider groove in prokaryotic NATs suggests that this subfamily may accommodate larger substrates than is the case for human NATs and may assist in the identification of potential endogenous substrates. It also suggests the cofactor-binding site as a unique subsite to target in drug design directed against NAT in mycobacteria.     

A Method for Screening and Validation of Resistant Mutations Against Kinase Inhibitors

The discovery of BCR/ABL as a driver oncogene in chronic myeloid leukemia (CML) resulted in the development of Imatinib, which, in fact, demonstrated the potential of targeting the kinase in cancers by effectively treating the CML patients. This observation revolutionized drug development to target the oncogenic kinases implicated in various other malignancies, such as, EGFR, B-RAF, KIT and PDGFRs. However, one major drawback of anti-kinase therapies is the emergence of drug resistance mutations rendering the target to have reduced or lost affinity for the drug. Understanding the mechanisms employed by resistant variants not only helps in developing the next generation inhibitors but also gives impetus to clinical management using personalized medicine. We reported a retroviral vector based screening strategy to identify the spectrum of resistance conferring mutations in BCR/ABL, which has helped in developing the next generation BCR/ABL inhibitors. Using Ruxolitinib and JAK2 as a drug target pair, here we describe in vitro screening methods that utilizes the mouse BAF3 cells expressing the random mutation library of JAK2 kinase.     

Ionizing radiation downregulates ASPM, a gene responsible for microcephaly in humans

Microcephaly is a malformation associated with in utero exposed atomic bomb survivors and can be induced in mice by fetal exposure to ionizing radiation (IR). The pathogenesis of IR-induced microcephaly, however, has not been fully understood. Our analyses of high-coverage expression profiling (HiCEP) demonstrated that the abnormal spindle-like microcephaly associated gene (ASPM) was down-regulated in irradiated human diploid fibroblasts. ASPM was recently reported as the causative gene for MCPH-5, the most common type of congenital microcephaly in humans. Here, we show that the expression of the Aspm gene was significantly reduced by IR in various human and murine cells. Additionally, Aspm was found downregulated in the irradiated fetal mouse brain, particularly in the ventricular zones. A similar suppression was observed in the irradiated neurosphere cultures. This is the first report suggesting that the suppression of Aspm by IR could be the initial molecular target leading to the future microcephaly formation.     

Secondary metabolites from Eurotium species, Aspergillus calidoustus and A. insuetus common in Canadian homes with a review of their chemistry and biological activities

As part of studies of metabolites from fungi common in the built environment in Canadian homes, we investigated metabolites from strains of three Eurotium species, namely E. herbariorum, E. amstelodami, and E. rubrum as well as a number of isolates provisionally identified as Aspergillus ustus. The latter have been recently assigned as the new species A. insuetus and A. calidoustus. E. amstelodami produced neoechinulin A and neoechinulin B, epiheveadride, flavoglaucin, auroglaucin, and isotetrahydroauroglaucin as major metabolites. Minor metabolites included echinulin, preechinulin and neoechinulin E. E. rubrum produced all of these metabolites, but epiheveadride was detected as a minor metabolite. E. herbariorum produced cladosporin as a major metabolite, in addition to those found in E. amstelodami. This species also produced questin and neoechinulin E as minor metabolites. This is the first report of epiheveadride occurring as a natural product, and the first nonadride isolated from Eurotium species. Unlike strains from mainly infection-related samples, largely from Europe, neither ophiobolins G and H nor austins were detected in the Canadian strains of A. insuetus and A. calidoustus tested, all of which had been reported from the latter species. TMC-120 A, B, C and a sesquiterpene drimane are reported with certainty for the first time from indoor isolates, as well as two novel related methyl isoquinoline alkaloids.     

Trypanosoma evansi: Pharmacological evidence of a nicotinic acetylcholine receptor

The role of calcium and its relevance have been deeply revised with respect to trypanosomatids, as the mechanism by which calcium enters trypanosomes was, until now, not well understood. There is evidence supporting the presence of a nAChR in another member of the trypanosomatidae family, Trypanosoma cruzi, these receptors being one entry path to calcium ions. The aims of this work were to determine if there was a nicotinic acetylcholine receptor (nAChR) in Trypanosoma evansi, and to subsequently perform a partial pharmacological characterization of this receptor.After being loaded with FURA-2AM, individual cells of T. evansi, were exposed to cholinergic compounds, and the cells displayed a dose-dependent response to carbachol. This observation indicated that a cholinergic receptor may be present in T. evansi. Although a dose-dependent response to muscarine could not be demonstrated, nicotine could promote an incremental dose-dependent response. The relative potency of this specific agonist of nAChR is in agreement with previous reports. The estimated affinity values were a K_d1 value of 29.6 ± 5.72 nM and a K_d2 value of 315.9 ± 26.6 nM, which is similar to the K_d value reported for the α4 nicotinic receptor. The Hill coefficients were determined to be an n₁ of 1.2 ± 0.3 and an n₂ of 4.2 ± 1.3. Finally, our calculations indicated that there are about 1020 receptors in each T. evansi parasite, which is approximately 15-fold lower than the number reported in Torpedo californica electric cells. These results suggest the presence of a nAChR in T. evansi, which is able to bind nicotinic ligands and induce calcium signals.     

Identification of novel PI3K inhibitors through a scaffold hopping strategy

A scaffold hopping strategy, including intellectual property availability assessment, was successfully applied for the discovery of novel PI3K inhibitors. Compounds were designed based on the chemical structure of the lead compound ETP-46321, a potent PI3K inhibitor, previously reported by our group. The new generated compounds showed good in vitro potency and selectivity, proved to inhibit potently the phosphorylation of AKT^Ser473 in cells and demonstrated to be orally bioavailable, thus becoming potential back-up candidates for ETP-46321.     

Design and synthesis of a novel, orally active, brain penetrant, tri-substituted thiophene based JNK inhibitor

The SAR of a series of tri-substituted thiophene JNK3 inhibitors is described. By optimizing both the N-aryl acetamide region of the inhibitor and the 4-position of the thiophene we obtained single digit nanomolar compounds, such as 47, which demonstrated an in vivo effect on JNK activity when dosed orally in our kainic acid mouse model as measured by phospho-c-jun reduction.     

Enhanced levels of plant cell cycle inhibitors hamper root-knot nematode-induced feeding site development

Root-knot nematodes (RKN) are highly specialized, obligatory plant parasites. These animals reprogram root cells to form large, multinucleate, and metabolically active feeding cells (giant cells) that provide a continuous nutrient supply during 3–6 weeks of the nematode’s life. The establishment and maintenance of physiologically fully functional giant cells are necessary for the survival of these nematodes. As such, giant cells may be useful targets for applying strategies to reduce damage caused by these nematodes, aiming the reduction of their reproduction. We have recently reported the involvement of cell cycle inhibitors of Arabidopsis, named Kip-Related Proteins (KRPs), on nematode feeding site ontogeny. Our results have demonstrated that this family of cell cycle inhibitors can be envisaged to efficiently disrupt giant cell development, based on previous reports which showed that alterations in KRP concentration levels can induce cell cycle transitions. Herein, we demonstrated that by overexpressing KRP genes, giant cells development is severely compromised as well as nematode reproduction. Thus, control of root-knot nematodes by modulating cell cycle-directed pathways through the enhancement of KRP protein levels may serve as an attractive strategy to limit damage caused by these plant parasites.     

Endosymbionts of Acanthamoeba Isolated from Domestic Tap Water in Korea

In a previous study, we reported our discovery of Acanthamoeba contamination in domestic tap water; in that study, we determined that some Acanthamoeba strains harbor endosymbiotic bacteria, via our molecular characterization by mitochondrial DNA restriction fragment length polymorphism (Mt DNA RFLP). Five (29.4%) among 17 Acanthamoeba isolates contained endosymbionts in their cytoplasm, as demonstrated via orcein staining. In order to estimate their pathogenicity, we conducted a genetic characterization of the endosymbionts in Acanthamoeba isolated from domestic tap water via 16S rDNA sequencing. The endosymbionts of Acanthamoeba sp. KA/WP3 and KA/WP4 evidenced the highest level of similarity, at 97% of the recently published 16S rDNA sequence of the bacterium, Candidatus Amoebophilus asiaticus. The endosymbionts of Acanthamoeba sp. KA/WP8 and KA/WP12 shared a 97% sequence similarity with each other, and were also highly similar to Candidatus Odyssella thessalonicensis, a member of the α-proteobacteria. The endosymbiont of Acanthamoeba sp. KA/WP9 exhibits a high degree of similarity (85-95%) with genus Methylophilus, which is not yet known to harbor any endosymbionts. This is the first report, to the best of our knowledge, to show that Methylophilus spp. can live in the cytoplasm of Acanthamoeba.     

Aromatase in human lung carcinoma

Lung cancer is the leading cause of cancer mortality in both women and men worldwide but gender differences exist in their clinical and biological manifestations. In particular, among life time non-smoker, female are far more likely to develop lung carcinoma than male. Recent studies demonstrated that estrogens are synthesized in situ in both male and female lung cancers through aromatase, suggesting that sex steroid may contribute to the pathogenesis and development of lung carcinoma. In addition, human lung carcinomas have been recently demonstrated to be frequently associated with expression of estrogen receptors in both male and female patients and a lower expression of aromatase was reported to be associated with better prognosis. Preclinical studies further demonstrated that aromatase inhibitor (AI) suppressed the lung tumor growth both in vitro and in vivo. These findings all suggest a potential role of intratumoral aromatase in biological behavior of non-small cell lung cancer (NSCLC), the most common form of human lung malignancy. Therefore, AIs may become viable therapeutic options for disease management in NSCLC patients but further studies are definitely required to obtain a better understanding of the potential roles of intratumoral aromatase expression as a predictive biomarker for clinical outcome in these NSCLC patients.     

Development of a prodrug of hydantoin based TACE inhibitor

Our research on hydantoin based TNF-α converting enzyme (TACE) inhibitors led to fused bi-heteroaryl hydantoin series that demonstrate sub-nanomolar potency (Ki) as well as excellent activity in human whole blood (hWBA). However, lead compound 2 posed some formulation challenges which prevented it for further development. A prodrug approach was investigated to address this issue. The pivalate prodrug 3 can be formulated as stable neutral form and demonstrated improved DMPK properties when compared with parent compound.     

Inhibition of Mycobacterium tuberculosis topoisomerase I by m-AMSA,a eukaryotic type II topoisomerase poison

m-AMSA, an established inhibitor of eukaryotic type II topoisomerases, exerts its cidal effect by binding to the enzyme–DNA complex thus inhibiting the DNA religation step. The molecule and its analogues have been successfully used as chemotherapeutic agents against different forms of cancer. After virtual screening using a homology model of the Mycobacterium tuberculosis topoisomerase I, we identified m-AMSA as a high scoring hit. We demonstrate that m-AMSA can inhibit the DNA relaxation activity of topoisomerase I from M. tuberculosis and Mycobacterium smegmatis. In a whole cell assay, m-AMSA inhibited the growth of both the mycobacteria.     

Characterization of field isolates of Trichoderma antagonistic against Rhizoctonia solani

The aim of the present study was to characterize sixteen isolates of Trichoderma originating from a field of sugar beet where disease patches caused by Rhizoctonia solani were observed. Use of both molecular and morphological characteristics gave consistent identification of the isolates. Production of water-soluble and volatile inhibitors, mycoparasitism and induced systemic resistance in plant host were investigated using in vitro and in vivo tests in both sterilized and natural soils. This functional approach revealed the intra-specific diversity as well as biocontrol potential of the different isolates. Different antagonistic mechanisms were evident for different strains. The most antagonistic strain, T30 was identified as Trichoderma gamsii. This is the first report of an efficient antagonistic strain of T. gamsii being able to reduce the disease in different conditions. The ability to produce water-soluble inhibitors or coil around the hyphae of the pathogen in vitro was not related to the disease reduction in vivo. Additionally, the strains collected from the high disease areas in the field were better antagonists. The antagonistic activity was not characteristic of a species but that of a population.