Hsp90 is essential in the filarial nematode Brugia pahangi

The development of a compound with activity against filarial nematodes (a 'macrofilaricide') has been a long-standing goal of the World Health Organization. However, adult filariae have proved remarkably difficult to kill. To some extent this reflects a lack of understanding of key pathways and processes in filarial nematodes that may be suitable targets for chemotherapy. In this paper we show that geldanamycin (GA), a specific inhibitor of the activity of the heat shock protein 90 (Hsp90) family, kills adult worms and microfilariae (Mf) of Brugia pahangi at nanomolar concentrations. In addition, release of Mf from adult worms is inhibited within 24 h of exposure to GA and is not recoverable, demonstrating that GA effectively sterilises the worm. Similar results were obtained with a second filarial worm Acanthocheilonema viteae. In contrast GA has no effect on the free-living nematode Caenorhabditis elegans despite a high degree of conservation between the nematode Hsp90 sequences. In keeping with these findings, Brugia Hsp90 binds GA in a solid phase pull-down assay while the binding of C. elegans Hsp90 to immobilised GA is undetectable. In other eukaryotes, GA is known to bind in the N-terminal ATP pocket of Hsp90, disrupting its interactions with client proteins which are then targeted for degradation via the proteasome pathway. Thus, Hsp90 or some of its client proteins may provide novel targets for the chemotherapy of filarial infection.     

Synthetic pathways of glycerophospholipids in adult Brugia pahangi and Brugia patei

The pathways of glycerophospholipid syntheses in adult Brugia pahangi and Brugia patei were examined by radioisotopic incorporation and demonstration of the enzymatic steps. Radiolabelling studies showed that l-U-¹⁴C-glycerol-3-phosphate was rapidly incorporated into glycerophospholipids of B. pahangi and B. patei, respectively, with the label distributed in phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine (PS), phosphatidylinositol (PI), phosphatidylglycerol (PG) and cardiolipin (CL) fractions. Crude extracts of these worms were found to contain significant activities of sn-glycerol-3-phosphate acyl-transferase (EC 2.3.1.15), phosphatidic acid phosphatase (EC 3.1.3.4), choline phosphotransferase (EC 2.7.8.2), ethanolamine phosphotransferase (EC 2.7.8.1), PE methyltransferase (EC 2.1.1.17), PS decarboxylase (EC 4.1.1.65), phosphatidylglycerolphosphate synthetase (EC 2.7.8.5), phosphatidylinositol synthetase (EC 2.7.8.11), and base exchange enzymes of ethanolamine, serine and inositol. These findings suggest that filarial worms can synthesize PC by two pathways, PE by three pathways, and PI by two pathways and fabricate PS, PG and CL.     

Tissue migration capability of larval and adult Brugia pahangi

Infection with mosquito-born filarial nematodes occurs when hosts are bitten by a vector carrying the infective third stage larvae (L3) of the parasites. These larvae, deposited on the skin by the feeding mosquito, are presumed to enter the skin via the vector-induced puncture wound. Larvae of Brugia spp. must then migrate from the entry site, penetrate various skin layers, and locate a lymphatic vessel that leads to their lymphatic predilection site. We have recently established an intradermal (ID) infection model using B. pahangi and the Mongolian gerbil, allowing us to investigate the migratory capability ofB. pahangi. Larval and adult parasites recovered from the peritoneal cavities of gerbils were capable of establishing an infection following ID (larvae) or subcutaneous (adult) injection. Third and fourth stage larvae both migrated away from the injection site within hours, although data suggest they localize to different lymphatic tissues at 3 days postinfection (DPI). Immature adult (28 day) B. pahangi also migrated away from their SC inoculation site within 7 DPI. Mature (45 day) adult B. pahangi displayed little migration away from the SC infection site, suggesting tissue migration may be limited to developing stages of the parasite.     

FS36作为新型人源Hsp90抑制剂的发现

热休克蛋白90(Hsp90)通过对几百种蛋白质底物(客户蛋白质)进行合理的折叠、成熟其构象并且激活,在肿瘤细胞的生长和繁殖中发挥重要作用．因此,Hsp90成为非常有吸引力、有前途的抗肿瘤药物靶点,并且超过20种抑制剂已经进入临床实验阶段．我们在这里设计并合成了一个小分子抑制剂：FS36．收集了Hsp90^N-FS36复合物晶体结构的X射线衍射实验数据．高分辨率X射线晶体结构表明,FS36在ATP结合位点上与Hsp90^N相互作用,并且FS36可能替代核苷酸与Hsp90^N结合．FS36和Hsp90^N的复合物晶体结构和相互作用为后期设计和优化新型抗肿瘤药物奠定基础．     

In vitro killing of microfilariae of Brugia pahangi and Brugia malayi by eosinophil granule proteins

Eosinophil infiltration and degranulation around the tissue-invasive stages of several species of helminths have been observed. Release of eosinophil granule contents upon the worms is supported by localization of two of the major granule proteins, major basic protein (MBP) and eosinophil peroxidase (EPO), on and around species of trematodes, nematodes, and cestodes. In the case of filarial worms, MBP is deposited on degenerating microfilariae (mf) of Onchocerca volvulus. Here, we performed in vitro assays of the toxicity of four purified eosinophil granule proteins, namely, MBP, EPO, eosinophil cationic protein (ECP), and eosinophil-derived neurotoxin (EDN), for the mf of Brugia pahangi and Brugia malayi. MBP, ECP, and EDN killed these worms in a dose-related manner although relatively high concentrations of EDN were necessary. EPO, in the presence of a H2O2-generating system and a halide, was the most potent toxin on a molar basis; here, the most potent halide was I- followed by Br- and Cl-. Surprisingly, EPO in the absence of H2O2 killed mf at concentrations comparable to those required for MBP and ECP. The toxicity of EPO + H2O2 + halide was inhibited by heparin, catalase, or 1% BSA, whereas the toxicity of EPO alone was inhibited only by heparin. Heparin also inhibited killing by both MBP and ECP. Despite the homology of ECP with certain RNases, placental RNasin, an RNase inhibitor, was unable to inhibit ECP-mediated toxicity. These results indicate that all of the eosinophil granule proteins are toxic to mf and they support the hypothesis that eosinophil degranulation causes death of mf in vivo.     

Targeting the Hsp90 C-terminal domain to induce allosteric inhibition and selective client downregulation

BackgroundInhibition of Hsp90 is desirable due to potential downregulation of oncogenic clients. Early generation inhibitors bind to the N-terminal domain (NTD) but C-terminal domain (CTD) inhibitors are a promising class because they do not induce a heat shock response. Here we present a new structural class of CTD binding molecules with a unique allosteric inhibition mechanism.MethodsA hit molecule, NSC145366, and structurally similar probes were assessed for inhibition of Hsp90 activities. A ligand-binding model was proposed indicating a novel Hsp90 CTD binding site. Client protein downregulation was also determined.ResultsNSC145366 interacts with the Hsp90 CTD and has anti-proliferative activity in tumor cell lines (GI₅₀ = 0.2–1.9 μM). NSC145366 increases Hsp90 oligomerization resulting in allosteric inhibition of NTD ATPase activity (IC₅₀ = 119 μM) but does not compete with NTD or CTD-ATP binding. Treatment of LNCaP prostate tumor cells resulted in selective client protein downregulation including AR and BRCA1 but without a heat shock response. Analogs had similar potencies in ATPase and chaperone activity assays and variable effects on oligomerization. In silico modeling predicted a binding site at the CTD dimer interface distinct from the nucleotide-binding site.ConclusionsA set of symmetrical scaffold molecules with bisphenol A cores induced allosteric inhibition of Hsp90. Experimental evidence and molecular modeling suggest that the binding site is independent of the CTD-ATP site and consistent with unique induction of allosteric effects.General significanceAllosteric inhibition of Hsp90 via a mechanism used by the NSC145366-based probes is a promising avenue for selective oncogenic client downregulation.     

Defining the requirements for Hsp40 and Hsp70 in the Hsp90 chaperone pathway

The Hsp90 chaperoning pathway and its model client substrate, the progesterone receptor (PR), have been used extensively to study chaperone complex formation and maturation of a client substrate in a near native state. This chaperoning pathway can be reconstituted in vitro with the addition of five proteins plus ATP: Hsp40, Hsp70, Hop, Hsp90, and p23. The addition of these proteins is necessary to reconstitute hormone-binding capacity to the immuno-isolated PR. It was recently shown that the first step for the recognition of PR by this system is binding by Hsp40. We compared type I and type II Hsp40 proteins and created point mutations in Hsp40 and Hsp70 to understand the requirements for this first step. The type I proteins, Ydj1 and DjA1 (HDJ2), and a type II, DjB1 (HDJ1), act similarly in promoting hormone binding and Hsp70 association to PR, while having different binding characteristics to PR. Ydj1 and DjA1 bind tightly to PR whereas the binding of DjB1 apparently has rapid on and off rates and its binding cannot be observed by antibody pull-down methods using either purified proteins or cell lysates. Mutation studies indicate that client binding, interactions between Hsp40 and Hsp70, plus ATP hydrolysis by Hsp70 are all required to promote conformational maturation of PR via the Hsp90 pathway.     

Brugia pahangi: uptake and incorporation of adenosine and thymidine.

The uptake and incorporation of adenosine and thymidine by infective larvae, 10-day-old juvenile, and adult stages of Brugia pahangi were investigated using scintillation counting and autoradiographic techniques. No evidence of thymidine incorporation by the worm was obtained in this study. Scintillation counting methods demonstrated that ¹⁴C-labeled adenosine was incorporated by all three stages of this filarial worm. Autoradiography, performed on worms incubated in [³H] adenosine from 5 min through 2 hr, revealed that following 5–15 min incubation the greatest degree of adenosine incorporation occurred in the hypodermis and somatic cords. Adenosine incorporation into the deeper body tissues, including the gut, increased significantly with longer periods of incubation. The results obtained further support the concept that nutrient uptake in B. pahangi occurs by a transcuticular route.     

Experimental Brugia pahangi and B. malayi infections of callitrichid primates

The callitrichid primates, Callithrix jacchus jacchus (the marmoset) and Saguinus labiatus (the tamarin) were inoculated with infective larvae of Brugia malayi and B. pahangi. Microfilaraemia at low levels developed in 3 out of 4 C.j. jacchus infected with B. malayi and living or dead adult worms found in all 4. Only one of 4 C.j. jacchus became microfilaraemic (mf + ve) when given B. pahangi and adults were found in two. Of 4 S. labiatus given B. pahangi one became very lightly mf + ve and adults were found in 3. It is concluded that these animals are not suitable hosts for chemotherapeutic experiments.     

Targeting Hsp90 prevents escape of breast cancer cells from tyrosine kinase inhibition

Recent studies have identified development of resistance to tyrosine kinase inhibition (TKI) as a significant roadblock to effective treatment. One mechanism of resistance recently appreciated involves 'oncogene switching', or the re-activation of signaling pathways by one or more redundant upstream activators. In breast cancer models, ErbB TKIs such as gefitinib have been shown to lose the ability to modulate ErbB-driven signaling pathways over time, even though ErbB inhibition is maintained. Although incomplete ErB inhibition has been proposed to underlie this phenomenon, our findings suggest that oncogene switching can also re-activate downstream signaling pathways in breast cancer cells, even when ErbB inhibition is complete. We find that ErbB TKI-induced Src activation mediates downstream signaling rebound in SKBR3 cells, and we show that combination of Src and ErbB inhibitors is more effective and longlasting than is either TKI alone. Finally, the Hsp90 inhibitor 17-AAG, by simultaneously and durably inhibiting multiple signaling activators including ErbB and Src kinases, does not permit oncogene switching and results in a more prolonged and robust inhibition of downstream signaling pathways in breast cancer cells than do individual TKIs. These data support the continued clinical evaluation of Hsp90 inhibitors in breast cancer.     

Brugia pahangi: susceptibility and macroscopic pathology of golden hamster.

Twenty male hamsters were inoculated with 95 to 150 infective larvae of B. pahangi via the subcutaneous route. Worms recovered from 19 hamsters averaged 14% (0–32) from 11 hamsters killed at 105–195 days after infection and 16% (5–19) from 8 hamsters examined at 23–45 days after infection. Approximately one-half of the worms recovered were from the lymphatic vessels of the testes, epididymis, and spermatic cord. A few were found in afferent or efferent vessels of regional lymph nodes. The remaining worms were from the heart and lungs. Low-level microfilaremias were observed in 10 of 12 hamsters held for over 100 days. The average prepatent period was 89 days (65–128). Worms were recovered for up to 3 weeks following inoculation of nine hamsters via the intraperitoneal route with 100–400 infective larvae of B. pahangi.Gross lymphatic pathologic lesions consisted of moderate to marked dilation of lymphatic vessels, enlargement of regional lymph nodes, and numerous lymphthrombi and emboli. Macroscopic changes were most consistent and severe in the lymphatic vessels of the testes, epididymis, and spermatic cord and were noted less frequently in the afferent or efferent vessels of various regional lymph nodes. Areas of reddish discoloration were observed frequently on the serosal surface of the lung in infected hamsters.     

Brugia malayi and Brugia pahangi: transmission blocking activity of ivermectin and brugian filarial infections in Aedes aegypti

Brugia malayi- or Brugia pahangi-infected, microfilaremic jirds (Meriones unguiculatus) were treated with ivermectin at a single dose of 200 micrograms/kg body weight, administered subcutaneously. After different time intervals, Aedes aegypti mosquitoes were fed on treated or untreated jirds. Sausage stage, L2, and L3 larvae failed to develop in mosquitoes that fed on jirds from 15 to 30 days post-treatment. After 1 month, the numbers of L3 larvae recovered from mosquitoes fed on treated B. pahangi jirds were comparable to controls. However, the number of L3's recovered from mosquitoes fed on B. malayi jirds remained significantly lower than controls, 2 and 3 months after treatment. This reduction suggests that ivermectin may be more effective in blocking transmission of B. malayi than B. pahangi. Ivermectin treatment had no effect on the mean number of circulating microfilariae in treated jirds. Therefore, mosquitoes ingested comparable numbers of microfilariae when compared to those mosquitoes fed on untreated controls. Only in the case of jirds infected with B. malayi did the circulating microfilarial counts fall 30 days after treatment. The failure of microfilariae to develop to the L3 stage in mosquitoes fed on jirds within 30 days of treatment was not due to failure of mosquitoes to ingest microfilariae. Brugia malayi microfilariae also failed to develop to L3 in mosquitoes that were allowed to feed on microfilaremic jird blood treated with ivermectin (50 ng/ml) in vitro, indicating its efficacy at low concentrations. In addition to N-acetyl glucosamine, microfilariae obtained for a period of 15 days from ivermectin-treated but not control jirds showed D-mannose, N-acetyl galactosamine, and L-fucose moieties on the surface of the sheath.(ABSTRACT TRUNCATED AT 250 WORDS)     

Brugia pahangi: effects upon the flight capability of Aedes aegypti.

Aedes aegypti mosquitoes were adversely affected by infections of the filarial worm Brugia pahangi. Infected mosquitoes flew significantly shorter distances and showed marked reductions in total flight time during 24-hr flight mill tests compared to uninfected controls. Total flight range and duration flown by infected mosquitoes remained relatively constant throughout the infection process, while control mosquitoes flew further and longer with increasing time after their blood meal. Furthermore, a significantly greater number of infected mosquitoes either died or were rendered incapable of flight. Of flying and nonflying mosquitoes with 6-day-old or older infections dissected for parasite burdens, the nonflying group contained significantly more worms. Results of this study indicate that developing filarial larvae within this mosquito vector reduce its ability to survive and to transmit its infection by reducing its flight capabilities. Conclusions from this study relate only to A. aegypti homozygous for the gene fm which is fully susceptible to this filarial parasite.     

Parasite-induced suppression of the immune response in Aedes aegypti by Brugia pahangi

The melanization response against intrathoracically inoculated Brugia pahangi and Dirofilaria immitis microfilariae (mff) isolated from vertebrate host blood was evaluated in both uninfected Aedes aegypti black-eyed Liverpool strain and in mosquitoes harboring a developing B. pahangi infection. The immune response against inoculated mff of either species was significantly reduced by 28-47% in infected as compared with uninfected mosquitoes. Attempts to passively transfer this suppression factor(s) by inoculating naive mosquitoes with 0.1-0.2 microliter of hemolymph from B. pahangi-infected mosquitoes produced equivocal results. The role this parasite-induced immune suppression might play in aiding parasite survival in compatible vectors is discussed.     

Brugia malayi and Brugia pahangi: inherent difference in immune activation in the mosquitoes Armigeres subalbatus and Aedes aegypti

The inherent ability of Brugia malayi and Brugia pahangi (Nematoda) to establish successful relationships with the mosquitoes Armigeres subalbatus and Aedes aegypti Liverpool strain was evaluated. Brugia pahangi microfilariae (mff) avoided the immune response and developed normally in A. subalbatus exposed to the parasite by an infective bloodmeal, whereas nearly 85% of B. malayi were destroyed by the immune response. Because A. aegypti supports the development of both filarial worm species but destroys intrathoracically inoculated B. pahangi isolated from jird blood, blood-isolated B. malayi were inoculated into A. aegypti, and the immune response was compared with that observed against B. pahangi. The response against B. malayi was significantly more rapid and effective than the response against B. pahangi. Similar results were obtained when blood-isolated B. pahangi or B. malayi were inoculated into A. subalbatus. Microfilariae of both species were able to avoid immune destruction in A. aegypti if they were allowed to penetrate the Liverpool midgut in vitro prior to inoculation. Most B. pahangi that had first penetrated an Armigeres midgut prior to inoculation into A. subalbatus were able to avoid the immune response, but by day 3 postinoculation, less than 40% of the B. malayi, treated in the same manner, were able to escape the immune response. Genetic susceptibility of mosquitoes to infection by filarial worms and potential mechanisms of immune evasion/suppression are discussed regarding B. malayi and B. pahangi.     

Possible direct effect of diethylcarbamazine on the infective larvae of Brugia pahangi

The direct action of diethylcarbamazine (DEC) on the infective larvae of Brugia pahangi was studied. The larvae were cultured in RPMI 1640 supplemented with foetal bovine serum and antibiotics for 22 days. Most of the larvae remained alive for 8 days, but survival rate of larvae decreased rapidly from day 10 onwards. The larvae did not grow in the culture system. The addition of DEC did not affect the morbidity of the larvae and no difference was observed in the morphological characteristics between the larvae cultured in the presence or absence of DEC. The infective larvae were cultured in vitro for 5 days in the presence or absence of DEC, and inoculated into jirds. The animals were necropsied at intervals, and developing larvae and adult worms were recovered. When the larvae were cultured without DEC and then inoculated subcutaneously into jirds, 29.8% of the inoculum was recovered 3-15 days, and 25% 19-22 weeks, post-inoculation. However, when the larvae were exposed to DEC in vitro and inoculated into jirds, the rate of recovery was reduced to 25% 3-15 days post-inoculation and 2% after 19-22 weeks. When the control larvae cultured in vitro were inoculated intraperitoneally into jirds, 41.3% of inoculum was recovered 3-15 days, and 42.8% 19-22 weeks, post-inoculation. Again the corresponding value for larvae exposed to DEC in vitro was reduced to 19.8% 3-15 days, and 8% 19-22 weeks, post-inoculation. It was observed that the larvae exposed to DEC in vitro were retarded in their development in jirds.(ABSTRACT TRUNCATED AT 250 WORDS)