Rational Identification of Hsp90 Inhibitors as Anticancer Lead Molecules by Structure Based Drug Designing Approach.

BACKGROUND: Heat shock protein 90 (Hsp90) is an encouraging anticancer target for the development of clinically significant molecules. Schiff bases play a crucial role in anticancer research because of their ease of synthesis and excellent antiproliferative effect against multiple cancer cell lines. Therefore, we started our research work with the discovery of resorcinol/4-chloro resorcinol derived Schiff bases as Hsp90 inhibitors, which resulted in the discovery of a viable anticancer lead molecule. OBJECTIVE: The objective of the study is to discover more promising lead molecules using our previously established drug discovery program, wherein the rational drug design is achieved by molecular docking studies. METHODS: The docking studies were carried out by using Surflex Geom X programme of Sybyl X-1.2 version software. The molecules with good docking scores were synthesized and their structures were confirmed by IR, 1H NMR and mass spectral analysis. Subsequently, the molecules were evaluated for their potential to attenuate Hsp90 ATPase activity by Malachite green assay. The anticancer effect of the molecules was examined on PC3 prostate cancer cell lines by utilizing 3-(4,5-dimethythiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay methodology. RESULTS: Schiff bases 11, 12, 20, 23 and 27 exhibiting IC50 value below 1µM and 15µM, in malachite green assay and MTT assay, respectively, emerged as viable lead molecules for future optimization. CONCLUSION: The research work will pave the way for the rational development of cost-effective Schiff bases as Hsp90 inhibitors as the method employed for the synthesis of the molecules is simple, economic and facile.

First Report of Peanut stripe virus (Family Potyviridae) in South Korea.

Severe mosaic symptoms resembling those reported for a blotch isolate of Peanut stripe virus (PStV) (1) were observed in the year 1998 in Suwon, South Korea, on several peanut cultivars. The incidence of the virus was as high as 100% in cv. Daekwang. The virus was seed transmitted to varying degrees depending on the cultivar and a maximum seed transmission of 15.7% was observed in cv. Aul. Electron microscopic examination of leaf dip preparations showed filamentous rods having modal length of 720 nm. Viral inclusion bodies in infected cells were of pinwheel, scroll, and laminated aggregates. The 3' terminal region of the viral genome was amplified using degenerate primers (2) and the resulting approximately 700 bp fragment was cloned and sequenced. GenBank searches using the 709 nucleotides consisting of the complete 3'-untranslated region and a part of the coat protein gene showed that the virus shared 98% sequence identity with the currently known PStV isolates. To our knowledge, this is the first report of PStV in the Republic of Korea. References: (1) J. W. Demski et al. Ann. Appl. Biol. 105:495, 1984. (2) S. S. Pappu et al. J. Virol. Methods 41:9, 1993.

Distribution and Characteristics of Groundnut Rosette Disease in Kenya.

Groundnut rosette is a major virus disease of peanut in sub-Saharan Africa. The disease is caused by a complex of three agents: GRAV (groundnut rosette assistor luteovirus), GRV (groundnut rosette umbravirus), and the associated satellite RNA (Sat-RNA). During the 1997 to 1998 crop season, the incidence of rosette in farmers' fields was estimated at 24 to 40% in western Kenya and 30% in the Rift Valley. Sequence analysis of Kenyan isolates revealed that GRAV-CP sequences shared 97 to 100% and 95 to 98% sequence homology at nucleotide and amino acid levels, respectively, amongst themselves and with the Malawian and Nigerian isolates. The ORFs 3 and 4 of GRV were similar, with a homology of 99% at the nucleotide and amino acid levels among Kenyan isolates. The GRV sequences of Kenyan isolates were closer to the Malawian (95 to 96%) than to the Nigerian (87 to 88%) isolates. Sat-RNA shared 89 to 94% nucleotide identity with those from Malawi and Nigeria. A closer sequence relationship was observed between Kenyan and Malawian isolates in all regions compared. This is the first report on the distribution and molecular characterization of groundnut rosette disease complex in East Africa.

Phylogenetic studies of tospoviruses (family: Bunyaviridae) based on intergenic region sequences of small and medium genomic RNAs.

Analysis of the intergenic region (IGR) of S and M RNAs of tospoviruses (Family Bunyaviridae) indicated their heterogeneity both in length and sequence. In general, IGRs of M RNA were shorter in length compared to the IGRs of their respective S RNA species. Percent identity among the S RNA IGR sequences of distinct tospovirus species varied from 42 to 57%, whereas it was 79 to 99% among isolates of the same species. Similarly, when IGRs of M RNAs were compared, there was higher sequence identity among isolates of the same tospovirus species (84 to 98%) than among distinct tospovirus species (46 to 59%). Percent nucleotide identities and maximum likelihood trees of IGR sequences of S and M RNAs indicated that their sequence divergence is similar to that of nucleocapsid gene at inter and intra-species levels. This is the first detailed sequence analysis of IGRs of S and M RNAs of known tospoviruses.

Occurrence of Cowpea aphid-borne mosaic virus in Peanut in Brazil.

Surveys of peanut crops in northeastern Brazil since 1995 showed the occurrence of a hitherto unreported virus disease. Characteristic leaf symptoms were ring spots and blotches. The virus was seed transmitted in peanut (1/610) and cowpea (47/796). Local and systemic symptoms were observed in cowpea (cv. TVu 3433) known to be susceptible to most Cowpea aphid-borne mosaic virus (CABMV) isolates. The virus was transmitted by aphids Toxoptera citricidus and Aphis gossypii. Using degenerate primers, the 3' terminal region of the viral genome was cloned and sequenced. Sequence analyses of the coat protein and the 3' untranslated region indicated that the potyvirus was most closely related to CABMV isolates from South Africa, Zimbabwe, and the United States. On the basis of genome analysis, the virus was identified as CABMV. The natural occurrence of CABMV on peanut has so far not been reported. The significance of this finding especially for germ plasm exchange is discussed.

Outbreak of Tomato yellow leaf curl virus (Family Geminiviridae) in Georgia.

Tomato yellow leaf curl virus (TYLCV) of the family Geminiviridae is a serious production constraint to tomato (3). In the southeastern United States the virus has been largely confined to Florida. The disease appeared in the southern most Georgia county (Decatur) in 1998, at an incidence rate of less than 1% (2). During the fall of 1999, tomato plants showing symptoms indicative of TYLCV were observed in commercial fields in Grady, Colquitt, and Lowndes counties and the experimental plots of the Coastal Plain Experiment Station in two locations in Tift County, GA. The 12-acre commercial field in Grady County had a disease incidence of 15%. In Tift County, in both experimental plots (≈5 miles apart), TYLCV incidence ranged from 15 to 20%. Bemisia argentifolii populations in southern Georgia, based on the observed high incidence of silverleaf symptoms in squash and the intensity of adult migrations during August and September, were the highest in more than 5 years. TYLCV infection was verified by polymerase chain reaction (PCR) amplification with degenerate primers (5'-GCC CAC ATY GTC TTY CCN GT-3' and 5' -GGC TTY CTR TAC ATR GG-3') specific to the DNA A component (4). A simplified and faster DNA extraction procedure was used to obtain PCR-ready templates. Leaf tissue was homogenized in 300 µl of extraction buffer (1), followed by one phenol and one chloroform/ isoamyl alcohol (24:1) extraction. The supernatant was purified using a QiaPrep MiniPrep purification kit (Qiagen, Valencia, CA) and was used in PCR amplification. The procedure yielded highly consistent PCR-quality template. The resulting ≈1.3-kb PCR product was cloned in pGEM-T vector (Promega Corp., Madison, WI) and completely sequenced. Sequence comparisons indicated 98% identity with known TYLCV isolates from Spain (GenBank Accession no. AJ223505), the Dominican Republic (GenBank Accession no. AF024715), and Israel (GenBank Accession no. X15656). Using PCR followed by restriction digestion analysis, three symptomatic plants from one field each in Colquitt and Lowndes counties were TYLCV positive. The higher incidence of TYLCV in the Georgia counties of Tift and Grady and its concurrent occurrence in Colquitt and Lowndes counties indicates its rapid spread in the southeastern United States. References:(1) I. B. Dry et al. J. Gen. Virol. 74:147, 1993. (2) M. T. Momol et al. Plant Dis. 83:487, 1999. (3) J. E. Polston et al. Plant Dis. 83:984, 1999. (4) M. R. Rojas et al. Plant Dis. 77:340, 1993.

Analysis of the intergenic region of tomato spotted wilt Tospovirus medium RNA segment.

The intergenic region (IGR) of the medium (M) RNA of tomato spotted wilt Tospovirus (TSWV) isolates naturally infecting peanut (groundnut), pepper, potato, stokesia, tobacco and watermelon in Georgia (GA) and a peanut isolate from Florida (FL) was cloned and sequenced. The IGR sequences were compared with one another and with respective M RNA IGRs of TSWV isolates from Brazil and Japan and other tospoviruses. The length of M IGR of GA and FL isolates varied from 271 to 277 nucleotides. The M IGRs of TSWV from potato and stokesia, and tobacco and watermelon were identical with each other in their length and sequence. IGR sequences were more conserved (95-100%) among the populations of TSWV from GA and FL, than when compared with those of TSWV isolates from other countries (83-94%). The conserved motif (CAAACTTTGG) present in the IGRs of both M and small (S) RNAs of a Brazilian isolate of TSWV was also conserved in the isolates studied. Cluster analysis of the IGR sequences showed that all GA and FL isolates are closely clustered and are distinct from the TSWV isolates from other countries as well as from other tospoviruses.

First Report of Natural Infection of Peanut (Groundnut) by Impatiens Necrotic Spot Tospovirus (Family Bunyaviridae).

Impatiens necrotic spot tospovirus (INSV) of the family Bunyaviridae is an important viral pathogen of ornamentals and a major constraint in the greenhouse industry (2). Evidence of natural infection of peanut (groundnut, Arachis hypogaea L.) by INSV was found in samples collected from three sites in Frio County, TX, and one site each in Mitchell and Tift counties, GA, during October 1998. Roots from several plants were tested by enzyme-linked immunosorbent assay for tomato spotted wilt tospovirus (TSWV) and INSV. Symptoms on individual mature plants positive for INSV were the same as those associated with late-season TSWV infections: plants appeared yellow and wilted, internal taproot and crown were necrotic, and plant death resulted (1). At one Texas site, three of five composite samples were positive only for INSV. One composite sample at a second Texas site was positive for both TSWV and INSV. Double infections were found in three of four TSWV-positive samples at a third Texas site. In Mitchell County, GA, three of four samples tested were positive only for TSWV, and one was positive for both TSWV and INSV. In Tift County, GA, 11 of 23 samples tested were positive only for INSV, whereas 4 were positive only for TSWV. Double infections were found in 5 of 23 samples. The presence of INSV in the sample from Mitchell County was verified by immunocapture-polymerase chain reaction (PCR) (4). The apparently low titer of INSV in the doubly infected plant necessitated two cycles of PCR for detection of INSV sequences. A primer pair that can amplify most tospoviruses was used for the first PCR cycle (3). Using the PCR product obtained, a second PCR cycle was performed with one tospovirus-specific and one INSV-specific primer. This approach resulted in a product of the expected size (≈298 bp). The PCR product was cloned in a pGEM-T vector and sequenced. Comparisons indicated the sequence obtained from the infected peanut sample from Georgia was 99% identical to the respective S RNA region from known INSV isolates. Serological and molecular sequence data suggest the peanut samples were infected by INSV. Future surveys and screenings of peanut plants for spotted wilt disease should include a test for INSV. References: (1) A. K. Culbreath et al. Plant Dis. 75:863, 1991. (2) M. Daughtrey et al. Plant Dis. 81:1220, 1997. (3) R. Dewey et al. Virus Genes 13:255, 1996. (4). R. K. Jain et al. Plant Dis. 82:900, 1998.

First Report of the Green Rosette Variant of Groundnut Rosette Disease in Kenya.

Groundnut (Arachis hypogaea L.) is an important food crop in sub-Saharan Africa. One of the major production constraints is groundnut rosette disease, which is caused by a complex of two viruses, groundnut rosette assistor luteovirus (GRAV) and groundnut rosette umbravirus (GRV) together with the associated satellite RNA (satRNA) (1). Two main forms of the disease have been described: chlorotic and the green rosette. Variants of the satRNA have been shown to be largely responsible for the different forms of the disease (1). Chlorotic rosette has been the predominant form in all of sub-Saharan Africa while green rosette has been reported in the western and southern regions of Africa (2). During the 1997-1998 crop season, disease surveys conducted in Kenya showed the incidence of the rosette disease in farmers' fields to be 24 to 40% in a total of 23 fields surveyed in the western regions of the country (Homabay, Kendubay, Kisumu) and 30% in 8 fields sampled in the Rift Valley (Cheplamus, Marigat) regions. Representative peanut plants showing rosette symptoms were analyzed for the presence of GRV by reverse transcription polymerase chain reaction (RT-PCR). With primers specific to a portion of ORF4 of GRV RNA (3), RT-PCR gave a product of expected size (approximately 300 bp). The PCR product was cloned in pGEM-T vector and sequenced. The sequenced region showed 89% nucleotide sequence identity with published GRV sequences. Green rosette was observed on groundnut cultivars Nyaela Red and Homabay Local in the Kendu Bay region. The incidence of the green rosette was 5.3% of the plants with rosette symptoms. References: (1) A. F. Murant and I. K. Kumar. Ann. Appl. Biol. 117:85, 1990. (2) R. A. Naidu et al. Ann. Appl. Biol. 132:525, 1998. (3). M. E. Taliansky et al. J. Gen. Virol. 77:2335, 1996.

Sequence characteristics of natural populations of tomato spotted wilt tospovirus infecting flue-cured tobacco in Georgia.

Using primers from conserved regions, the nucleocapsid (Nc) gene sequences of naturally occurring tomato spotted wilt tospovirus (TSWV) isolates from flue-cured tobacco (Nicotiana tabacum) grown in several Georgia counties were amplified by immunocapture-reverse transcription-polymerase chain reaction. The resulting amplicons were cloned and sequenced. Sequence analyses showed a high degree of sequence conservation among the Nc genes of the tobacco isolates, and those reported from other parts of the world. However, distinct differences that were unique to these tobacco isolates as well as the previously studied peanut, tomato and pepper isolates from Georgia were present. The Georgia isolates formed a distinct cluster that was clearly resolved from the rest of the TSWV isolates based on sequence phylograms.

Watermelon bud necrosis tospovirus is a distinct virus species belonging to serogroup IV.

The nucleocapsid protein gene of a tospovirus infecting watermelon in India was cloned and sequenced. Sequence analyses showed that the gene was most closely related to those of watermelon silver mottle tospovirus (WSMV) from Taiwan and peanut bud necrosis tospovirus (PBNV) from India, the two definitive species of serogroup IV. Amino acid sequence similarity was 84% and 82% with WSMV and PBNV, respectively. On the basis of the sequence divergence and the previously determined host range differences, the watermelon tospovirus, designated as watermelon bud necrosis tospovirus, should be considered as a distinct species belonging to serogroup IV.

Differentiation of Biologically Distinct Peanut Stripe Potyvirus Strains by a Nucleotide Polymorphism-Based Assay.

A necrotic strain of peanut stripe potyvirus (PStV-Ts) was used to design and test strain-differentiating oligonucleotides. The 3' region of PStV-Ts, including a part of the NIb region, the complete coat protein (CP) gene, and the 3'-untranslated region, was cloned and sequenced. PStV-Ts had a high degree of sequence identity (92 to 95%) to the known non-necrotic (blotch) strains both at the nucleotide and amino acid sequence levels. Nucleotide sequence differences unique to the necrotic strain were identified when compared to the available non-necrotic isolates of PStV. Nucleotide polymorphism in the CP gene sequences was utilized in designing oligonucleotides that were specific to the necrotic strain, and were employed in an assay to differentiate the necrotic strain from non-necrotic. The 3' end mismatch in the oligonucleotides contributed in particular to the differentiation of the strains. This approach facilitated rapid, sensitive, and reliable detection and differentiation of PStV strains.

First Report of Tomato Spotted Wilt Tospovirus Infection of Watermelon in Georgia.

In 1996, volunteer watermelon plants in a tobacco field in Coffee County, GA, exhibited foliar symptoms that included necrotic ring spots and veinal necrosis. Watermelon plants from experimental plots of the Coastal Plain Experiment Station in Tifton, GA, similarly showed necrotic lesions, often resulting in necrotic ring spots during the late summer of 1997. Out of 16 samples tested for the presence of tomato spotted wilt tospovirus (TSWV) with a commercially available enzyme-linked immunosorbent assay (ELISA) kit (Agdia, Elkhart, IN), six were positive for TSWV. Primers specific to the nucleocapsid gene of TSWV were used in a reverse transcription-polymerase chain reaction assay (RT-PCR) (1) to verify the presence of TSWV. RT-PCR gave an expected PCR product of approximately 350 bp. The amplicon was cloned in pGEM-T vector and the recombinant clone was sequenced. The sequence of the cloned PCR product confirmed the identity of TSWV, thus verifying TSWV infection of watermelon. The potential impact of TSWV on watermelon crop in Georgia will be investigated. This is the first report of natural infection of watermelon by TSWV in Georgia. Reference: (1) H. R. Pappu et al. Tobacco Sci. 40:74, 1996.

Molecular Diagnosis of Tomato Spotted Wilt Tospovirus Infection of Peanut and Other Field and Greenhouse Crops.

Tomato spotted wilt tospovirus (TSWV) is a major disease constraint to peanut, tomato, pepper, and tobacco production in Georgia. Rapid molecular diagnosis of TSWV infection in peanut and its molecular studies were severely hampered by the lack of practical and rapid procedures for the extraction and amplification of the genomic nucleic acid. To circumvent this technical constraint, we adapted an immunocapture-procedure (ICP) for enriching the peanut tissue extracts for TSWV, and combined the ICP with a single-buffer, one-tube reverse transcription polymerase chain reaction (RT-PCR) to achieve rapid and reliable amplification of TSWV sequences from peanut. Both leaf and root tissue of peanut provided PCR-quality templates. Immunocapture, RT, and PCR were done in the same tube, allowing higher throughput. The technique was applicable to a wide range of TSWV-susceptible crops such as tomato, pepper, tobacco, gloxinia, and impatiens. Primers derived from the nucleocapsid protein gene as well as from the large RNA of the viral genome were able to amplify the target sequences in a highly specific and reproducible manner. This approach facilitated rapid molecular typing of natural populations of TSWV in Georgia.

Characterization of the 3' proximal gene of the citrus tristeza closterovirus genome.

The 3' proximal open reading frame (ORF 11) in the citrus tristeza virus (CTV) genome potentially encodes a protein of 209 amino acids with an estimated molecular weight of 23 kDa (p23). The p23 ORF from the severe Florida strain T36 of CTV was expressed in Escherichia coli, and the expressed protein was used to raise polyclonal antibodies in a rabbit. Using these antisera on a Western blot, a protein of expected size (23 kDa) was detected in tissue extracts from CTV-infected citrus but not from uninfected citrus. Most of the p23 protein was found in the soluble, cytoplasmic fraction. Comparison of the sequence of p23 genes from several biologically and geographically diverse CTV isolates indicated a high degree of conservation for this gene and for the RNA binding motif in particular. A cluster dendrogram of the deduced amino acid sequences correlated with the biological properties of the isolates, forming distinct groups of mild, quick decline on stem pitting-inducing isolates. Therefore it is possible that, in addition to the capsid protein gene, the p23 gene also may serve as an indicator for disease severity.

Molecular characterization and interviral homologies of a potyvirus infecting sesame (Sesamum indicum) in Georgia.

Phylogenetic analyses of the 3' terminal region of a potyvirus naturally infecting sesame (Sesamum indicum) in Georgia indicated that the virus belongs to the passionfruit woodiness potyvirus subgroup, and is most closely related to cowpea aphid-borne mosaic potyvirus (CABMV) and South African passiflora potyvirus. Based on the sequence identity, this virus is a strain of CABMV. Use of degenerate primers, reverse transcription-polymerase chain reaction, cloning and sequence analysis allowed us to rapidly determine the taxonomic status of this potyvirus.

A robust point-matching algorithm for autoradiograph alignment.

We present a novel method for the geometric alignment of autoradiographs of the brain. The method is based on finding the spatial mapping and the one-to-one correspondences (or homologies) between point features extracted from the images and rejecting non-homologies as outliers. In this way, we attempt to account for the local, natural and artifactual differences between the autoradiograph slices. We have used the resulting automated algorithm on a set of left prefrontal cortex autoradiograph slices, specifically demonstrated its ability to perform point outlier rejection, validated its robustness property using synthetically generated spatial mappings and provided an anecdotal visual comparison with the well-known iterated closest-point (ICP) algorithm. Visualization of a stack of aligned left prefrontal cortex autoradiograph slices is also provided.

The diverged copy of the citrus tristeza virus coat protein is expressed in vivo.

Nucleotide sequence analysis of a portion of the citrus tristeza closterovirus (CTV) genome revealed an open reading frame immediately upstream of the coat protein gene that can encode a protein with a calculated M(r) of 27,360 (p27). The deduced amino acid sequence indicated that this putative nonstructural gene product is highly homologous to the coat protein. To investigate whether p27 was expressed in CTV-infected plants, a fusion protein of p27 produced in Escherichia coli was used to raise polyclonal antibodies. Western blot analysis using the p27 antibodies indicated that p27 is expressed in CTV-infected citrus, but not in uninfected plants. Tissue fractionation studies revealed that p27 accumulates in cell wall enriched fractions.

Nucleotide sequence and organization of eight 3' open reading frames of the citrus tristeza closterovirus genome.

The citrus tristeza closterovirus (CTV) RNA genome was cloned as cDNA generated from both CTV-specific double-stranded RNA and genomic RNA, and the sequence of the 3' 7292 nucleotides was determined. The sequenced portion contained eight open reading frames potentially encoding, in the 5' to 3' direction, proteins with the apparent molecular weights of 65, 61, 27, 25 (capsid protein, CP), 18, 13, 20, and 23 kDa, and a potential noncoding region of 277 nucleotides. The 65-kDa protein is a viral homolog of cellular hsp70 heat shock proteins (hsp), the 61-kDa protein is distantly related to the hsp90 proteins, and the 27-kDa protein is a diverged copy of the CP. Database searches did not identify any protein sequences of significant similarity to the remaining four ORFs downstream of the CP. A specific four-gene module consisting of the hsp70 protein, the hsp90-related protein, the diverged copy of the CP, and the CP itself was found to be common in organization between CTV and beet yellows closterovirus. All four proteins in this module were highly conserved, indicating that these viruses probably have evolved from a common ancestor.

Variability in the length of the amino terminal sequence contributes to the capsid protein diversity among dasheen mosaic potyvirus isolates.

Western blot analysis of several dasheen mosaic virus isolates revealed molecular weight differences among their capsid proteins (CPs). Sequence analysis of the CP gene showed that the CP of isolate TEN was 15 amino acids shorter than that of isolate LA due to a 57-base deletion and a 12-base insertion into the 5' end of the TEN CP gene. Our data suggest that frequent deletions and insertions are responsible for the CP size diversity among DMV isolates.