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Quantitative RT-PCR assay for high-throughput screening (HTS) of drugs against the growth of Cryptosporidium parvum in vitro.

Zhang H, Zhu G - Front Microbiol (2015)

Bottom Line: However, conventional qRT-PCR protocol is not very amendable to high-throughput analysis when total RNA needs to be purified by lengthy, multi-step procedures.Recently, several commercial reagents are available for preparing cell lysates that could be directly used in downstream qRT-PCR analysis (e.g., Ambion Cell-to-cDNA kit and Bio-Rad iScript sample preparation reagent).This qRT-PCR protocol is able to give a >150-fold linear dynamic range using samples isolated from cells infected with various numbers of parasites.

View Article: PubMed Central - PubMed

Affiliation: Department of Veterinary Pathobiology, College of Veterinary Medicine & Biomedical Sciences, Texas A&M University College Station, TX, USA.

ABSTRACT
Our laboratory has previously developed a qRT-PCR assay to assess drug efficacy on the growth of Cryptosporidium parvum in vitro by detecting the levels of parasite 18S rRNA. This approach displayed up to four orders of magnitude of linear dynamic range and was much less labor-intensive than the traditional microscopic methods. However, conventional qRT-PCR protocol is not very amendable to high-throughput analysis when total RNA needs to be purified by lengthy, multi-step procedures. Recently, several commercial reagents are available for preparing cell lysates that could be directly used in downstream qRT-PCR analysis (e.g., Ambion Cell-to-cDNA kit and Bio-Rad iScript sample preparation reagent). Using these reagents, we are able to adapt the qRT-PCR assay into high-throughput screening of drugs in vitro (i.e., 96-well and 384-well formats for the cultivation of parasites and qRT-PCR detection, respectively). This qRT-PCR protocol is able to give a >150-fold linear dynamic range using samples isolated from cells infected with various numbers of parasites. The new assay is also validated by the NIH-recommended intra-plate, inter-plate, and inter-day uniformity tests. The robustness and effectiveness of the assay are also confirmed by evaluating the anti-cryptosporidial efficacy of paromomycin and by a small scale screening of compounds.

No MeSH data available.


In vitro anti-cryptosporidial activities of 48 small molecules (C001–C048). (A) Efficacies of all 48 compounds (10 μM each) obtained from primary screening (solid teal round dots). A second test was performed on seven top hits (red circles), in which six compounds displayed similar levels of efficacy between the two tests as shown by overlaps or near distances of their corresponding teal dots and red circles. Only one compound showed significantly different efficacies between the two tests (highlighted by a yellow line to link the corresponding teal dot and red circle). Compound codes are given on the seven top hits and one of the potential parasite growth enhancer as discussed in the text. (B) Dose-response curves on the anti-cryptosporidial activities of C004 and C028 compounds. Paromomycin at 100 μg/ml (140 μM) was used as a positive control in this assay. Bars represent standard errors of the mean (SEM) derived from at least two biological replicates.
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Figure 5: In vitro anti-cryptosporidial activities of 48 small molecules (C001–C048). (A) Efficacies of all 48 compounds (10 μM each) obtained from primary screening (solid teal round dots). A second test was performed on seven top hits (red circles), in which six compounds displayed similar levels of efficacy between the two tests as shown by overlaps or near distances of their corresponding teal dots and red circles. Only one compound showed significantly different efficacies between the two tests (highlighted by a yellow line to link the corresponding teal dot and red circle). Compound codes are given on the seven top hits and one of the potential parasite growth enhancer as discussed in the text. (B) Dose-response curves on the anti-cryptosporidial activities of C004 and C028 compounds. Paromomycin at 100 μg/ml (140 μM) was used as a positive control in this assay. Bars represent standard errors of the mean (SEM) derived from at least two biological replicates.

Mentions: In the primary screening of 48 small molecules (10 μM each), two compounds (C012 and C020) displayed cytotoxicity on HCT-8 cells, while others displayed a wide range of effects on the C. parvum growth in vitro (i.e., from −86.6 to 98.8% inhibition) (Figure 5). The negative inhibition values suggested that a few compounds might actually promote the parasite growth and/or affected more on host cells than on parasites, which could be evaluated by looking at the Hs18S and Cp18S CT values. For example, for C023 with −86.6% inhibition, the mean CT values for Hs18S and Cp18S were 13.38 and 18.08 (vs. 13.63 and 19.17 in the DMSO control), suggesting that this compound likely promoted the parasite growth more than reducing the host cell growth. Because we were interested only in identifying anti-cryptosporidial inhibitors in this study, we repeated the primary testing on the seven most efficacious compounds, in which six compounds exhibited similar efficacies (Figure 5A, red circles). We also performed dose-response experiments on C004 and C028, in which both compounds at 10 μM displayed the same efficacy as observed in the two primary testing experiments (Figure 5B). Their IC50 values were at 0.98 and 1.33 μM, respectively, suggesting that these two compounds might be further explored for drug development or serve as leads. Experiments to further characterize these two compounds and other top hits from the 48 compounds are still ongoing as part of our larger effort in discovering new anti-cryptosporidial drugs, which will be reported separately. Nonetheless, these observations conformed the robustness and suitability of the assay in high-throughput screening of drugs by directly evaluating their anti-cryptosporidial efficacy in vitro.


Quantitative RT-PCR assay for high-throughput screening (HTS) of drugs against the growth of Cryptosporidium parvum in vitro.

Zhang H, Zhu G - Front Microbiol (2015)

In vitro anti-cryptosporidial activities of 48 small molecules (C001–C048). (A) Efficacies of all 48 compounds (10 μM each) obtained from primary screening (solid teal round dots). A second test was performed on seven top hits (red circles), in which six compounds displayed similar levels of efficacy between the two tests as shown by overlaps or near distances of their corresponding teal dots and red circles. Only one compound showed significantly different efficacies between the two tests (highlighted by a yellow line to link the corresponding teal dot and red circle). Compound codes are given on the seven top hits and one of the potential parasite growth enhancer as discussed in the text. (B) Dose-response curves on the anti-cryptosporidial activities of C004 and C028 compounds. Paromomycin at 100 μg/ml (140 μM) was used as a positive control in this assay. Bars represent standard errors of the mean (SEM) derived from at least two biological replicates.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4585199&req=5

Figure 5: In vitro anti-cryptosporidial activities of 48 small molecules (C001–C048). (A) Efficacies of all 48 compounds (10 μM each) obtained from primary screening (solid teal round dots). A second test was performed on seven top hits (red circles), in which six compounds displayed similar levels of efficacy between the two tests as shown by overlaps or near distances of their corresponding teal dots and red circles. Only one compound showed significantly different efficacies between the two tests (highlighted by a yellow line to link the corresponding teal dot and red circle). Compound codes are given on the seven top hits and one of the potential parasite growth enhancer as discussed in the text. (B) Dose-response curves on the anti-cryptosporidial activities of C004 and C028 compounds. Paromomycin at 100 μg/ml (140 μM) was used as a positive control in this assay. Bars represent standard errors of the mean (SEM) derived from at least two biological replicates.
Mentions: In the primary screening of 48 small molecules (10 μM each), two compounds (C012 and C020) displayed cytotoxicity on HCT-8 cells, while others displayed a wide range of effects on the C. parvum growth in vitro (i.e., from −86.6 to 98.8% inhibition) (Figure 5). The negative inhibition values suggested that a few compounds might actually promote the parasite growth and/or affected more on host cells than on parasites, which could be evaluated by looking at the Hs18S and Cp18S CT values. For example, for C023 with −86.6% inhibition, the mean CT values for Hs18S and Cp18S were 13.38 and 18.08 (vs. 13.63 and 19.17 in the DMSO control), suggesting that this compound likely promoted the parasite growth more than reducing the host cell growth. Because we were interested only in identifying anti-cryptosporidial inhibitors in this study, we repeated the primary testing on the seven most efficacious compounds, in which six compounds exhibited similar efficacies (Figure 5A, red circles). We also performed dose-response experiments on C004 and C028, in which both compounds at 10 μM displayed the same efficacy as observed in the two primary testing experiments (Figure 5B). Their IC50 values were at 0.98 and 1.33 μM, respectively, suggesting that these two compounds might be further explored for drug development or serve as leads. Experiments to further characterize these two compounds and other top hits from the 48 compounds are still ongoing as part of our larger effort in discovering new anti-cryptosporidial drugs, which will be reported separately. Nonetheless, these observations conformed the robustness and suitability of the assay in high-throughput screening of drugs by directly evaluating their anti-cryptosporidial efficacy in vitro.

Bottom Line: However, conventional qRT-PCR protocol is not very amendable to high-throughput analysis when total RNA needs to be purified by lengthy, multi-step procedures.Recently, several commercial reagents are available for preparing cell lysates that could be directly used in downstream qRT-PCR analysis (e.g., Ambion Cell-to-cDNA kit and Bio-Rad iScript sample preparation reagent).This qRT-PCR protocol is able to give a >150-fold linear dynamic range using samples isolated from cells infected with various numbers of parasites.

View Article: PubMed Central - PubMed

Affiliation: Department of Veterinary Pathobiology, College of Veterinary Medicine & Biomedical Sciences, Texas A&M University College Station, TX, USA.

ABSTRACT
Our laboratory has previously developed a qRT-PCR assay to assess drug efficacy on the growth of Cryptosporidium parvum in vitro by detecting the levels of parasite 18S rRNA. This approach displayed up to four orders of magnitude of linear dynamic range and was much less labor-intensive than the traditional microscopic methods. However, conventional qRT-PCR protocol is not very amendable to high-throughput analysis when total RNA needs to be purified by lengthy, multi-step procedures. Recently, several commercial reagents are available for preparing cell lysates that could be directly used in downstream qRT-PCR analysis (e.g., Ambion Cell-to-cDNA kit and Bio-Rad iScript sample preparation reagent). Using these reagents, we are able to adapt the qRT-PCR assay into high-throughput screening of drugs in vitro (i.e., 96-well and 384-well formats for the cultivation of parasites and qRT-PCR detection, respectively). This qRT-PCR protocol is able to give a >150-fold linear dynamic range using samples isolated from cells infected with various numbers of parasites. The new assay is also validated by the NIH-recommended intra-plate, inter-plate, and inter-day uniformity tests. The robustness and effectiveness of the assay are also confirmed by evaluating the anti-cryptosporidial efficacy of paromomycin and by a small scale screening of compounds.

No MeSH data available.