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Drug target mining and analysis of the Chinese tree shrew for pharmacological testing.

Zhao F, Guo X, Wang Y, Liu J, Lee WH, Zhang Y - PLoS ONE (2014)

Bottom Line: The discovery of new drugs requires the development of improved animal models for drug testing.The Chinese tree shrew is considered to be a realistic candidate model.Target validation also demonstrated that the constitutive expression of the proteinase-activated receptors of tree shrew platelets is similar to that of human platelets but differs from that of mouse platelets.

View Article: PubMed Central - PubMed

Affiliation: Key Laboratory of Animal Models and Human Disease Mechanisms of the Chinese Academy of Sciences & Yunnan Province, Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming, Yunnan, PR China; Kunming College of Life Science, University of Chinese Academy of Sciences, Kunming, Yunnan, PR China.

ABSTRACT
The discovery of new drugs requires the development of improved animal models for drug testing. The Chinese tree shrew is considered to be a realistic candidate model. To assess the potential of the Chinese tree shrew for pharmacological testing, we performed drug target prediction and analysis on genomic and transcriptomic scales. Using our pipeline, 3,482 proteins were predicted to be drug targets. Of these predicted targets, 446 and 1,049 proteins with the highest rank and total scores, respectively, included homologs of targets for cancer chemotherapy, depression, age-related decline and cardiovascular disease. Based on comparative analyses, more than half of drug target proteins identified from the tree shrew genome were shown to be higher similarity to human targets than in the mouse. Target validation also demonstrated that the constitutive expression of the proteinase-activated receptors of tree shrew platelets is similar to that of human platelets but differs from that of mouse platelets. We developed an effective pipeline and search strategy for drug target prediction and the evaluation of model-based target identification for drug testing. This work provides useful information for future studies of the Chinese tree shrew as a source of novel targets for drug discovery research.

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PAR1, 4 of Chinese tree shrews for antithrombotic testing in vitro.(A) Protein sequences alignment of PAR1 and 4 from humans, guinea pig and the tree shrew. The data focused on the “tethered ligand” and “conserved 2nd extracellular loop” (ligand binding region) of PAR1 and 4. (B) PAR1 and 4 of the tree shrew can be greatly activated and aggregated by using various concentrations of PAR1-AP (tPAR1-AP: 30 µM, 90 µM, 300 µM ) or PAR4-AP (tPAR4-AP: 150 µM, 300 µM, 600 µM) . Platelet treated with 3 µg/ml collagen and 0.3 U/ml thrombin as positive control; platelet treated with 1 µl buffer B as negative control. (C) Vorapaxar inhibited thrombin-induced human or tree shrew platelet aggregation. Thrombin-induced human platelet aggregation was significantly inhibited with vorapaxar (p < 0.001), and only a slight inhibition (p  =  0.1019) in the tree shrew. Platelet treated with 0.08U thrombin as the control.
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pone-0104191-g007: PAR1, 4 of Chinese tree shrews for antithrombotic testing in vitro.(A) Protein sequences alignment of PAR1 and 4 from humans, guinea pig and the tree shrew. The data focused on the “tethered ligand” and “conserved 2nd extracellular loop” (ligand binding region) of PAR1 and 4. (B) PAR1 and 4 of the tree shrew can be greatly activated and aggregated by using various concentrations of PAR1-AP (tPAR1-AP: 30 µM, 90 µM, 300 µM ) or PAR4-AP (tPAR4-AP: 150 µM, 300 µM, 600 µM) . Platelet treated with 3 µg/ml collagen and 0.3 U/ml thrombin as positive control; platelet treated with 1 µl buffer B as negative control. (C) Vorapaxar inhibited thrombin-induced human or tree shrew platelet aggregation. Thrombin-induced human platelet aggregation was significantly inhibited with vorapaxar (p < 0.001), and only a slight inhibition (p  =  0.1019) in the tree shrew. Platelet treated with 0.08U thrombin as the control.

Mentions: In humans, PARs (PAR1 and PAR4) are responsible for thrombin-induced platelet activation. Platelet activation and aggregation assays were therefore performed to elicit this function using human PAR activated peptides (PAR-APs; Figure 6A). As shown in Figure 6B, tree shrew platelets could be activated by human PAR1-AP and PAR4-AP, whereas mouse platelets were activated only by human PAR4-AP. In particular, when 30 µM PAR1-AP was added, human platelets displayed robust activation with a steep aggregation curve; in mouse and tree shrew platelets, there was no obvious activation and the curve was smooth. When 90 µM PAR1-AP was added, tree shrew platelets exhibited slight activation and a gradually rising curve, whereas no activation was observed in mouse platelets. Both mouse and tree shrew platelets were activated with the addition of PAR4-AP at 150 and 300 µM (Figure 6B). This surprising difference between the tree shrew and mouse platelet response can be attributed to the extracellular N-terminal end of PAR4 that acts as a tethered ligand. Moreover, a significant difference between mouse and Chinese tree shrew was observed on the histogram maked according to the max aggregation rate of human, mouse and Chinese tree shrew platelets (Figure 6C). The aggregation rate of mouse platelets stimulated with PAR1-AP(90 µM) was significant decreased (79.43%) compared to Chinese tree shrew platelets stimulated with PAR1-AP(90 µM). Meanwhile, using the synthesized PAR-APs of Chinese tree shrews, according to alignment results of “tethered ligand region” of PAR1 and PAR4 from humans and Chinese tree shrews (Figure 7A), we observed Chinese tree shrews' platelets had a greater aggregation with tPAR1-AP (90 µM) or tPAR4-AP (300 µM) than stimulated with human PAR1-AP or PAR4-AP (Figure 7B). Under 0.08U thrombin-induced human platelet aggregation assay, a very significant inhibition (p < 0.01) was observed after platelets pretreated with 33 nM human PAR1 antagonist vorapaxar for 5 min at 37°C, while in Chinese tree shrews a little weak inhibition ( p  =  0.1019 ) was observed (Figure 7C ). These findings shown PAR1 was expressed on the platelet of Chinese tree shrew and was similar with humans, though some differences was also existed between human and Chinese tree shrew PAR1, especially the tethered ligand region and extracellular loop II ligand binding region of PAR1 likely led to the aggregation diversities, some drug testing in platelet could be drawn on the the tree shrews, but not on mice.


Drug target mining and analysis of the Chinese tree shrew for pharmacological testing.

Zhao F, Guo X, Wang Y, Liu J, Lee WH, Zhang Y - PLoS ONE (2014)

PAR1, 4 of Chinese tree shrews for antithrombotic testing in vitro.(A) Protein sequences alignment of PAR1 and 4 from humans, guinea pig and the tree shrew. The data focused on the “tethered ligand” and “conserved 2nd extracellular loop” (ligand binding region) of PAR1 and 4. (B) PAR1 and 4 of the tree shrew can be greatly activated and aggregated by using various concentrations of PAR1-AP (tPAR1-AP: 30 µM, 90 µM, 300 µM ) or PAR4-AP (tPAR4-AP: 150 µM, 300 µM, 600 µM) . Platelet treated with 3 µg/ml collagen and 0.3 U/ml thrombin as positive control; platelet treated with 1 µl buffer B as negative control. (C) Vorapaxar inhibited thrombin-induced human or tree shrew platelet aggregation. Thrombin-induced human platelet aggregation was significantly inhibited with vorapaxar (p < 0.001), and only a slight inhibition (p  =  0.1019) in the tree shrew. Platelet treated with 0.08U thrombin as the control.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4126716&req=5

pone-0104191-g007: PAR1, 4 of Chinese tree shrews for antithrombotic testing in vitro.(A) Protein sequences alignment of PAR1 and 4 from humans, guinea pig and the tree shrew. The data focused on the “tethered ligand” and “conserved 2nd extracellular loop” (ligand binding region) of PAR1 and 4. (B) PAR1 and 4 of the tree shrew can be greatly activated and aggregated by using various concentrations of PAR1-AP (tPAR1-AP: 30 µM, 90 µM, 300 µM ) or PAR4-AP (tPAR4-AP: 150 µM, 300 µM, 600 µM) . Platelet treated with 3 µg/ml collagen and 0.3 U/ml thrombin as positive control; platelet treated with 1 µl buffer B as negative control. (C) Vorapaxar inhibited thrombin-induced human or tree shrew platelet aggregation. Thrombin-induced human platelet aggregation was significantly inhibited with vorapaxar (p < 0.001), and only a slight inhibition (p  =  0.1019) in the tree shrew. Platelet treated with 0.08U thrombin as the control.
Mentions: In humans, PARs (PAR1 and PAR4) are responsible for thrombin-induced platelet activation. Platelet activation and aggregation assays were therefore performed to elicit this function using human PAR activated peptides (PAR-APs; Figure 6A). As shown in Figure 6B, tree shrew platelets could be activated by human PAR1-AP and PAR4-AP, whereas mouse platelets were activated only by human PAR4-AP. In particular, when 30 µM PAR1-AP was added, human platelets displayed robust activation with a steep aggregation curve; in mouse and tree shrew platelets, there was no obvious activation and the curve was smooth. When 90 µM PAR1-AP was added, tree shrew platelets exhibited slight activation and a gradually rising curve, whereas no activation was observed in mouse platelets. Both mouse and tree shrew platelets were activated with the addition of PAR4-AP at 150 and 300 µM (Figure 6B). This surprising difference between the tree shrew and mouse platelet response can be attributed to the extracellular N-terminal end of PAR4 that acts as a tethered ligand. Moreover, a significant difference between mouse and Chinese tree shrew was observed on the histogram maked according to the max aggregation rate of human, mouse and Chinese tree shrew platelets (Figure 6C). The aggregation rate of mouse platelets stimulated with PAR1-AP(90 µM) was significant decreased (79.43%) compared to Chinese tree shrew platelets stimulated with PAR1-AP(90 µM). Meanwhile, using the synthesized PAR-APs of Chinese tree shrews, according to alignment results of “tethered ligand region” of PAR1 and PAR4 from humans and Chinese tree shrews (Figure 7A), we observed Chinese tree shrews' platelets had a greater aggregation with tPAR1-AP (90 µM) or tPAR4-AP (300 µM) than stimulated with human PAR1-AP or PAR4-AP (Figure 7B). Under 0.08U thrombin-induced human platelet aggregation assay, a very significant inhibition (p < 0.01) was observed after platelets pretreated with 33 nM human PAR1 antagonist vorapaxar for 5 min at 37°C, while in Chinese tree shrews a little weak inhibition ( p  =  0.1019 ) was observed (Figure 7C ). These findings shown PAR1 was expressed on the platelet of Chinese tree shrew and was similar with humans, though some differences was also existed between human and Chinese tree shrew PAR1, especially the tethered ligand region and extracellular loop II ligand binding region of PAR1 likely led to the aggregation diversities, some drug testing in platelet could be drawn on the the tree shrews, but not on mice.

Bottom Line: The discovery of new drugs requires the development of improved animal models for drug testing.The Chinese tree shrew is considered to be a realistic candidate model.Target validation also demonstrated that the constitutive expression of the proteinase-activated receptors of tree shrew platelets is similar to that of human platelets but differs from that of mouse platelets.

View Article: PubMed Central - PubMed

Affiliation: Key Laboratory of Animal Models and Human Disease Mechanisms of the Chinese Academy of Sciences & Yunnan Province, Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming, Yunnan, PR China; Kunming College of Life Science, University of Chinese Academy of Sciences, Kunming, Yunnan, PR China.

ABSTRACT
The discovery of new drugs requires the development of improved animal models for drug testing. The Chinese tree shrew is considered to be a realistic candidate model. To assess the potential of the Chinese tree shrew for pharmacological testing, we performed drug target prediction and analysis on genomic and transcriptomic scales. Using our pipeline, 3,482 proteins were predicted to be drug targets. Of these predicted targets, 446 and 1,049 proteins with the highest rank and total scores, respectively, included homologs of targets for cancer chemotherapy, depression, age-related decline and cardiovascular disease. Based on comparative analyses, more than half of drug target proteins identified from the tree shrew genome were shown to be higher similarity to human targets than in the mouse. Target validation also demonstrated that the constitutive expression of the proteinase-activated receptors of tree shrew platelets is similar to that of human platelets but differs from that of mouse platelets. We developed an effective pipeline and search strategy for drug target prediction and the evaluation of model-based target identification for drug testing. This work provides useful information for future studies of the Chinese tree shrew as a source of novel targets for drug discovery research.

Show MeSH
Related in: MedlinePlus