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Sakamoto,H., et al. 2011. CH5424802, a Selective ALK Inhibitor Capable of Blocking the Resistant Gatekeeper Mutant. Cancer Cell 19:679-690.

Part A: Sakamoto, et al, 2011. Short answer. Please write your answer in the space provided. Bulleted points are fine.

1. Introduction & methods: Sakamoto and colleagues discuss selectivity of kinase inhibitors and development of resistance as key issues for improved cancer therapies.

a. The authors describe the importance of potency and selectivity of kinase inhibitors. They cite as an example the first and second generation BCR-ABL inhibitors imatinib and nilotinib. What strategy for kinase inhibitors (e.g., ALK inhibitors) do they suggest is needed to achieve higher efficacy and safety?

b. What type of resistance do they identify as a serious problem for long-term cancer treatment? Name one mutation of the EML4-ALK fusion oncogene they identify as potentially conferring resistance to ALK inhibitors.

c. Methods (end of paper) – Kinase inhibitory assays in vitro: What method did the authors use to measure the inhibitory ability of Alectinib (CH5424802) against kinases (except MEK1 and Raf-1)?

d. Methods – What EML4-ALK variant and mutant did the authors express in Ba/F3 cells? How were the transfected cells generated and confirmed?


2. Results – Potent and selective inhibition of ALK Activity (Table 1 and Fig. 1):

a. What property/properties of CH5424802 (alectinib) does table 1 show?

b. What is the ratio (roughly) of the IC50 value for inhibition INSR (insulin receptor) compared to the IC50 for inhibition of ALK?

c. What concentrations of alectinib (CH5424802) inhibited phosphorylation of ALK (Fig. 1B)?

3. Results – Selective antitumor activity in ALK-positive cancers (Fig. 1 D, E; Fig. 2):

a. Alectinib (CH5424802) inhibited the growth of NCI-H2228 cells but not HCC827 cells (Figure 1D). In contrast, gefitinib inhibited the growth of HCC827 cells but not NCI-H2228 cells (Figure 1D). What do these results indicate about the selectivity of the antitumor activity of alectinib and gefitinib?

b. What does Figure 1E show? What is the significance of this finding?

c. What effect did alectinib have on the growth (measured as tumor volume) of NCI-H2228 and A549 tumors (Fig. 2A, upper panels)? Did alectinib shrink the tumors (regression)?

d. What effect did alectinib have on body weight over 28 days of treatment (Fig. 2A (lower panels)?

4. Results – Potency of Alectinib (CH5424802) against the L1196M Gatekeeper Mutation of ALK (Figures 4 and 5).

a. What does Figure 4B show about alectinib (CH5424802) and crizotinib (PF-02341066) in terms of kinase inhibitory Ki for native (wild type) and L1196M mutant ALK?

b. What does Figure 4D show in terms of 50% inhibitory concentration (IC50) for cells with native ALK or L1196M mutant ALK?

c. Figure 5 shows results from an in vivo tumor growth study using Ba/F3 cells expressing either native (wild type) or mutant (L1196M) ALK. What do the upper panels of Figure 5 demonstrate in terms of antitumor activity of crizotinib (PF-02341066) and alectinib (CH5424802)?

d. In Figure 5, did alectinib cause tumor stasis (no growth but no shrinkage) or tumor regression (tumor shrinkage)?


5. Discussion – Advantages and features of alectinib (CH5424802).

a. What do the authors claim is a remarkable characteristic of alectinib (CH5424802)?


b. At a molecular level (crystal structure analysis) what interaction of CH5424802 with ALK do the authors highlight as key for achieving selectivity for ALK? How is this different than other ALK inhibitors (according to the authors)?


c. The authors note that other ALK inhibitors also inhibit IGF-1R and insulin receptor (INSR). They state that selective ALK inhibitors (e.g., CH5424802 – Alectinib) would exhibit sufficiently wide therapeutic windows in patients with ALK-activated cancers. What is their reasoning/logic for this?


d. The authors focused on the gatekeeper mutation in ALK for this study. What was their reasoning for this? What data in the paper support this claim (briefly list figures or tables supporting activity vs the gatekeeper mutation)? How do these results enable them to hypothesize in the last paragraph “We expect that CH5424802 (Alectinib) might provide therapeutic opportunities for patients with acquired resistance to PF-02340166 (Crizotinib)?”


e. BONUS: Drug Label – Both Zykadia (ceritinib) and Alecensa (Alectinib) are approved for treating ALK-positive metastatic NSCLC in patients who have progressed on crizotinib. In the label under mechanisms of action, ceritinib is characterized as an inhibitor of ALK, insulin-like growth factor 1 receptor (IGF-1R), insulin receptor (InsR) and ROS1. In the Alecensa label the same section say “Alectinib is a tyrosine kinase inhibitor that targets ALK and RET.” Ceritinib has more and more strongly worded warnings and precautions (e.g., hyperglycemia, pancreatitis, severe gastrointestinal toxicity) than does alectinib. Briefly explain (two reasons or examples) how the selectivity differences between the two may underpin the safety differences.

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