High Consistency of Structure-Based Design and X-Ray Crystallography: Design, Synthesis, Kinetic Evaluation and Crystallographic Binding Mode Determination of Biphenyl-N-acyl-β-d-Glucopyranosylamines as Glycogen Phosphorylase Inhibitors
, Efthimios Kyriakis 2, George A. Stravodimos 2, Vassiliki T. Skamnaki 2, Panagiota G.V. Liggri 2,3, Spyros E. Zographos 3, Rainer Riedl 1,* and Demetres D. Leonidas 2,*
Round 1
Reviewer 1 Report
The manuscript by Fischer et al describes the design, synthesis and combined kinetic and structural characterization of two novel inhibitors of glycogen phosphorylase. The methods, results and interpretations are clearly described in sufficient detail for high quality studies in this field. The modest decrease in Ki for the two compounds compared to the reference compound, the agreement of experimentally determined binding modes with modes generated by docking studies, and a few of the structural details provide sufficient interest and novelty to warrant publication.
The writing and presentation are generally excellent, and I only have a few minor suggestions or corrections:
1. The paper references an earlier paper with regards to crystallization conditions, but the Kyriakis paper also does not give specific precipitant, pH, etc. Instead, it refers to yet another paper. It is unreasonable to expect a reader to chase down these basic experimental conditions through a chain of references. I insist that these critical experimental details be repeated here. pH is particular important for interpreting some of the details of interactions between His residues, etc. with the bound ligands. The final steps in the preparation of the protein needs to be added as well. The compounds appear quite hydrophobic. Was any additional DMSO or other solvents added during soaking above the 10% stated for the soaking solution?
2. Figure 6 does not provide a sufficiently clear view of the two loops to understand the nature of the conformational difference. I suggest that the authors prepare a stereoscopic diagram or a movie to more clearly show the differences between the two structures.
3. The last sentence on page 6, right above Fig. 6 is not clear:
"This conformation is further stabilized through formation of a hydrogen bond between Asp283 and Phe285."
What functional group in Asp283 is forming a hydrogen bond with which functional group of Phe285? Improving the clarity of Fig. 6 would also help, but I think that both the sentence and the figure need to be improved.
4. In the second-last paragraph on page 7, I think that another point should be made:
"Furthermore, despite the fact that the two terminal phenyl groups of 3 and 4 are inclined by ~15° to each other, both are involved in π-stacking interactions to the imidazole ring of His341 (figure 5)."
If His341 is protonated (and the likelihood of this depends on the pH of the soaking solution, which I was not able to determine), perhaps a favourable cation-pi interaction might also be present.
5. The very last sentence of the manuscript before the methods is very poorly written. It is a reasonable point, but the sentence is grammatically incorrect and contains a spelling/word use error (“rationale”).
"Given the somehow unexpected significant difference in the potency of the two inhibitors the present study highlights the importance in obtaining structural data in every step of the inhibitor optimization process, to guide it and provide the rational for the next step."
6. The legend for the symbols in Figure S1 are incomprehensible and must be fixed. I think that both graphs and insets also need to be expanded in size for clarity.
7. The unit cell dimensions need to be added to the crystallographic statistics table S1.
Author Response
1. The paper references an earlier paper with regards to crystallization conditions, but the Kyriakis paper also does not give specific precipitant, pH, etc. Instead, it refers to yet another paper. It is unreasonable to expect a reader to chase down these basic experimental conditions through a chain of references. I insist that these critical experimental details be repeated here. pH is particular important for interpreting some of the details of interactions between His residues, etc. with the bound ligands. The final steps in the preparation of the protein needs to be added as well. The compounds appear quite hydrophobic. Was any additional DMSO or other solvents added during soaking above the 10% stated for the soaking solution?
Response:
Following the reviewer’s suggestion we have now included all details of the crystallization process to section 4.5. The final steps for the preparation of the protein for crystallization are also included.
No additional DMSO or other solvents were added during the soaking above the 10% stated in the manuscript.
2. Figure 6 does not provide a sufficiently clear view of the two loops to understand the nature of the conformational difference. I suggest that the authors prepare a stereoscopic diagram or a movie to more clearly show the differences between the two structures.
Response:
We have now replaced Figure 6 with a stereo diagram. A movie is included in the supporting information file.
3. The last sentence on page 6, right above Fig. 6 is not clear:
"This conformation is further stabilized through formation of a hydrogen bond between Asp283 and Phe285."
What functional group in Asp283 is forming a hydrogen bond with which functional group of Phe285? Improving the clarity of Fig. 6 would also help, but I think that both the sentence and the figure need to be improved.
Response:
We have now modified the sentence, following the reviewer’s suggestion to “This conformation is further stabilized through the formation of a hydrogen bond between the main chain carbonyl oxygen of Asp283 and the main chain amide of Phe285.”
4. In the second-last paragraph on page 7, I think that another point should be made:
"Furthermore, despite the fact that the two terminal phenyl groups of 3 and 4 are inclined by ~15° to each other, both are involved in π-stacking interactions to the imidazole ring of His341 (figure 5)."
If His341 is protonated (and the likelihood of this depends on the pH of the soaking solution, which I was not able to determine), perhaps a favourable cation-pi interaction might also be present.
Response:
We thank the reviewer for his/her comment. The interaction between the phenyl groups of 3 and 4 and that of the imidazole ring of His341 is a cation-pi interaction. We now mention this in the manuscript.
5. The very last sentence of the manuscript before the methods is very poorly written. It is a reasonable point, but the sentence is grammatically incorrect and contains a spelling/word use error (“rationale”).
"Given the somehow unexpected significant difference in the potency of the two inhibitors the present study highlights the importance in obtaining structural data in every step of the inhibitor optimization process, to guide it and provide the rational for the next step."
Response:
We have now rephrased the very last sentence to “Given the unexpected significant difference in the potency of the two inhibitors, which can be explained by the X-ray crystallography results, the present study highlights the importance of obtaining structural data in every step of the inhibitor optimization process.”
6. The legend for the symbols in Figure S1 are incomprehensible and must be fixed. I think that both graphs and insets also need to be expanded in size for clarity.
Response:
Following reviewers’ suggestion graphs in Figure S1 are now expanded for clarity and the legend has been fixed to be more comprehensible.
7. The unit cell dimensions need to be added to the crystallographic statistics table S1.
Response:
Unit cell dimensions were now added to the crystallographic statistics table S1.
Reviewer 2 Report
This is a concise and interesting article describing a full story about structure-based inhibitor discovery. From background to rational design, docking study to chemistry synthesize, the biochemical assay to structural analysis, each work is solid and well-done. Also, it is a well-prepared manuscript with an elegant demonstration. The author provides sufficient experimental details and the conclusions are supported and are well justified. The importance of the finding, good quality of data and the subject are of sufficiently wide interest to merit publication in Molecules. I suggest publishing this manuscript with minor revision.
Some point needs to be considered:
Do you perform any in silico screen when designing the warhead in attaching to the phenyl group?
In Fig. 3, what is the docking score of these poses?
Please show the residue (Tyr280, Asp339, and Ala383) discussed in the second paragraph of 2.1. Molecular Modeling to figure 3
It would be better if the authors can briefly describe how the assay determines the inhibition constant in the main text.
Author Response
Do you perform any in silico screen when designing the warhead in attaching to the phenyl group?
Response:
No, we visually inspected the space given in the pocket next to the original ligand and placed a generic phenyl group. The postulated molecules were docked and consecutively synthesized.
In Fig. 3, what is the docking score of these poses?
Response:
We added the three docking scores to the figure caption of figure 3.
Please show the residue (Tyr280, Asp339, and Ala383) discussed in the second paragraph of 2.1. Molecular Modeling to figure 3
Response:
We added the three residues (Tyr280, Asp339 and Ala383) to figure 3.
It would be better if the authors can briefly describe how the assay determines the inhibition constant in the main text.
Response:
We have now explained in more detail how from the enzymatic assay we determine inhibition constant values in the main text.
