Structure–function insights reveal the human ribosome as a cancer target for antibiotics

Many antibiotics in clinical use target the bacterial ribosome by interfering with the protein synthesis machinery. However, targeting the human ribosome in the case of protein synthesis deregulations such as in highly proliferating cancer cells has not been investigated at the molecular level up to now. Here we report the structure of the human 80S ribosome with a eukaryote-specific antibiotic and show its anti-proliferative effect on several cancer cell lines. The structure provides insights into the detailed interactions in a ligand-binding pocket of the human ribosome that are required for structure-assisted drug design. Furthermore, anti-proliferative dose response in leukaemic cells and interference with synthesis of c-myc and mcl-1 short-lived protein markers reveals specificity of a series of eukaryote-specific antibiotics towards cytosolic rather than mitochondrial ribosomes, uncovering the human ribosome as a promising cancer target.

In this manuscript, the authors disclosed for the first time structures of human 80S ribosome in complex with E-site tRNA and the translation inhibitor cycloheximide, respectively. A comparison of the structures offers a molecular rationale for the effective competition of cycloheximide against the much larger tRNA and revealed subtle but important differences in molecular interactions with cycloheximide between human and yeast ribosome, suggesting future directions for modifying existing inhibitors to potentially improve potency. Separately, the authors tested a number of translation elongation inhibitors against a panel of leukemia cells and demonstrated anti-leukemic activity via inhibition of translation as reflected in the expression levels of c-Myc and Mcl-1.
Although the authors have previously reported the Cryo-EM structure of the human 80S ribosome and the structure of the complex between yeast ribosome and cycloheximide was reported by others, there are interesting and significant new insights revealed in this manuscript as described above. As such, the manuscript deserves consideration for publication in Nature Communications.
There are a couple of issues that the authors need to address/clarify to further improve the manuscript.
(1) In the introduction, the authors stated that "targeting the human ribosome has not yet been envisaged yet...". This is simply not true. In fact, homoharringtonine that was tested against various cancer cell lines by the author is the first approved drug targeting translation for the treatment of CML. And eukaryotic translation has been pursued as a general cancer drug target for a long time by many groups.
(2) The average resolution for the structure was reported to be 3.6 Å with some parts having higher resolution. It was unclear what is the resolution for the cycloheximide and the confidence level on the different bonding interactions described for its interaction with the ribosome.
Reviewer #2 (Remarks to the Author) Myasnikov et al.
The manuscript entitled "Structure-function insights reveal the human ribosome as a cancer target for antibiotics" by authors Myasnikov et al. presents the results on the antibiotic CHX in two indirectly related parts of a study. One is a cryo-EM study of the human ribosome in either presence or absence of CHX along with their atomic models. The other is a study of the role of CHX in anti-proliferative dose response in leukemic cells and in interference with synthesis of cmyc and mcl-1 short-lived protein markers. This manuscript concludes that CHX specifically targets the ribosome in leukemic cells. The following comments specifically address the cryo-EM part of the study only.
The MS does not fulfill the minimum requirements for showing the evidence from which conclusions are drawn. The only two cryo-EM maps that are shown, apo, vs. CHX-bound, are uninformative as presented. Furthermore, no details of atomic modeling are presented, and the validity of structural details related to the binding of the ligand and accompanying conformational changes cannot be assessed/verified since the critical parts of the structure are not shown in the context of map density. As such, the MS is unsuited for serious consideration in this journal. only shown in Figure 1 in a size little larger than thumbnails. None of the panels shows the correspondence between the maps and inferred structures. The atomic models themselves lack explicit validation.
2. In Results (pages 4-5), the authors write: "The overall binding site corresponds to that observed in the yeast ribosome (8) but the ligand is slightly rotated, has the piperidine-2,6-dione moiety flipped and exhibits an additional interaction with the 2-keto group of C4342. Specific interactions in the ligand pocket (Fig. 1c)..." . The questions are: (a) If this is the structural base to claim that CHX specifically targets the human ribosome, Figure  1 does not show the differences in the binding of CHX with either the human ribosome or the ribosome in yeast. 4. On page 6, the authors write: "When tested on normal peripheral blood lymphocytes (PBLs), anisomycin and verrucarin-A displayed some toxicity for normal cells (36 and 24% increase in cell death, respectively) but only at very high doses (30 and 1 μM, respectively), while CHX did not trigger cell death in normal cells compared to leukemic cell lines (Suppl. data Fig. 5).". -Are there any indications that the human ribosome is different in normal cells and in leukemic cells? 5. In Methods (page 9), the authors write: "Both complexes contain E-site tRNA but the rotated 80S particles have eEF2, while the non-rotated particles contain no factor." But the map showing eEF2 was ignored without obvious reason. It would be interesting to see if CHX is present in that map.
6. In Methods (pages 9-10), the authors write: "3D sorting of non-rotated states showed (after exclusion of bad 3D class containing 1899 particles) a class without E-site tRNA (19026 particles) with CHX bound and the rest contained E-site tRNA (50311 particles).". But they reported one resolution value only, 3.6 Angstroms ("The resolution was estimated in Relion and IMAGIC at 0.143 FSC and half-bit criteria (16,18,19), indicating an average resolution of 3.6 Å". It is difficult to see how these two maps, including 1899 and 50311 particles, respectively, could end up with the same resolution. An explanation is needed. 7. In Methods (page 10), the authors write: "Local resolution estimation with ResMap (20) shows that many regions reach ~3.1 Å resolution in the CHX 80S complex." -this is the map including fewer particles. It is necessary to show the local resolution map in the MS.
Minor points 1. A lot of abbreviations are used in the MS, which may not be clear to many readers. At one place, a list of the abbreviations with the full meaning for each is needed. 2. The writing at a number of places needs to be clarified. There are a number of poor, ambiguous constructions, poor word choices and missing information. For example" (a) on page 3 "The ribosome is the molecular machinery at the heart of protein synthesis, a highly regulated function which is tightly wired to cell activation and proliferation ..." (b) on page 7: "The tRNA complex exhibits no density for CHX...."

Reviewer #1 (Remarks to the Author):
In this manuscript, the authors disclosed for the first time structures of human 80S ribosome in complex with E-site tRNA and the translation inhibitor cycloheximide, respectively. A comparison of the structures offers a molecular rationale for the effective competition of cycloheximide against the much larger tRNA and revealed subtle but important differences in molecular interactions with cycloheximide between human and yeast ribosome, suggesting future directions for modifying existing inhibitors to potentially improve potency. Separately, the authors tested a

number of translation elongation inhibitors against a panel of leukemia cells and demonstrated anti-leukemic activity via inhibition of translation as reflected in the expression levels of c-Myc and Mcl-1.
Although the authors have previously reported the Cryo-EM structure of the human 80S ribosome and the structure of the complex between yeast ribosome and cycloheximide was reported by others, there are interesting and significant new insights revealed in this manuscript as described above. As such, the manuscript deserves consideration for publication in Nature Communications.
We thank the referee for the very positive feedback and the detailed insights.
There are a couple of issues that the authors need to address/clarify to further improve the manuscript.
(1) In the introduction, the authors stated that "targeting the human ribosome has not yet been envisaged yet...". This is simply not true. In fact, homoharringtonine that was tested against various cancer cell lines by the author is the first approved drug targeting translation for the treatment of CML. And eukaryotic translation has been pursued as a general cancer drug target for a long time by many groups.
We have now toned down the statement and rephrased this sentence. Homoharringtonine is indeed a very good example and illustrates the potential for cancer research of protein synthesis inhibitors. We thank the referee for these comments that help clarifying this point.
(2) The average resolution for the structure was reported to be 3.6 Å with some parts having higher resolution. It was unclear what is the resolution for the cycloheximide and the confidence level on the different bonding interactions described for its interaction with the ribosome. As suggested, we have now estimated the local resolution and show the atomic model together with the cryo-EM. To describe this in more detail we have created an additional figure (Fig. 2) and also modified Fig. 1 to show the cryo-EM map of the L1 region.

Reviewer #2 (Remarks to the Author):
The manuscript entitled "Structure-function insights reveal the human ribosome as a cancer target for antibiotics" by authors Myasnikov et al. presents the results on the antibiotic CHX in two indirectly related parts of a study. One is a cryo-EM study of the human ribosome in either presence or absence of CHX along with their atomic models. The other is a study of the role of CHX in anti-proliferative dose response in leukemic cells and in interference with synthesis of c-myc and mcl-1 short-lived protein markers. This manuscript concludes that CHX specifically targets the ribosome in leukemic cells. The following comments specifically address the cryo-EM part of the study only.
The MS does not fulfill the minimum requirements for showing the evidence from which conclusions are drawn. The only two cryo-EM maps that are shown, apo, vs. CHX-bound, are uninformative as presented. Furthermore, no details of atomic modeling are presented, and the validity of structural details related to the binding of the ligand and accompanying conformational changes cannot be assessed/verified since the critical parts of the structure are not shown in the context of map density. As such, the MS is unsuited for serious consideration in this journal.
We thank the referee for the constructive comments and for suggesting to show more details on the cryo-EM map and the derived atomic model. This is now addressed in detail by modifying Fig. 1 and  by creating a new figure (Fig. 2) as described below.

Major points in detail:
1. The cryo-EM maps are among the major results of this study, but, paradoxically, the maps are only shown in Figure 1 in a size little larger than thumbnails. None of the panels shows the correspondence between the maps and inferred structures. The atomic models themselves lack explicit validation.
We have now added several images to show the cryo-EM map and the derived atomic model. To describe this in more detail we have created an additional figure (Fig. 2) which shows the atomic model together with the cryo-EM of the ligand-binding pocket region and a local resolution estimation. Moreover, we modified Fig. 1 to also show the cryo-EM map of the L1 region and describe the accompanying conformational changes. In addition, we provide an additional figure in the Suppl. Data (Suppl. Data Fig. 2) to show the overall quality of the cryo-EM map and the atomic model. Finally, the geometric parameters of the derived atomic model show a very good model quality as described in the methods section, further refined while the manuscript was under review and also improved over our previously published human 80S ribosome structure (Khatter et al., 2015). , the authors write: "The overall binding site corresponds to that observed in the yeast ribosome (8) but the ligand is slightly rotated, has the piperidine-2,6-dione moiety flipped and exhibits an additional interaction with the 2-keto group of C4342. Specific interactions in the ligand pocket (Fig. 1c)..." . The questions are: (a) If this is the structural base to claim that CHX specifically targets the human ribosome, Figure 1 does not show the differences in the binding of CHX with either the human ribosome or the ribosome in yeast.

In
We now show a comparison of the human and yeast ribosome structures with CHX (new Fig. 2d) (b) What is the reason and proposed mechanism for the flip?
The reason for the flip could be the presence of a Mg 2+ ion that is absent in the yeast ribosome CHX complex. We have added this in the text now. (c) Can the slight flip result from the specific binding of CHX to the human ribosome?