In this study, we identified a subset of the migrastatin family of glutarimide-containing natural products, including LTM and isomigrastatin, as potent inhibitors of eukaryotic translation elongation. Despite their structural similarity to the cell migration inhibitor migrastatin, LTM and isomigrastatin act by a completely different mechanism and their ability to inhibit cell migration very likely is only secondary to their effect on translation elongation. It is quite interesting to compare the structure and activity of migrastatin, isomigrastatin, dorrigocin and LTM. Although migrastatin, isomigrastatin and dorrigocin B share the same constituents and the glutarimide moiety, isomigrastatin features a 12-membered macrocycle, which can be readily converted to either the 14-membered migrastatin or the linear dorrigocins. Yet, the three natural products have completely distinct biological properties. While migrastatin inhibits cell migration, isomigrastatin inhibits translation and the dorrigocins possess neither activity. The shared inhibitory capacity on eukaryotic protein translation between isomigrastatin and LTM highlights the importance of the 12-membered macrolides for this activity. The lack of activity in the linear analogs including 8
is somewhat surprising. Streptimidone, which has a structure similar to that of CHX, essentially consisting of only the glutarimide and a linker without the macrolide present in LTM, also inhibits translation 2
. However, extension of the linker region with a flexible chain in dorrigocin B (6
) abolished any inhibitory activity.
The structural similarity between LTM and CHX and their common effect on eukaryotic translation elongation offered an opportunity to deconvolute their mechanisms of action. A systematic examination of their effects on different steps of translation elongation revealed that both inhibitors share a similar mechanism of action by blocking translation elongation through binding to the same position in the E site of the large ribosomal subunit. In addition, these experiments also revealed some subtle but definitive differences between LTM and CHX at their physiologically active concentrations. First, LTM is over ten-fold more potent than CHX for the inhibition of protein synthesis both in vitro and in vivo. Second, while LTM caused a significant depletion of polysomes in cell culture, CHX had little effect on the polysome profile. Third, LTM stalled the ribosome on the initiating AUG codon, but CHX appeared to allow one round of translocation such that the ribosome stalled on the second codon of the template mRNA. Both similarities and differences between the two structurally related inhibitors can now be reconciled based on the new observations made in this study.
The footprint at C3993 generated by both LTM and CHX, together with the cross-resistance to the yeast L28 (mammalian L27a) mutant towards both inhibitors, provides for the first time three key points defining a common binding pocket for both inhibitors in the ribosome. Taking the reports on L36a into account, this binding site lies between C3993 at the base of hairpin 88 of the 28S rRNA, the 38th
amino acid of L27a and proline 54 of L36a. All three points of reference lie in vicinity of one another and in proximity to the two 3’-terminal nucleotides of the E-site tRNA (Supplementary Fig. 8
). The location of the footprint corresponds to the same nucleotide that was protected by the 3’-end of the E-site tRNA in bacterial ribosomes34
. Under the same conditions as used for the E. coli
ribosome, we observed a footprint of Phe-tRNA on rabbit ribosomes as well, confirming the conserved function of the location.
It has been previously proposed that CHX likely acts via the E-site3
. In this study, we offer direct experimental evidence corroborating the proposed model. The lower affinity of CHX compared to LTM alone, however, could not account for the differences in polysome profile and toeprinting pattern. The underlying cause of these differences likely stems from the larger size of LTM due to its unique 12-membered macrolide, which CHX lacks. When de-acylated tRNA was 3’ end-labeled with [32
P], we observed a decrease in tRNA binding at increasing concentrations of LTM but not CHX. Only at a concentration of 10 mM did CHX cause a detectable decrease in the amount of bound tRNA. It is unclear, though, whether the effect of CHX at 10 mM is solely due to its binding to the E site or to some non-specific interactions with other sites of the ribosome or the de-acylated tRNA. It had been previously observed that CHX treatment of ribosomes decreases their ability to release deacylated tRNA2
. With its 12-membered macrocycle, LTM is significantly larger in size and thus takes up considerably more space than the smaller CHX. Both molecules bind to the same pocket of the ribosome and given their structural similarity around the glutarimide moiety, it is tempting to speculate that the observed footprint is the binding site of the glutarimide portion of each inhibitor. In bacteria, interaction between the tRNA 3’-OH and the E-site is necessary to stimulate translocation36
. This is consistent with our observation that both LTM and CHX inhibited eEF2-mediated translocation from the A to the P site and reduced the rate of tripeptide formation.
Taking all existing experimental results into consideration, we propose a mechanistic model for the action of both CHX and LTM (). In this model, both CHX and LTM share a largely similar mechanism through binding to the E site of the 60S ribosome. The binding of CHX and LTM to the E site blocks eEF2-mediated tRNA translocation. This model explains the similar effects of CHX and LTM on translational elongation and tripeptide formation (). A difference between CHX and LTM lies in the ability of CHX to bind the E site together with the E-site tRNA while the larger LTM occludes deacylated tRNA from the E site. Thus, binding of CHX to the E site alone does not affect translocation while occupation of the E site by both CHX and deacylated tRNA stalls translocation, leading to an arrest of the ribosome on the second codon (). This is in agreement with the observation by others that CHX allows for 2 rounds of translocation on a CrPV IRES template, because the cricket paralysis virus element initiates translation without initiator tRNA and begins translation from the A-site3
. Consequently it takes two translocation events before deacylated tRNA reaches the E site. While LTM binds to the same site, its sheer size occludes deacylated tRNA. Thus LTM blocks the very first round of elongation and prevents the ribosome from leaving the start site. Unlike LTM, the smaller CHX presumably can also bind to actively translocating ribosomes, providing a plausible explanation for the distinct polysome profiles for LTM and CHX. In contrast to CHX, the mutually exclusive binding of LTM and deacylated tRNA to the E site leads to preferential binding of LTM to empty E site immediately after initiation, allowing dipeptide formation but blocking the translocation of the newly formed deacylated initiating tRNA from translocating from the P to E site, stalling the ribosome at the AUG start codon. Once elongation has been initiated and the E site is occupied by deacylated tRNA, it will be more difficult for LTM to gain access to the E site, leading to polysome depletion (). Unlike LTM, CHX can interrupt translation elongation at any time, as its binding to the E site is independent of the occupancy of E site by de-acylated tRNA. Thus, its polysome profile does not significantly differ from that of an untreated cell (). We note that this model does not seem to account for the effect of CHX on eEF-2 mediated translocation assay indirectly measured by phenylalanyl puromycin formation at first sight. However, there were plenty of deacylated tRNA present in the translocation assay, whose co-occupation of the E site may explain the inhibition seen by CHX.
Mechanistic models for inhibition of translation elongation by CHX and LTM
Increasing evidence points to a connection between protein synthesis and cancer cell growth. Didemnin B and homoharringtonine, two small molecule inhibitors of translation, have advanced to clinical trials37,38
. Inhibitors of translation elongation in conjunction with an established chemotherapeutic agent such as doxorubicin have been shown to sensitize the tumor to therapy19
. Furthermore, the development of drug resistance necessitates expression of anti-apoptotic proteins or drug transporters. Inhibition of translation should therefore greatly suppress the occurrence of resistance. Given LTM’s specificity for transformed cell lines and effect on in vivo
tumor growths, our findings seem to corroborate a potential therapeutic use of translation elongation inhibitors in cancer treatment.
LTM furthermore extends the molecular toolbox for inhibiting a specific step in eukaryotic translation. Together with CHX, LTM makes it possible to dissect translation at the first and at the second translocation step. A comparison between LTM and CHX reveals how the CHX core structure is further elaborated through addition of a 12-member macrocycle to enhance its affinity for the E site of the ribosome and increase its potency against tumor cell lines. It remains to be determined which structural element of the E site of the ribosome, be it ribosomal RNA or protein, interacts with the macrocycle portion of LTM to confer higher potency. Deeper insights into the interaction between LTM and the E site of the ribosome may further our mechanistic understanding of translocation and guide the design of future small molecule inhibitors of eukaryotic translation. It is possible that chemical modifications of the macrolide portion of LTM and isomigrastatin will further enhance the potency and specificity of this family of natural products.