Synthesis and single-molecule imaging reveal stereospecific enhancement of binding kinetics by the antitumour eEF1A antagonist SR-A3

Abstract

Ternatin-family cyclic peptides inhibit protein synthesis by targeting the eukaryotic elongation factor-1α. A potentially related cytotoxic natural product (‘A3’) was isolated from Aspergillus, but only 4 of its 11 stereocentres could be assigned. Here, we synthesized SR-A3 and SS-A3—two out of 128 possible A3 epimers—and discovered that synthetic SR-A3 is indistinguishable from naturally derived A3. Relative to SS-A3, SR-A3 exhibits an enhanced residence time and rebinding kinetics, as revealed by single-molecule fluorescence imaging of elongation reactions catalysed by eukaryotic elongation factor-1α in vitro. An increased residence time—stereospecifically conferred by the unique β-hydroxyl in SR-A3—was also observed in cells. Consistent with its prolonged duration of action, thrice-weekly dosing with SR-A3 led to a reduced tumour burden and increased survival in an aggressive Myc-driven mouse lymphoma model. Our results demonstrate the potential of SR-A3 as a cancer therapeutic and exemplify an evolutionary mechanism for enhancing cyclic peptide binding kinetics via stereospecific side-chain hydroxylation.

Main

All living organisms rely on protein synthesis mediated by the ribosome and its associated translation factors. Bacterial ribosomes have long been targeted by small-molecule antimicrobials¹, while the human ribosome and translation factors have recently emerged as promising drug targets for cancer and viral infections^2,3. Eukaryotic elongation factor-1α (eEF1A) is an essential component of the translation machinery⁴. GTP-bound eEF1A delivers aminoacyl-transfer RNAs (aa-tRNAs) to the ribosomal A site during the elongation phase of protein synthesis. Base pairing between the A-site messenger RNA (mRNA) codon and the aa-tRNA anticodon promotes GTP hydrolysis by eEF1A, releasing the aa-tRNA from eEF1A and allowing its accommodation into the ribosome. The growing protein chain is subsequently transferred from the P-site peptidyl tRNA to the A-site aa-tRNA, extending it by one amino acid through ribosome-catalysed peptide bond formation.

Tumour cells and viruses hijack the protein synthesis machinery to elicit growth and replication. Specific eEF1A inhibitors—all of which are macrocyclic natural products—have been evaluated as potential anticancer and antiviral drugs⁵. Didemnin B^6,7, cytotrienin A⁸, nannocystin A⁹ and cordyheptapeptide A¹⁰ are examples of structurally diverse macrocycles that bind eEF1A and inhibit translation elongation. Dehydro-didemnin B (plitidepsin) is approved in Australia for the treatment of relapsed/refractory multiple myeloma¹¹ and is efficacious in SARS-CoV-2 infection models¹².

The cyclic heptapeptide A3 was isolated from an Aspergillus strain on the basis of its ability to inhibit cancer cell proliferation at low nanomolar concentrations¹³. Although the amino acid composition, sequence and N-methylation pattern of A3 were deduced, only four out of 11 stereocentres could be assigned (Fig. 1). Motivated by its potent anti-proliferative activity and unknown mechanism of action, we sought to determine which of the 128 possible stereoisomers (based on seven unassigned stereocentres) corresponds to A3. Based on our observation that the amino acid sequence and N-methylation pattern of A3 and ternatin are similar¹⁴, we had previously designed and synthesized ternatin-4, which incorporates the dehydromethyl leucine (dhML) and pipecolic acid residues found in A3, yet lacks the β-hydroxy group attached to N-Me-Leu (Fig. 1). We discovered that ternatin-4 inhibits cancer cell proliferation and SARS-CoV-2 replication by targeting eEF1A^15,16. However, the precise step(s) of eEF1A-catalysed elongation blocked by ternatin-family cyclic peptides—as well as the structure of A3 and the role of its unique β-hydroxy group—all remained unknown.

Fig. 1: Partially determined structure of the natural product A3.

We hypothesized that A3 corresponds to one of two epimers—SR-A3 or SS-A3—related to ternatin, a natural product, and ternatin-4, an A3-inspired synthetic compound¹⁴. Pip, pipecolic acid.

Here, we report the total syntheses of two A3 epimers, SR-A3 and SS-A3 (Fig. 1), along with cellular and single-molecule biophysical studies focused on quantifying drug-target residence times. Synthetic SR-A3 potently inhibited cell proliferation and protein synthesis by targeting eEF1A and was spectroscopically and biologically indistinguishable from the natural product A3. Transient exposure of cells to SR-A3 resulted in long-lasting inhibitory effects, whereas similarly prolonged effects were not observed with SS-A3 or ternatin-4. To gain mechanistic insight into these differences, we assessed eEF1A-catalysed translation via single-molecule fluorescence resonance energy transfer (smFRET) imaging. These experiments directly revealed SR-A3’s comparatively long duration of eEF1A blockade on the ribosome, enabled in part by its enhanced capacity to rebind following dissociation. Preclinical studies in a mouse model of human Burkitt lymphoma revealed that SR-A3, but not ternatin-4, exhibits potent antitumour activity. Our data thus reveal a striking and stereospecific enhancement in eEF1A binding kinetics conferred by a single oxygen atom appended to a cyclic peptide.

Results

Synthesis of SR-A3 and SS-A3 via an expeditious route to dhML

We speculated that dhML in the natural product A3 has the same stereochemistry as in the synthetic compound, ternatin-4 (Fig. 1). Because our original six-step synthesis of dhML methyl ester was low yielding and required a costly chiral auxiliary, we developed a more efficient, second-generation synthesis suitable for preparing gram quantities of Fmoc-dhML (Fmoc, the fluorenylmethoxycarbonyl protecting group).

Copper(I)-promoted S_N2′ reaction of a serine-derived organozinc reagent with allylic electrophiles has been previously used to synthesize amino acids that contain a γ-stereogenic centre^17,18. This method was appealing because it would provide dhML (as the tert-butoxycarbony (Boc) methyl ester) in only two steps from the inexpensive chiral building block, Boc-(S)-serine-OMe¹⁷. After extensive optimization aimed at improving S_N2′ versus S_N2 selectivity and conversion (Extended Data Fig. 1), we obtained Boc-dhML-OMe 3 in 43% isolated yield (1.6 g) through the use of 50 mol% CuBr·DMS (DMS, dimethyl sulfide) and two equivalents of crotyl chloride (Fig. 2a). Boc to Fmoc exchange, followed by ester hydrolysis, provided Fmoc-dhML 5, which was incorporated into the linear heptapeptide as described below.

Fig. 2: Expeditious synthesis of dhML, ternatin-4 and A3 epimers.

a, Scheme for synthesis of Fmoc-dhML 5. Su, succinimide; THF, tetrahydrofuran; r.t., room temperature; TMSCl, trimethylsilyl chloride; DMF, N, N-Dimethylformamide; DCE, 1,2-Dichloroethane. b, Identification of alternative macrocyclization site B. c, Scheme for solid-phase synthesis of linear heptapeptide precursors, followed by solution-phase cyclization to provide ternatin-4, SR-A3 and SS-A3 (Supplementary Information for details). AA, amino acid; HATU, 1-[Bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxid hexafluorophosphate; DIPEA, N,N-Diisopropylethylamine; HFIP, 1,1,1,3,3,3-Hexafluoro-2-propanol; DCM, dichloromethane.

A solid-phase route was previously employed to synthesize a linear heptapeptide precursor of ternatin, followed by solution-phase cyclization¹⁴. However, this strategy involved macrocyclization between the secondary amine of N-Me-Ala7 and the carboxylic acid of Leu1 (Fig. 2b, site A), which we found to be low yielding in the context of peptides containing dhML¹⁵. Thus, we sought to identify an alternative cyclization site using the ternatin-related cyclic peptide 6 as a model system (Fig. 2c). Linear heptapeptide precursors were synthesized on the solid phase, deprotected and cleaved from the resin, and cyclized in solution (Supplementary Information for details). Gratifyingly, cyclization at site B provided 6 in 63% overall yield (including the solid-phase linear heptapeptide synthesis), whereas cyclization at site A was less efficient (46% overall yield). By synthesizing the linear heptapeptide precursor on the solid phase and cyclizing in solution at site B, we were able to prepare ternatin-4 in three days and 70% overall yield (27 mg), a substantial improvement over our previous route (Fig. 2c). Most importantly, by incorporating Fmoc-protected (S,R)- and (S,S)-N-Me-β-OH-Leu, we completed a total syntheses of SR-A3 (21 mg, 35% overall yield) and SS-A3 (5 mg, 21% overall yield).

Synthetic SR-A3 is indistinguishable from naturally derived A3

With synthetic SR-A3 and SS-A3 in hand (Fig. 3a), we first compared their HPLC elution profiles with an authentic sample of the Aspergillus-derived natural product A3. SR-A3 and naturally derived A3 had identical retention times, whereas SS-A3 eluted later in the gradient (Fig. 3b). Furthermore, the ¹H and ¹³C NMR spectra of SR-A3 were identical to the corresponding spectra of natural A3 (Fig. 3c). Finally, SR-A3 and naturally derived A3 blocked proliferation of HCT116 cancer cells with superimposable dose–response curves (Fig. 3d; IC₅₀ ≈ 0.9 nM (IC₅₀, half maximal inhibitory concentration)), whereas SS-A3 was about threefold less potent (IC₅₀ ≈ 2.7 nM). Similar trends were observed in four additional cancer cell lines (Extended Data Fig. 2). Together, these data are consistent with our stereochemical hypothesis and strongly suggest that synthetic SR-A3 and natural A3 have the same structure.

Fig. 3: SR-A3 is indistinguishable from naturally derived A3.

a, Chemical structures of SS-A3 and SR-A3. b, HPLC elution profiles. c, Overlay of ¹H (top) and ¹³C (bottom) NMR spectra in acetone-d₆. f1, the resolution of the indirect dimension. d, Concentration-dependent anti-proliferative effects in HCT116 cells after 72 h. Cell proliferation (percent DMSO control) was quantified using alamarBlue. Data points (percent DMSO control) are mean values ± s.d. (n = 3).

Source data

N-Me-β-OH-Leu stereospecifically confers increased cellular residence time

We previously demonstrated that ternatin-4 is inactive towards a human cancer cell line that is homozygous for an Ala399Val mutation in eEF1A (encoded by the EEF1A1 gene)¹⁵. These cells were also resistant to SR-A3 (IC₅₀ ≫ 1 μM), providing strong genetic evidence that eEF1A is the relevant target (Fig. 4a). Consistent with this interpretation, treatment of cells with SR-A3 for 24 h inhibited global protein synthesis with an IC₅₀ of ~20 nM (Fig. 4b), as measured by a clickable puromycin (O-propargyl puromycin, OPP) incorporation assay (Extended Data Fig. 3)¹⁹. Under these conditions—24 h of continuous treatment prior to a 1 h pulse with OPP—SR-A3 behaved identically to ternatin-4, whereas SS-A3 was slightly less potent. Based on these cellular data, it remained unclear as to whether N-Me-β-OH-Leu in SR-A3 confers any advantage over the biosynthetically less ornate N-Me-Leu found in ternatin and ternatin-4.

Fig. 4: N-Me-β-OH-Leu stereospecifically endows SR-A3 with increased cellular residence time.

a, Wild-type (WT) and eEF1A-mutant (A399V) HCT116 cells were treated with SR-A3 for 72 h. Cell proliferation (percent DMSO control) was quantified using alamarBlue. Data points are mean values ± s.d. (n = 3). b, HCT116 cells were treated with the indicated compounds for 24 h and protein synthesis was quantified after pulse labelling with OPP for 1 h (Methods). Data points (percent DMSO control) are mean values ± s.d. (n = 3). c, HCT116 cells were treated with the indicated compounds (100 nM) or DMSO for 4 h, followed by wash-out into compound-free media. At the indicated time points post-wash-out, cells were pulse-labelled with OPP (1 h), and OPP incorporation was quantified. Normalized data (percent DMSO control) are mean values ± s.d. (n = 3). Statistical significance was determined by one-way analysis of variance (ANOVA) followed by Sidak’s multiple comparisons test. ***P < 0.001; ****P < 0.0001. The P value for SR-A3 versus ternatin-4 is 0.0001. The P value for SR-A3 versus SS-A3 is <0.0001. d, HCT116 cells were treated with the indicated compounds (100 nM) or DMSO for 4 h, followed by wash-out into compound-free media. At the indicated time points post-wash-out, cell proliferation was quantified using the CellTiter-Glo assay. Normalized data (log₂ fold change versus DMSO control at time t = 0 h post-wash-out) are mean values ± s.d. (n = 3). Statistical significance was determined by one-way ANOVA followed by Sidak’s multiple comparisons test. ****P < 0.0001. The P value for SR-A3 versus ternatin-4 is <0.0001. The P value for SR-A3 versus SS-A3 is <0.0001.

Source data

Drug-target residence time, which reflects not only the biochemical off-rate, but also the rebinding rate and local target density in vivo, has emerged as a critical kinetic parameter in drug discovery^20,21. To test for potential differences in cellular residence time, we treated HCT116 cells with 100 nM SR-A3, SS-A3 or ternatin-4 for 4 h, followed by wash-out into drug-free media. At various times post-wash-out, cells were pulse-labelled with OPP for 1 h. Whereas protein synthesis rates partially recovered in cells treated with ternatin-4 or SS-A3 (~30% of dimethyl sulfoxide (DMSO) control levels, 24 h post-wash-out), transient exposure of cells to SR-A3 resulted in more prolonged inhibition (Fig. 4c). To confirm the extended duration of action observed with SR-A3, we assessed cell proliferation during a 72 h wash-out period. Strikingly, cell proliferation over 72 h was sharply reduced after 4 h treatment with 100 nM SR-A3, followed by rigorous wash-out. By contrast, cell proliferation rates recovered nearly to DMSO control levels after transient exposure to 100 nM ternatin-4 or SS-A3 (Fig. 4d). These results demonstrate that the (R)-β-hydroxy group attached to N-Me-Leu endows SR-A3 with a substantial kinetic advantage over SS-A3 and ternatin-4, as reflected by the wash-out resistance and increased cellular residence time associated with inhibition of protein synthesis and cell proliferation.

Single-molecule FRET imaging reveals enhanced residence time and rebinding kinetics of SR-A3

To gain mechanistic insight into SR-A3’s kinetic advantage in cells relative to ternatin-4 and SS-A3, we employed smFRET imaging of biochemically reconstituted, eEF1A-catalysed translation reactions²². In this assay, tRNA in the P site of surface-immobilized human ribosomes is labelled with a donor fluorophore (Cy3) (Fig. 5a). Aminoacylated tRNA labelled with an acceptor fluorophore (LD655) is then stopped-flow delivered as a ternary complex (TC) with eEF1A and GTP. The resulting reaction is monitored in real time using a home-built total internal reflection microscope²³. The FRET efficiency between the two fluorophores—inversely related to the distance between the two tRNAs—increases through a series of well-characterized states (Fig. 5a): initiation complex (IC), GTPase-activated (GA) and fully accommodated (AC)^22,24.

Fig. 5: Single-molecule FRET imaging reveals increased residence time and rebinding kinetics of SR-A3.

a, Schematic showing predominant long-lived reaction species (IC, GA and AC) detected by the experimental set-up. E, exit; P, peptidyl; A, aminoacyl. b, Population FRET histograms of reactions initiated from IC by delivering the TC and either DMSO or the indicated drug (10 µM) at the start of data acquisition. N, number of observed molecules. c, Representative smFRET trace from a chase experiment initiated from drug-stalled elongation complexes (GA) by pre-incubation with SR-A3, followed by wash-out into drug-free buffer at the start of data acquisition. Blue circles represent the measured FRET efficiency for each video frame; the solid red line represents a hidden Markov model idealization of the data. d, Cumulative drug dissociation time distributions constructed from several thousand FRET traces as in c after wash-out into drug-free buffer. Data points represent bootstrap means (±s.e.m.) from all FRET traces acquired in a single video. The solid lines represent fits of single-exponential functions to the data (n = 3). e, Plots of inhibition time (prior to accommodation) as a function of drug concentration in the wash-out buffer were used to determine drug residence times and rebinding constants. Inhibition times were derived from curves shown in d, after wash-out into buffer containing the indicated drug concentrations. Each data point represents the mean of three independent experiments (±s.e.m., n = 3). Solid lines are linear fits to the data, with adjusted R² = 0.90 (ternatin-4), 0.98 (SS-A3) and 0.99 (SR-A3). R², coefficient of determination.

Before TC addition, no FRET is observed (Fig. 5a, IC; Fig. 5b). When TC is introduced to the microscope flow cell, it binds rapidly to the ribosomal A site, where codon–anticodon recognition occurs (Fig. 5a, GA), resulting in GTP hydrolysis and subsequent dissociation of eEF1A from aa-tRNA and the ribosome. The ribosome complex exhibits similar time-averaged FRET efficiencies of ~0.5 at both GA states (before and immediately after eEF1A dissociation). Upon eEF1A dissociation, aa-tRNA accommodates into the ribosomal peptidyl transferase centre (Fig. 5a, AC), as revealed by the marked increase in FRET efficiency to ~0.7 in DMSO control reactions (Fig. 5b, left). By contrast, when TC was delivered in the presence of 10 µM ternatin-4, SS-A3 or SR-A3, aa-tRNA accommodation was strongly inhibited, whereas formation of the initial TC/ribosome intermediate was unaffected (Fig. 5b). These results suggest that all three compounds similarly stall elongating ribosomes bound to TC (Fig. 5a, GA), presumably by preventing conformational changes in eEF1A (either before or after GTP hydrolysis), which are required for its dissociation from aa-tRNA and the ribosome.

Since SR-A3 exhibited a greater cellular residence time than ternatin-4 or SS-A3 (Fig. 4e), we designed a series of in vitro chase experiments to quantify differences in dissociation rates and rebinding constants between these closely related analogues. We first generated a population of drug-stalled elongation complexes by incubating immobilized ribosomes with TC and 10 µM of each drug for 30 seconds. Excess TC and drugs were then washed out of the microscope flow cell with buffer containing 0, 2.5, 5, 7.5 or 10 µM drug at the start of data acquisition. Under these wash-out conditions, drug-stalled elongation complexes can gradually transition into the fully accommodated high-FRET state (Fig. 5c, example trace showing wash-out into drug-free buffer). We estimated the average time required for aa-tRNA accommodation to occur in each wash-out condition (t_acc) from cumulative dwell-time distributions by calculating the fraction of ribosomes that reached the high-FRET state at each time point and fitting exponential functions to the resulting data (Fig. 5d and Extended Data Fig. 4a; Methods for details).

Given the assumption that drug dissociation must occur prior to aa-tRNA accommodation, the drug residence times and rebinding constants can then be determined from the chase series (Fig. 5e; Methods for details)^25,26. Based on the measured inhibition time after wash-out into drug-free buffer, the residence time of SR-A3 (82 s) is about 60% longer than that of ternatin-4 (56 s) or SS-A3 (51 s). In addition, the rebinding constant, which corresponds to the drug concentration in the wash-out buffer required to double the inhibition time (relative to drug-free buffer) through drug rebinding events, strongly favours SR-A3 (Fig. 5e). This implies that SR-A3 rebinds to the stalled eEF1A–ribosome complex (GA or related states; Fig. 5a) twice as fast as SS-A3 and four times as fast as ternatin-4 (Fig. 5e and Extended Data Fig. 4b). Taken together, and consistent with our cell-based findings, our smFRET data show that (S,R)-N-Me-β-OH-Leu stereospecifically endows SR-A3 with a longer residence time and faster target rebinding kinetics upon dissociation.

SR-A3 treatment significantly extends survival of Eµ-Myc tumour-bearing mice

The oncogenic transcription factor Myc is dysregulated in >50% of human cancers²⁷. Structural alterations of the MYC gene cause B-cell lymphoma in humans and mice^28,29,30,31, and a Myc-dependent increase in protein synthesis is a key oncogenic determinant³². The Eμ-Myc transgenic mouse²⁸, in which Myc is specifically overexpressed in B lymphocytes, has been employed as a preclinical model of human Burkitt lymphoma and other Myc-driven B-cell malignancies. Hence, the Eμ-Myc model is a paradigm for testing whether inhibition of protein synthesis downstream of Myc oncogenic activity confers therapeutic benefit. Given its enhanced residence time and rebinding kinetics, as well as improved metabolic stability (Extended Data Fig. 5a), we selected SR-A3 for a preclinical trial using the Eμ-Myc lymphoma allograft model. After intravenous injection of wild-type immunocompetent mice with mouse Eμ-Myc/+ lymphoma cells and waiting until tumours were palpable (approximately two weeks after injection), we began treatment with either vehicle or SR-A3 (dosed three times per week, 1.5 or 2.0 mg kg^–1, by intraperitoneal injection). Treatment of tumour-bearing mice with single-agent SR-A3 dramatically prolonged survival in a dose-dependent manner (Fig. 6a). Moreover, SR-A3 was well tolerated in both dose groups, and no notable body weight loss was observed (Extended Data Fig. 5b).

Fig. 6: SR-A3 extends overall survival and reduces tumour burden in the Eµ-Myc mouse lymphoma model.

a, Kaplan–Meier survival curves for mice dosed with vehicle or the indicated doses of SR-A3 (intraperitoneal injection, three times per week, n = 5 per dose group). Day 0 indicates the beginning of treatment. Statistical significance was determined by logrank test. ***P < 0.001; ****P < 0.0001. The P value for 2 mg per kg (mpk) SR-A3 versus vehicle is <0.0001. The P value for 2 mpk SR-A3 versus 1.5 mpk SR-A3 is 0.0001. The P value for 1.5 mpk SR-A3 versus vehicle is 0.0002. b, Mouse plasma concentrations of SR-A3 or ternatin-4 after intraperitoneal injection at 2 mg kg^–1 (mean ± s.d., n = 3 mice per time point). c, Tumour weights from Eµ-Myc mice after two weeks of treatment with vehicle, ternatin-4 (4.2 mg kg^–1) or SR-A3 (2 mg kg^–1; n = 5 per group). Graph represents mean value ± s.d. Statistical significance was determined by one-way ANOVA followed by Sidak’s multiple comparisons test. NS, not significant; ****P < 0.0001. The P value for SR-A3 versus vehicle is <0.0001. The P value for SR-A3 versus ternatin-4 is <0.0001. The P value for ternatin-4 versus vehicle is 0.0708.

Source data

Having established an efficacious and well-tolerated dosing regimen for SR-A3, we sought to test whether its enhanced residence time relative to ternatin-4 translates to improved antitumour activity in vivo. To ensure a fair comparison in the head-to-head antitumour study, we first assessed the pharmacokinetics of each compound in mice (2 mg kg^–1, single intraperitoneal injection). The area under the plasma concentration-time curve (AUC_inf) for SR-A3 was ~2.1-fold higher than for ternatin-4 (Fig. 6b), and the maximum plasma concentration (C_max) was ~1.7-fold higher (Extended Data Fig. 6a). We therefore treated Eμ-Myc tumour-bearing mice with either vehicle, 4.2 mg kg^–1 ternatin-4 or 2 mg kg^–1 SR-A3 (three times per week, n = 5 mice per treatment arm). After two weeks of treatment, tumours from each mouse were collected and weighed. Strikingly, SR-A3 treatment significantly reduced tumour burden, whereas ternatin-4 had no significant effect (Fig. 6c and Extended Data Fig. 6b,c). These results demonstrate the clear improvement in antitumour efficacy conferred by (R)-β-OH-N-Me-Leu and suggest that SR-A3 may be a viable preclinical candidate for the treatment of Myc-driven B lymphoid tumours.

Discussion

In this study, we first developed an improved synthetic route to dhML-containing ternatin variants, culminating in the total syntheses of SR-A3 and SS-A3 (Fig. 2). Our work provides spectroscopic, chromatographic and pharmacological evidence that synthetic SR-A3 (and not SS-A3) is identical to the fungal natural product ‘A3’ (Fig. 3), confirming the previous partial structure elucidation and providing a complete stereochemical assignment of this potent eEF1A antagonist.

SR-A3 differs structurally from the previously reported eEF1A inhibitor, ternatin-4, by the addition of a single oxygen atom into the side chain of N-Me-Leu (Fig. 1). We speculate that A3 is evolutionarily related to ternatin via acquisition of biosynthetic modules for (R)-pipecolic acid and (S,R)-dhML, as well as stereospecific hydroxylation of N-Me-Leu by the A3-producing fungus. Although amino acid β-hydroxylation is a common biosynthetic modification in cyclic peptide natural products, its stereospecific functions are mostly unknown. An unexpected finding from our work is that the N-Me-Leu β-hydroxyl in SR-A3 has little effect on cellular potency under continuous treatment conditions, as compared with ternatin-4. Rather, the β-hydroxyl in SR-A3, but not SS-A3, confers a dramatic increase in drug-target residence time and rebinding kinetics, as revealed by wash-out experiments in cells and reconstituted eEF1A-catalysed elongation reactions monitored by smFRET (Fig. 4c,d and Fig. 5). We note that ‘drug-target residence time’ measured in cells and tissues is determined by both drug dissociation and rebinding kinetics. Moreover, the intracellular concentration (or local density) of the target can play a major role in drug rebinding kinetics²⁰. This can potentially explain the much longer residence time in cells, where [eEF1A] is ~35 µM (ref. ³³), as compared to the smFRET flow cell, where [eEF1A] is ≪10 nM post-wash-out. The structural basis of SR-A3’s enhanced binding kinetics will likely require cryo-electron microscopy analysis of stalled SR-A3/eEF1A/ribosome complexes at atomic resolution. Although the precise molecular mechanism by which the β-hydroxyl confers this kinetic advantage awaits further investigation, our study nevertheless reveals the power of smFRET imaging to illuminate differences in drug dissociation and rebinding kinetics, both of which can contribute to drug-target residence time and therapeutic efficacy²⁰.

Dysregulation of the transcription factor Myc underlies multiple human cancers²⁷. Nevertheless, Myc is still considered ‘undruggable’ and no direct Myc inhibitors have advanced into clinical trials. Because the oncogenic activity of Myc relies on its ability to promote ribosome biogenesis and protein synthesis, an alternative approach is to attenuate protein synthesis rates. The Eμ-Myc lymphoma model has been employed to test various protein synthesis inhibition strategies, often in combination with other cytotoxic drugs. Targeting the protein synthesis machinery in Eμ-Myc mice either genetically³² or pharmacologically^34,35,36 has been shown to suppress tumour growth and prolong overall survival. However, to the best of our knowledge, no translation elongation inhibitors have shown single-agent efficacy in this aggressive B-cell lymphoma model.

Our preclinical trial revealed that intermittent, low doses of SR-A3 (1.5–2.0 mg kg^–1, three times per week), but not the des-OH (no hydroxyl) variant ternatin-4, profoundly reduced tumour burden and extended the survival of Eµ-Myc mice without obvious toxicity (Fig. 6). SR-A3 thus shows therapeutic potential for the treatment of Myc-driven B-cell lymphoma. Our study of SR-A3 chemistry and biology provides a compelling illustration of how a ‘ligand efficient’ side-chain modification (one oxygen atom) can be exploited to alter the pharmacological properties and residence time of a cyclic peptide natural product.

Methods

Cell culture

HCT116 cells (ATCC) were maintained in McCoy’s 5A media (Gibco) supplemented with 10% foetal bovine serum (FBS; Axenia Biologix), 100 units ml^–1 penicillin and 100 µg ml^–1 streptomycin (Gibco). H929 cells (ATCC) were maintained in advanced RPMI 1640 media (Gibco) supplemented with 6% FBS, 2 mM glutamine, 100 units ml^–1 penicillin and 100 mg ml^–1 streptomycin. MM1S, Jurkat and Ramos cells (ATCC) were maintained in RPMI 1640 media (Gibco) supplemented with 10% FBS, 100 units ml^–1 penicillin and 100 mg ml^–1 streptomycin. All cells were cultured at 37 °C in a 5% CO₂ atmosphere.

The natural product A3 was purified as described previously¹³.

Proliferation assay

Adherent cells were briefly trypsinized and repeatedly pipetted to produce a homogeneous cell suspension. 2,500 cells were seeded in 100 µl complete growth media per well in 96-well clear-bottom plates. Suspension cells were repeatedly pipetted to produce a homogeneous cell suspension. 10,000 cells were seeded in 100 µl complete growth media per well in 96-well clear-bottom plates. After allowing cells to grow/adhere overnight, cells were treated with 25 µl per well of ×5 drug stocks (0.1% DMSO final) and incubated for 72 hours. AlamarBlue (Life Technologies) was used to assess cell viability per the manufacturer’s instructions. Briefly, 12.5 µl alamarBlue reagent was added to each well, and plates were incubated at 37 °C. Fluorescence intensity was measured every 30 min to determine the linear range for each assay (excitation (Ex), 545 nm; emission (Em), 590 nm; SPARK, Tecan Austria). Proliferation curves were generated by first normalizing fluorescence intensity in each well to the DMSO-treated plate average. Normalized fluorescence intensity was plotted in GraphPad Prism (GraphPad), and IC₅₀ values were calculated from nonlinear regression curves. The reported IC₅₀ values represent the average of at least three independent determinations (±s.d.).

Wash-out proliferation assay

Adherent cells were briefly trypsinized and repeatedly pipetted to produce a homogeneous cell suspension. 2,500 cells were seeded in 100 µl complete growth media per well in 96-well clear-bottom plates. After allowing cells to grow/adhere overnight, cells were treated with ternatin analogues (100 nM, 0.1% DMSO final) and incubated for the indicated times. The growth medium was carefully removed, cells were washed with warm phosphate buffered saline (PBS) twice (×2 short wash), followed by 5 min incubation in warm media at 37 °C (long wash). This ‘short–long’ washing cycle was repeated three times. After indicated times post-wash-out, CellTiter-Glo (Promega) was used to assess cell viability per the manufacturer’s instructions. Briefly, after adding 100 µl CellTiter-Glo reagent to each well, the plate was rocked at room temperature for 5–10 min, and the luminescence intensity was measured. Proliferation curves were generated by first normalizing luminescence intensity in each well to the average values from the t = 0 time point. Normalized luminescence intensity was plotted in GraphPad Prism (GraphPad). The reported values represent the average of at least three independent determinations (±s.d.). Statistical significance was determined by one-way ANOVA followed by Sidak’s multiple comparisons test.

OPP incorporation assay

HCT116 cells at 60% confluency in 12-well plates were incubated with the indicated concentrations of ternatin analogues for 10 min or 24 h at 37 °C. After the indicated times, OPP (30 µM final concentration) was added, and the cells were incubated for 1 hour at 37 °C. Subsequently, the medium was removed, and the cells were trypsinized, collected and washed twice with ice-cold PBS before transferring to a 96-well V-bottom plate. 100 µl Zombie Green (BioLegend) solution was added to each well and incubated for 30 min at room temperature in the dark. Cells were then washed with 2% FBS in PBS before fixation with 200 µl of 4% paraformaldehyde (PFA) in PBS for 15 min on ice in the dark. After washing the cells with 2% FBS in PBS, 200 µl permeabilization buffer (3% FBS, 0.1% saponin in PBS) was added to each well, and the cells were incubated for 5 min at room temperature in the dark. Cells were then washed and resuspended in 25 µl permeabilization buffer. 100 µl click chemistry mix (50 mM HEPES buffer (pH 7.5), 150 mM NaCl, 400 µM tris(2-carboxyethyl)phosphine (TCEP), 250 µM tris((1-benzyl-4-triazolyl)methyl)amine (TBTA), 5 µM CF647-Azide red dye (Biotium) and 200 µM CuSO₄) was added to each well, and cells were incubated at room temperature in the dark. After overnight incubation, the cells were washed with permeabilization buffer followed by Flow Cytometry Staining Buffer (FACS buffer) (2% FBS, 1% penicillin/streptomycin (P/S) and 2 mM ethylenediaminetetraacetic acid (EDTA), in PBS without Ca/Mg). Cells were then resuspended in 200 µl FACS buffer and filtered before FACS analysis (CytoFLEX, Beckman-Coulter; FlowJo v.10.7.1, BD). Supplementary Fig. 1 shows the gating strategy. Protein synthesis inhibition curves were generated by gating for single live cells and plotting mean fluorescence intensity relative to the DMSO control values using GraphPad Prism (GraphPad). IC₅₀ values were calculated from nonlinear regression curves. The reported values represent the average of at least three independent determinations (±s.d.).

Wash-out OPP assay

HCT116 cells at 60% confluency in 12-well plates were incubated with compounds at 100 nM for 4 h at 37 °C. The medium was carefully removed, and cells were washed with warm PBS twice (×2 short wash), followed by 5 min incubation in warm media at 37 °C (long wash). After repeating the short–long wash-out cycle three times, cells were resuspended in warm media and incubated at 37 °C. After the indicated times post-wash-out, OPP (30 µM final concentration) was added, and the cells were incubated for an additional 1 h at 37 °C. The medium was removed, and the cells were trypsinized, collected, and washed twice with ice-cold PBS before transferring to a 96-well V-bottom plate. 100 µl Zombie Green (BioLegend) solution was added to each well and incubated for 30 min at room temperature in the dark. Cells were then washed with 2% FBS in PBS before fixation with 200 µl of 4% PFA in PBS for 15 min on ice in the dark. After washing the cells with 2% FBS in PBS, 200 µl permeabilization buffer (3% FBS, 0.1% saponin in PBS) was added to each well, and cells were incubated for 5 min at room temperature in the dark. Cells were then washed and resuspended in 25 µl permeabilization buffer. 100 µl click chemistry mix (50 mM HEPES (pH 7.5), 150 mM NaCl, 400 µM TCEP, 250 µM TBTA, 5 µM CF647-Azide (Biotium) and 200 µM CuSO₄) was added to each well, and cells were incubated at room temperature in the dark. After the overnight incubation, cells were washed with permeabilization buffer followed by FACS buffer (2% FBS, 1% P/S and 2 mM EDTA, in PBS without Ca/Mg). Cells were then resuspended in 200 µl FACS buffer and filtered before FACS analysis as described above. Statistical significance was determined by one-way ANOVA followed by Sidak’s multiple comparisons test.

The smFRET data collection

Ribosomes from HEK293T cells, elongation factor eEF1A and fluorescence-labelled tRNAs were prepared using the protocol described previously³⁷. All smFRET experiments were carried out at 25 °C in human polymix buffer (20 mM HEPES (pH 7.5), 5 mM MgCl₂, 140 mM KCl, 10 mM NH₄Cl, 2 mM spermidine, 5 mM putrescine and 1.5 mM 2-mercaptoethanol) containing 500 µM cycloheximide and a mixture of triplet-state quenchers (1 mM trolox, 1 mM 4-nitrobenzyl alcohol, 1 mM cyclooctatetraene) and an enzymatic oxygen scavenging system (2 µM 3,4-dihydroxybenzoic acid, 0.02 units ml^–1 protocatechuate 3,4-dioxygenase). The time evolution of the FRET signal was recorded using a home-built total-internal-reflection-based fluorescence microscope at ~0.1 kW cm^–2 laser illumination (532 nm). Videos were recorded either in time-lapse mode with one 500 ms frame acquired every 10 seconds or continuously at a time resolution of 500 ms. Donor and acceptor fluorescence intensities were extracted from the recorded videos, and FRET efficiency traces were calculated using the SPARTAN software package²³. FRET traces were selected for further analysis according to the following criteria: a single catastrophic photobleaching event, at least 8:1 signal/background-noise ratio and 6:1 signal/signal-noise ratio, less than four donor-fluorophore blinking events and a correlation coefficient between donor and acceptor of <0.5. The resulting smFRET traces were analysed using hidden Markov model idealization methods as implemented in the SPARTAN software package²³.

Ternary complex real-time delivery experiments

Some 80S initiation complexes containing Met-tRNA^fMet-Cy3 and displaying the codon UUC in the A site were immobilized on passivated quartz slides as described previously^37,38. This was followed by delivery of 10 nM eEF1A ternary complex containing Phe-tRNA^Phe-LD655 together with 1 mM GTP and either DMSO or 10 µM drug at the start of data acquisition.

Drug chase experiments

Stalled pre-accommodation complexes were formed by immobilization of 80S initiation complexes containing Met-tRNA^fMet-Cy3 and displaying the codon UUC in the A site to passivated quartz slides as described previously^37,38, followed by delivery of 10 nM eEF1A ternary complex containing Phe-tRNA^Phe-LD655 together with 1 mM GTP and 10 µM drug. After 30 s of incubation, the chase was started by delivery of buffer containing either 0, 2.5, 5, 7.5 or 10 µM drug concurrent with the start of data acquisition.

The smFRET data kinetic analysis

To construct cumulative distribution plots suitable for estimation of kinetic parameters, FRET traces were first idealized using hidden Markov model analysis to a model with three FRET states (FRET efficiencies, −0.0043 ± 0.06, 0.4485 ± 0.06 and 0.7070 ± 0.06) and then the cumulative sum of molecules that had arrived at the highest (0.7070) FRET state in each video frame was calculated. To estimate reaction mean times and their associated uncertainties, 1,000 bootstrap samples were generated from each experimental replicate and mean times were estimated by fitting of single-exponential functions to these data (equation (1)), taking into account unobserved events due to photobleaching of the fluorophores or dissociation of the intact ternary complex from the ribosomal A site by multiplying the estimated mean time with the inverse of the fraction of traces where accommodation was observed at the end of the process²⁵:

$$f_{{\mathrm{acc}}}\left( t \right) = \left( {1 - {\mathrm{e}}^{\frac{{ - 1}}{{\frac{1}{{f_{{\mathrm{acc}}}\left( \infty \right)}}\tau _{{\mathrm{acc}}}}}t}} \right)$$

(1)

Mean rates and standard errors were calculated as the weighted averages of two to three experimental replicates for each drug concentration (f_acc, fraction of reactions reached the accommodation state; τ_acc, time required to reach the accommodation state). For estimation of drug residence times and rebinding constants, these estimated mean times were plotted against drug concentration ([Drug]) and equation (2) was fitted to the data^25,26.

$$\tau _{\mathrm{I}}\left( {\left[ {{\mathrm{Drug}}} \right]} \right) = \tau _0\left( {1 + \frac{{\left[ {{\mathrm{Drug}}} \right]}}{{K_{\mathrm{I}}}}} \right)$$

(2)

In equation (2), τ_I is the observed inhibition mean time as a function of drug concentration, τ₀ is the drug residence time each time it binds and K_I is the rebinding constant corresponding to the drug concentration required to double the inhibition time through drug rebinding events; it can be interpreted as the ratio between the drug association rate constant and the rate of the process that renders the ribosome immune to drug inhibition, in this case conformational changes in eEF1A.

Animal experiments

All animal experiments were approved by the University of California San Francisco Institutional Animal Care and Use Committee. Eμ-Myc/+ transgenic mice were purchased from the Jackson Laboratory (stock no. 002728). Primers used for genotyping were as follows: 5′-CCG AGG TGA GTG TGA GAG G-3′; 5′-AAA CAG TAA TAG CGC AGC A-3′. For Eμ-Myc clonal B-cell line collection, Eμ-Myc/+ mice harbouring lymphoma were euthanized according to Institutional Animal Care and Use Committee guidelines. Lymph nodes were collected immediately on ice, minced and passed through a 40 μm cell strainer in cold PBS with 2% FBS. Cells were centrifuged at 300g for 5 min. Cells were then resuspended in cold erythrocyte lysis solution ACK (Thermo Fisher A1049201) for 1 min. Isolated lymphoma cells were centrifuged at 300g for 5 min and washed in PBS before freezing in cell cryopreservation medium and storing in liquid nitrogen. For the lymphoma preclinical trial, Eμ-Myc/+ lymphoma cells were thawed and washed once in PBS. One million cells were injected intravenously into eight-week-old male C57BL/6 mice. Mice were monitored for lymphoma development by palpation every other day. Once the lymphoma tumours became palpable (approximately two weeks after tumour cell injection), mice (five per group) were dosed by intraperitoneal injection with either SR-A3 or vehicle (10% EtOH/Kolliphor EL in water) three times per week (every other day) until the survival end point (moribund mice were sacrificed; statistical significance of Kaplan–Meier survival curves was determined by logrank test) or for two weeks, at which time the tumours were dissected and weighed (statistical significance was determined by one-way ANOVA). The maximum allowable tumour size (2 cm in diameter) was not exceeded in any study. No animals or data points were excluded from the analyses.

Chemical synthesis (general)

All reactions in non-aqueous media were conducted under a positive pressure of dry argon in glassware that had been dried in an oven prior to use, unless noted otherwise. Anhydrous solutions of reaction mixtures were transferred via an oven-dried syringe or cannula. All solvents were dried prior to use unless noted otherwise. Thin layer chromatography was performed using precoated silica gel plates (EMD Chemical; 60 g F254 plates). Flash column chromatography was performed on a CombiFlash Rf 200i system (Teledyne Isco). The ¹H and ¹³C NMR spectra were obtained on a Varian Inova 400 MHz spectrometer recorded in parts per million (ppm; chemical shift, δ) downfield of TMS (δ = 0) in CDCl₃ unless noted otherwise. NMR spectra were analysed using MestReNova v.14.1.1 (Mestrelab Research). Signal splitting patterns were described as singlet (s), doublet (d), triplet (t), quartet (q), quintet (quint) or multiplet (m), with coupling constants (J) in hertz. High-resolution mass spectra were obtained on a Waters Xevo G2-XS quadrupole time-of-flight liquid-chromatography/mass-spectrometry system, eluting with a water/MeCN (+0.1% formic acid) gradient at 0.6 ml min^–1. The Supplementary Information contains more details.

Statistics and reproducibility

The statistical significance of the difference between experimental groups was determined by one-way ANOVA, followed by Sidak’s multiple comparisons test. P values indicate statistical significance denoted by *P < 0.05, **P < 0.01, ***P < 0.005 and ****P < 0.0001, and not significant by P > 0.05. Data were organized using Microsoft Excel 2017, and graphing and statistical analyses were performed using Prism 8.4.0. All biological experiments (except Fig. 6d) were repeated at least twice with similar results. The experiment in Fig. 6d was performed once with five mice allocated to each dose group. Descriptions of the error bars and the number of replicates within each experiment are provided in the figure legends.

Reporting summary

Further information on research design is available in the Nature Research Reporting Summary linked to this article.