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.

Extended Data Fig. 1. Screening conditions for Cu(I)-promoted S_N2’ reaction.

Extended Data Fig. 2. Effects on cancer cell proliferation.

The indicated cell lines were treated with DMSO or increasing concentrations of synthetic SR-A3 and SS-A3 and natural A3. After 72 h, cell proliferation was quantified using alamarBlue (% DMSO control, mean ± SD, n = 3). GraphPad Prism was used to calculate IC50 values.

Extended Data Fig. 3. Measuring effects on cellular protein synthesis rates.

(a) General workflow for measuring protein synthesis rates in cells using O-Propargyl-Puromycin (OPP). See “OPP incorporation assay” in the methods section for details. (b) OPP is an aminoacyl-tRNA mimic (clickable puromycin derivative) that, like puromycin itself, is incorporated into ribosome-associated nascent polypeptides by reacting with peptidyl-tRNAs in the ribosomal P site. After fixing and permeabilizing cells, click chemistry is used to conjugate the OPP alkyne with a fluorophore azide, and the intracellular fluorescence intensity (proportional to the amount of actively translating ribosomes) is measured by flow cytometry. (c) General workflow for washout-OPP and washout-proliferation experiments.

Extended Data Fig. 4. Time and concentration-dependent effects on aa-tRNA accommodation revealed by smFRET.

(a) Cumulative dissociation time distributions for ternatin-4, SS-A3, and SR-A3 at the indicated concentrations. Distributions are constructed as described in the main text and the methods. Error bars represent SEM derived from 1000 bootstrap replicates. (b) Tabulated kinetic parameters based on data in Figure 5 and Extended Data Fig. 4a.

Extended Data Fig. 5. Microsome stability and effects on mouse body weight.

(a) Human and mouse liver microsome stability results. Percent remaining of each analog was quantified by LC/MS after incubating at 1 μM in the presence of human or mouse liver microsomes (with NADPH) for 30 min at 37°C. This study was performed by the contract research organization, Bioduro-Sundia (San Diego, CA). (b) Average body weight (± SD, relative to day 0) during the efficacy study in Eμ-Myc mice (n = 5 per arm, Figure 6a). Day 0 indicates the beginning of treatment.

Extended Data Fig. 6. Pharmacokinetics and effects of ternatin-4 and SR-A3 on mouse body weight and tumor size.

(a) Mean pharmacokinetic (PK) parameters of ternatin-4 and SR-A3. Mice (n = 3) were intraperitoneally injected with ternatin-4 or SR-A3 (2 mg/kg), and plasma concentrations were quantified at various time points (Figure 6b). PK data were acquired and analyzed by the contract research organization, Bioduro-Sundia (San Diego, CA). (b) Average mouse body weight (± SD, relative to day 0) during the Eμ-Myc tumor study (Figure 6c). Day 0 indicates the beginning of treatment (n = 5 per arm). (c) Photographs of tumors from each mouse after two weeks of treatment (n =5 per arm).

Figure 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 compound. Pip, pipecolic acid.

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

(a) Scheme for synthesis of Fmoc-dhML 5. (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 (see 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.

Figure 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 and ¹³C NMR spectra in acetone-d₆. (d) Concentration-dependent antiproliferative effects in HCT116 cells after 72 h. Cell proliferation (% DMSO control) was quantified using alamarBlue. Data points (% DMSO control) are mean values ± SD (n = 3).

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

(a) Wild-type and eEF1A-mutant (A399V) HCT116 cells were treated with SR-A3 for 72 h. Cell proliferation (% DMSO control) was quantified using alamarBlue. Data points are mean values ± SD (n = 3). (b) HCT116 cells were treated with the indicated compounds for 24 h and protein synthesis was quantified after pulse labeling with O-propargyl puromycin for 1 h (see Methods). Data points (% DMSO control) are mean values ± SD (n = 3). (c) HCT116 cells were treated with the indicated compounds (100 nM) or DMSO for 4 h, followed by washout into compound-free media. At the indicated time points post-washout, cells were pulse-labeled with OPP (1 h), and OPP incorporation was quantified. Normalized data (% DMSO control) are mean values ± SD (n = 3). Statistical significance was determined by one-way 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 washout into compound-free media. At the indicated time points post-washout, cell proliferation was quantified using the CellTiter-Glo assay. Normalized data (log2 fold change vs. DMSO control at t = 0 h post-washout) are mean values ± SD (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.

Figure 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 setup. (b) Population FRET histograms of reactions initiated from IC by delivering the ternary complex (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 washout into drug-free buffer at the start of data acquisition. Blue circles represent the measured FRET efficiency for each movie 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 washout into drug-free buffer. Data points represent bootstrap means (± SEM) from all FRET traces acquired in a single movie. 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 washout buffer were used to determine drug residence times and rebinding constants. Inhibition times were derived from curves exemplified in (d), after washout into buffer containing the indicated drug concentrations. Each data point represents the mean of three independent experiments (± SEM, n = 3). Solid lines are linear fits to the data, adjusted R² = 0.90 (ternatin-4), 0.98 (SS-A3), and 0.99 (SR-A3). R², coefficient of determination.

Figure 6.. SR-A3 extends overall survival and reduces tumor 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, 3 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 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 (mean ± SD, n = 3 mice per time point). (c) Tumor weights from Eμ-Myc mice after 2 weeks of treatment with vehicle, ternatin-4 (4.2 mg/kg), or SR-A3 (2 mg/kg) (n = 5 per group). Graph represents mean value ± SD. 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.