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-tRNAfMet-Cy3 and displaying the codon UUC in the A site were immobilized on passivated quartz slides as described previously37,38. This was followed by delivery of 10 nM eEF1A ternary complex containing Phe-tRNAPhe-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-tRNAfMet-Cy3 and displaying the codon UUC in the A site to passivated quartz slides as described previously37,38, followed by delivery of 10 nM eEF1A ternary complex containing Phe-tRNAPhe-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:

$$f_}}\left( t \right) = \left( }^}}}\left( \infty \right)}}\tau _}}}}t}} \right)$$

(1)

Mean rates and standard errors were calculated as the weighted averages of two to three experimental replicates for each drug concentration (facc, fraction of reactions reached the accommodation state; τacc, 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 data25,26.

$$\tau _}\left( }} \right]} \right) = \tau _0\left( }} \right]}}}}}} \right)$$

(2)

In equation (2), τI is the observed inhibition mean time as a function of drug concentration, τ0 is the drug residence time each time it binds and KI 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.