Resistance to Enediyne Antitumor Antibiotics by Sequestration

Abstract

The enediynes, microbial natural products with extraordinary cytotoxicities, have been translated into clinical drugs. Two self-resistance mechanisms are known in the enediyne producers-apoproteins for the nine-membered enediynes and self-sacrifice proteins for the ten-membered enediyne calicheamicin. Here we show that: (1) tnmS1, tnmS2, and tnmS3 encode tiancimycin (TNM) resistance in its producer Streptomyces sp. CB03234, (2) tnmS1, tnmS2, and tnmS3 homologs are found in all anthraquinone-fused enediyne producers, (3) TnmS1, TnmS2, and TnmS3 share a similar β barrel-like structure, bind TNMs with nanomolar K_D values, and confer resistance by sequestration, and (4) TnmS1, TnmS2, and TnmS3 homologs are widespread in nature, including in the human microbiome. These findings unveil an unprecedented resistance mechanism for the enediynes. Mechanisms of self-resistance in producers serve as models to predict and combat future drug resistance in clinical settings. Enediyne-based chemotherapies should now consider the fact that the human microbiome harbors genes encoding enediyne resistance.

Keywords: anthraquinone-fused enediyne; antibody-drug conjugate; anticancer drug; biosynthesis; enediyne; resistance; sequence similarity network; sequestration; the human microbiome; tiancimycin.

Figure 1.. Bioinformatics Analysis Revealing Candidates for Self-Resistance Genes in the Biosynthetic Gene Clusters of Anthraquinone-Fused Enediynes

(A) Structures of representative anthraquinone-fused enediynes DYN, UCM, TNM A and TNM C, and YPM. (B) The biosynthetic gene clusters of DYN, UCM, TNM, and YPM. Genes are color-coded based on GNN annotation (see Figure S1 for details). The 5.5-kb fragment harboring tnmS1 to tnmS3 in the tnm biosynthetic gene cluster is highlighted by a red box. (C) Selected clusters from the enediyne GNN (Figure S1) that highlight apoproteins and self-sacrifice proteins conferring enediyne resistance by the two known mechanisms and reveal new candidates conferring resistance to anthraquinone-fused enediynes by an unprecedented mechanism. The genes in the same color are homologous proteins.

Figure 2.. The tnmS1, tnmS2, tnmS3 Genes, and their Homologs Encoding Resistance to Anthraquinone-Fused Enediynes as Demonstrated by the Disk Diffusion Assays

(A) The ΔtnmS1-tnmS3 mutant strain SB20003 is sensitive to TNM A, in comparison with the wild-type strain CB03234 as a positive control, establishing that TnmS1, TnmS2, and TnmS3 are necessary and sufficient to confer TNM resistance in its producer. (B) E. coli recombinant strains SB20004 to SB20013, expressing tnmS1, tnmS2, tnmS3, and their homologs from the UCM, DYN, and YPM producers, are resistant to TNM A, UCM, DYN, and YPM, respectively, in comparison with E. coli BL21(DE3)/pET28a as a negative control, demonstrating that each of these gene products is sufficient to confer resistance to anthraquinone-fused enediynes in their respective producers. (C) E. coli recombinant strains SB20015 to SB20018, expressing the five homologs from the human microbiome, show resistance to TNM A, in comparison withE. coli BL21(DE3)/pET28a as a negative control, revealing that the human microbiome might already possess resistant elements to anthraquinone-fused enediynes. Depicted next to the disks are the amounts of DYN, UCM, TNM A, and YMP, respectively, loaded to each set of the diffusion assays.

Figure 3.. The Crystal Structures of TnmS1, TnmS2, and TnmS3 Revealing a β Barrel-like Structure that Forms Two Large Concavities to Accommodate TNMs

(A) The overall structures of TnmS1, TnmS2, and TnmS3 shown in ribbon diagram. The two chains of TnmS1, TnmS2, and TnmS3 are shown by green/blue, pale green/orange, and yellow/red, respectively. (B) Solvent accessible surfaces of the binding cavities of TnmS1, TnmS2, and TnmS3, highlighting a common mode for enediyne binding. The binding modes of TNM A in TnmS1 and TnmS2 were modeled based on the structure of TnmS3 in complex with TNM A.

Figure 4.. The Crystal Structure of TnmS3 in Complex with TNMs Revealing the Molecular Details for Resistance to the Anthraquinone-Fused Enediynes by Sequestration

(A) The overall structure of TnmS3 in complex with TNM A shown in ribbon diagram. Two molecules of TNM A are bound by one TnmS3 dimer. (B) Local view showing that TnmS3 undergoes a slight conformational change upon TNM A binding, as depicted by the side chains of Trp100 and Gln103 flipping into new conformations to interact with TNM A, with the apo-TnmS3 structure shown in gray. (C) Local view of the enediyne binding cavity highlighting the residues involved in interactions between TnmS3 and TNM A. Hydrogen bonds are shown in red dashed lines. (D) The local view of the enediyne binding cavity highlighting the residues involved in interactions between TnmS3 and TNM C. While TNM A and TNM C share many of the same interactions with TnmS3, the side chain at C-26 of TNM C reveals no major interaction. The 2F_o–F_c electron density maps are contoured at 1s shown as white mesh. The σ-A-weighted difference (mF_o–DF_c) omit maps of TnmS3 in complex with TNM A and TNM C, respectively, are shown in Figure S5.

Figure 5.. Sequence Similarity Network of Proteins from the InterPro Family IPR029068 Revealing Homologs of TnmS1, TnmS2, and TnmS3 Are Widely Distributed in Nature, Including the Human Microbiome

The SSN of the IPR029068 family was generated using the online Enzyme Function Initiative-Enzyme Similarity Tool (Gerlt et al., 2015) and visualized with Cytoscape with an e value threshold of 10⁻²⁰ (~30% sequence identity over 80 amino acids). Each node represents proteins with above 75% sequence identities. The nodes are colored by the taxonomy of the sources of these proteins. The TnmS1, TnmS2, TnmS3, and their homologs characterized in this study are highlighted in larger circles and labeled with orange dashed lines. Other characterized proteins from the IPR029068 family are also highlighted in large circles and labeled with their respective PDB numbers. PDB: 1NPB, 4JH8, and 4NAZ are thiol transferases; PDB: 1F9Z, 2C21, 2ZA0, and 4MTS are glyoxalases-I; PDB: 1BYL, 4IAG, 1QTO, and 1EWJ are BLM binding proteins; PDB: 1JC5 is epimerase; PDB: 1KLL is mitomycin binding protein; PDB: 1R9C is epoxide hydrolase; and PDB: 3ITW is thiocoraline binding protein.