Engineered production of cancer targeting peptide (CTP)-containing C-1027 in Streptomyces globisporus and biological evaluation

Abstract

Conjugation of cancer targeting peptides (CTPs) with small molecular therapeutics has emerged as a promising strategy to deliver potent (but typically nonspecific) cytotoxic agents selectively to cancer cells. Here we report the engineered production of a CTP (NGR)-containing C-1027 and evaluation of its activity against selected cancer cell lines. C-1027 is an enediyne chromoprotein produced by Streptomyces globisporus, consisting of an apo-protein (CagA) and an enediyne chromophore (C-1027). NGR is a CTP that targets CD13 in tumor vasculature. S. globisporus SB1026, a recombinant strain engineered to encode CagA with the NGR sequence fused at its C-terminus, directly produces the NGR-containing C-1027 that is equally active as the native C-1027. Our results demonstrate the feasibility to produce CTP-containing enediyne chromoproteins by metabolic pathway engineering and microbial fermentation and will inspire efforts to engineer other CTP-containing drug binding proteins for targeted delivery.

Keywords: C-1027; Cancer; Cancer targeting peptide (CTP); Enediyne; Streptomyces globisporus.

Figure 1

Schematic representation of an NGR-containing C-1027 chromoprotein. (A) The CagA-aromatized C-1027 chromophore complex structure (PDB code 1HZL), with its C-terminus depicted with the engineered NGR motif. (B) The C-1027 chromophore undergoing Bergman cyclization to yield a benzenoid diradical that causes DNA cleavage and cell death and the aromatized C-1027 chromophore.

Figure 2

Engineering the S. globisporus SB1026 strain that produces NGR-containing C-1027 chromoprotein. (A) Fusion of the NGR motif (red with the extra two Gly as linker in blue) at the C-terminus of CagA (only the C-terminus shown). (B) Constructions of the cagA mutant strain SB1024 and the NGR-containing C-1027 producing SB1026 strain. (C) Construction of the cagA mutant variant SB1025. Apr^R, apramycin resistant; Apr^S, apramycin sensitive; Ery^R, erythromycin resistant; Ery^S, erythromycin sensitive. (D) Southern analysis confirming the genotypes of SB1025 (lanes 2, 3) and SB1024 (lanes 4, 5) with the S. globisporus wild-type as control (lane 1). Genomic DNAs were digested with SacI, and the 4.3-kb SacI fragment was used as a probe. M, molecular weight standards.

Figure 3

Production of NGR-containing C-1027 chromoprotein by S. globisporus SB1026. (A) C-1027 chromoprotein production as evaluated by bioassay against M. luteus. (B) CagA production as evaluated by 15% SDS-PAGE analysis. M, molecular weight standards. Crude (lane 5) and purified NGR-containing CagA (lanes 1, 2) from SB1026; crude (lane 6) and purified native CagA (lanes 3, 4) from wild-type. The predicted molecular weights of native CagA and NGR-containing CagA are 10,501.5 Da and 11,149.2 Da, respectively. (C) C-1027 chromophore (◆) production as evaluated by HPLC analysis.