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Abstract Chemotherapy-induced drug resistance remains a major cause of cancer recurrence and patient mortality. ATP binding cassette subfamily B member 1 (ABCB1) transporter overexpression in tumors contributes to resistance, yet current ABCB1 inhibitors have been unsuccessful in clinical trials. To address this challenge, we propose a new strategy using tryptophan as a lead molecule for developing ABCB1 inhibitors. Our idea stems from our studies on bat cells, as bats have low cancer incidences and high ABCB1 expression. We hypothesized that potential ABCB1 substrates in bats could act as competitive inhibitors in humans. By molecular simulations of ABCB1-substrate interactions, we generated a benzylatedCyclo-tryptophan (C3N-Dbn-Trp2) that inhibits ABCB1 activity with efficacy comparable to or better than the classical inhibitor, verapamil. C3N-Dbn-Trp2 restored chemotherapy sensitivity in drug-resistant human cancer cells with no adverse effect on cell proliferation. Our unique approach presents a promising lead toward developing effective ABCB1 inhibitors to treat drug-resistant cancers. 

Abstract The emergence of drug resistance in cancer cells eventually causing relapse is a serious threat that demands new advances. Upregulation of the ATP‐dependent binding cassette (ABC) transporters, such as ABCB1, significantly contributes to the emergence of drug resistance in cancer. Despite more than 30 years of therapeutic discovery, and several generations of inhibitors against P‐gp, the search for effective agents that minimize toxicity to human cells, while maintaining efflux pump inhibition is still underway. Leads derived from natural product scaffolds are well‐known to be effective in various therapeutic approaches. Inspired by the biosynthetic pathway to Nocardioazine A, a marine alkaloid known to inhibit the P‐gp efflux pump in cancer cells, we devised a regioselective pathway to create structurally unique indole‐C3‐benzylcyclo‐L‐Trp‐L‐Trp diketopiperazines (DKPs). Using bat cells as a model to derive effective ABCB1 inhibitors for targeting human P‐gp efflux pumps, we have recently identifiedexo‐C3‐N‐Dbn‐Trp2 (13)as a lead ABCB1 inhibitor. This C3‐benzylated lead inhibited ABCB1 better than Verapamil.^[21]Additionally,C3‐N‐Dbn‐Trp2restored chemotherapy sensitivity in drug‐resistant human cancer cells and had no adverse effect on cell proliferation in cell cultures. For a clearer structure‐activity relationship, we developed a broader screen to test C3‐functionalized pyrroloindolines as ABCB1 inhibitors and observed that C3‐benzylation is outperforming respective isoprenylated derivatives. Results arising from the molecular docking studies indicate that the interactions at the access tunnel between ABCB1 and the inhibitor result in a powerful predictor for the efficacy of the inhibitor. Based on fluorescence‐based assays, we conclude that the most efficacious inhibitor is thep‐cyano‐derivedexo‐C3‐N‐Dbn‐Trp2 (33 a), closely followed by thep‐nitro substituted analogue. By combining assay results with molecular docking studies, we further correlate that the predictions based on the inhibitor interactions at the access tunnel provide clues about the design of improved ABCB1 inhibitors. As it has been well documented that ABCB1 itself is powerfully engaged in multi‐drug resistance, this work lays the foundation for the design of a new class of inhibitors based on the endogenous amino acid‐derivedcyclo‐L‐Trp‐L‐Trp DKP scaffold. 

Abstract The 2,5-diketopiperazines are a prominent class of bioactive molecules. The nocardioazines are actinomycete natural products that feature a pyrroloindoline diketopiperazine scaffold composed of two D-tryptophan residues functionalized byN- andC-methylation, prenylation, and diannulation. Here we identify and characterize the nocardioazine B biosynthetic pathway from marineNocardiopsissp. CMB-M0232 by using heterologous biotransformations, in vitro biochemical assays, and macromolecular modeling. Assembly of thecyclo-L-Trp-L-Trp diketopiperazine precursor is catalyzed by a cyclodipeptide synthase. A separate genomic locus encodes tailoring of this precursor and includes an aspartate/glutamate racemase homolog as an unusualD/Lisomerase acting upon diketopiperazine substrates, a phytoene synthase-like prenyltransferase as the catalyst of indole alkaloid diketopiperazine prenylation, and a rare dual function methyltransferase as the catalyst of bothN- andC-methylation as the final steps of nocardioazine B biosynthesis. The biosynthetic paradigms revealed herein showcase Nature’s molecular ingenuity and lay the foundation for diketopiperazine diversification via biocatalytic approaches. 

The first stereoselective total synthesis of (−)-griseocazine D2 and D3 is reported. 

Nocardioazines A and B are prenylated, bioactive pyrroloindoline natural products, isolated from Nocardiopsis, with a desymmetrized cyclo-D-Trp-D-Trp DKP core. Based on our deeper biosynthetic understanding, a biomimetic total synthesis of (+)-nocardioazine B is accomplished in merely seven steps and 23.2% overall yield. This pathway accesses regio- and stereoselectively C3-isoprenylated analogs of (+)-nocardioazine B, using the same number of steps and in similar efficiency. The successful strategy mandated that the biomimetic C3-prenylation step be executed early. The use of an unprotected carboxylic acid of Trp led to high diastereoselectivity toward formation of key intermediates exo-12a, exo-12b, and exo-12c (>19:1). Evidence shows that N1methylation causes the prenylation reaction to bifurcate away to result in a C2-normal-prenylated isomer. Nocardioazine A, possessing an isoprenoidal-epoxide bridge, inhibits P-glycoprotein (P-gp)-mediated membrane efflux, in multidrug-resistant mammalian colon cancer cells. As several P-gp inhibitors have failed due to their toxicity effects, endogenous amino-acid-derived noncytotoxic inhibitors (from the nocardioazine core) are worthy leads toward a rejuvenated strategy against resistant carcinomas. This total synthesis provides direct access to Trp-derived isoprenylated DKP natural products and their derivatives. 

Microorganisms are remarkable chemists, with enzymes as their tools for executing multi-step syntheses to yield myriad natural products. Microbial synthetic aptitudes are illustrated by the structurally diverse 2,5-diketopiperazine (DKP) family of bioactive nonribosomal peptide natural products. Nonribosomal peptide synthetases (NRPSs) have long been recognized as catalysts for formation of DKP scaffolds from two amino acid substrates. Cyclodipeptide synthases (CDPSs) are more recently recognized catalysts of DKP assembly, employing two aminoacyl-tRNAs (aa-tRNAs) as substrates. CDPS-encoding genes are typically found in genomic neighbourhoods with genes encoding additional biosynthetic enzymes. These include oxidoreductases, cytochrome P450s, prenyltransferases, methyltransferases, and cyclases, which equip the DKP scaffold with groups that diversify chemical structures and confer biological activity. These tailoring enzymes have been characterized from nine CDPS-containing biosynthetic pathways to date, including four during the last year. In this review, we highlight these nine DKP pathways, emphasizing recently characterized tailoring reactions and connecting new developments to earlier findings. Featured pathways encompass a broad spectrum of chemistry, including the formation of challenging C–C and C–O bonds, regioselective methylation, a unique indole alkaloid DKP prenylation strategy, and unprecedented peptide-nucleobase bond formation. These CDPS-containing pathways also provide intriguing models of metabolic pathway evolution across related and divergent microorganisms, and open doors to synthetic biology approaches for generation of DKP combinatorial libraries. Further, bioinformatics analyses support that much unique genetically encoded DKP tailoring potential remains unexplored, suggesting opportunities for further expansion of Nature's biosynthetic spectrum. Together, recent studies of DKP pathways demonstrate the chemical ingenuity of microorganisms, highlight the wealth of unique enzymology provided by bacterial biosynthetic pathways, and suggest an abundance of untapped biosynthetic potential for future exploration.