Screening and characterization of novel specific peptides targeting MDA-MB-231 claudin-low breast carcinoma by computer-aided phage display methodologies.

BACKGROUND: Claudin-low breast carcinoma represents 19% of all breast cancer cases and is characterized by an aggressive progression with metastatic nature and high rates of relapse. Due to a lack of known specific molecular biomarkers for this breast cancer subtype, there are no targeted therapies available, which results in the worst prognosis of all breast cancer subtypes. Hence, the identification of novel biomarkers for this type of breast cancer is highly relevant for an early diagnosis. Additionally, claudin-low breast carcinoma peptide ligands can be used to design powerful drug delivery systems that specifically target this type of breast cancer. METHODS: In this work, we propose the identification of peptides for the specific recognition of MDA-MB-231, a cell line representative of claudin-low breast cancers, using phage display (both conventional panning and BRASIL). Binding assays, such as phage forming units and ELISA, were performed to select the most interesting peptides (i.e., specific to the target cells) and bioinformatics approaches were applied to putatively identify the biomarkers to which these peptides bind. RESULTS: Two peptides were selected using this methodology specifically targeting MDA-MB-231 cells, as demonstrated by a 4 to 9 log higher affinity as compared to control cells. The use of bioinformatics approaches provided relevant insights into possible cell surface targets for each peptide identified. CONCLUSIONS: The peptides herein identified may contribute to an earlier detection of claudin-low breast carcinomas and possibly to develop more individualized therapies.

Selection of Novel Peptides Homing the 4T1 CELL Line: Exploring Alternative Targets for Triple Negative Breast Cancer.

The use of bacteriophages to select novel ligands has been widely explored for cancer therapy. Their application is most warranted in cancer subtypes lacking knowledge on how to target the cancer cells in question, such as the triple negative breast cancer, eventually leading to the development of alternative nanomedicines for cancer therapeutics. Therefore, the following study aimed to select and characterize novel peptides for a triple negative breast cancer murine mammary carcinoma cell line- 4T1. Using phage display, 7 and 12 amino acid random peptide libraries were screened against the 4T1 cell line. A total of four rounds, plus a counter-selection round using the 3T3 murine fibroblast cell line, was performed. The enriched selective peptides were characterized and their binding capacity towards 4T1 tissue samples was confirmed by immunofluorescence and flow cytometry analysis. The selected peptides (4T1pep1 -CPTASNTSC and 4T1pep2-EVQSSKFPAHVS) were enriched over few rounds of selection and exhibited specific binding to the 4T1 cell line. Interestingly, affinity to the human MDA-MB-231 cell line was also observed for both peptides, promoting the translational application of these novel ligands between species. Additionally, bioinformatics analysis suggested that both peptides target human Mucin-16. This protein has been implicated in different types of cancer, as it is involved in many important cellular functions. This study strongly supports the need of finding alternative targeting systems for TNBC and the peptides herein selected exhibit promising future application as novel homing peptides for breast cancer therapy.

Triple Negative Breast Cancer: Nanosolutions for a Big Challenge.

Triple negative breast cancer (TNBC) is a particular immunopathological subtype of breast cancer that lacks expression of estrogen and progesterone receptors (ER/PR) and amplification of the human epidermal growth factor receptor 2 (HER2) gene. Characterized by aggressive and metastatic phenotypes and high rates of relapse, TNBC is the only breast cancer subgroup still lacking effective therapeutic options, thus presenting the worst prognosis. The development of targeted therapies, as well as early diagnosis methods, is vital to ensure an adequate and timely therapeutic intervention in patients with TNBC. This review intends to discuss potentially emerging approaches for the diagnosis and treatment of TNBC patients, with a special focus on nano-based solutions that actively target these particular tumors.

Mechanistic dissection of the enzyme complexes involved in biosynthesis of lacticin 3147 and nisin.

The thioether rings in the lantibiotics lacticin 3147 and nisin are posttranslationally introduced by dehydration of serines and threonines, followed by coupling of these dehydrated residues to cysteines. The prepeptides of the two-component lantibiotic lacticin 3147, LtnA1 and LtnA2, are dehydrated and cyclized by two corresponding bifunctional enzymes, LtnM1 and LtnM2, and are subsequently processed and exported via one bifunctional enzyme, LtnT. In the nisin synthetase complex, the enzymes NisB, NisC, NisT, and NisP dehydrate, cyclize, export, and process prenisin, respectively. Here, we demonstrate that the combination of LtnM2 and LtnT can modify, process, and transport peptides entirely different from LtnA2 and that LtnT can process and transport unmodified LtnA2 and unrelated peptides. Furthermore, we demonstrate a higher extent of NisB-mediated dehydration in the absence of thioether rings. Thioether rings apparently inhibited dehydration, which implies alternating actions of NisB and NisC. Furthermore, certain (but not all) NisC-cyclized peptides were exported with higher efficiency as a result of their conformation. Taken together, these data provide further insight into the applicability of Lactococcus lactis strains containing lantibiotic enzymes for the design and production of modified peptides.

Influence of shifting positions of Ser, Thr, and Cys residues in prenisin on the efficiency of modification reactions and on the antimicrobial activities of the modified prepeptides.

Since the recent discovery that the nisin modification and transport machinery can be used to produce and modify peptides unrelated to nisin, specific questions arose concerning the specificity of the modification enzymes involved and the limits of their promiscuity with respect to the dehydration and cyclization processes. The nisin leader peptide has been postulated to fulfill a recognition and binding function required for these modifications. Here, we investigated whether the relative positions of the modifiable residues in the nisin prepeptide, with respect to the leader peptide, could influence the efficiency of their modification. We conducted a systematic study on the insertion of one to four alanines in front of either ring A or ring D to change the "reading frame" of modifiable residues, resulting in altered distance and topology of the modifiable residues relative to the leader. The insertion of N-terminal and hinge-located Ala residues had only a modest influence on the modification efficiency, demonstrating that the "phasing" of these residues relative to the leader peptide is not a critical factor in determining modification. However, in all cases, but especially with the N-terminal insertions, the antimicrobial activities of the fully modified nisin species were decreased.

NisC, the cyclase of the lantibiotic nisin, can catalyze cyclization of designed nonlantibiotic peptides.

Nisin is a pentacyclic peptide antibiotic active against Gram-positive bacteria. Its thioether rings are formed by two enzymatic steps: nisin dehydratase (NisB)-mediated dehydration of serines and threonines followed by nisin cyclase (NisC)-catalyzed enantioselective coupling of cysteines to the formed dehydroresidues. Here, we report the in vivo activity of NisC to cyclize a wide array of unrelated and designed peptides that were fused to the nisin leader peptide. To assess the role of NisC, leader peptide fusions, secreted by Lactococcus lactis cells containing NisBT with or without NisC were compared. In hexapeptides, a dehydroalanine could spontaneously react with a more C-terminally located cysteine. In contrast, peptides containing dehydrobutyrines require NisC for cyclization. In agreement with in silico predictions NisC could efficiently cyclize the hexapeptides ADhbVECK and IDhbPGCK, but ADhbVWCE was not cyclized. Interestingly, NisC could efficiently catalyze the synthesis of peptides with intertwined rings and of a designed polyhexapeptide containing four thioether rings. Taken together the data demonstrate that NisC can be widely applied for the cyclization and stabilization of nonlantibiotic peptides.

Production of dehydroamino acid-containing peptides by Lactococcus lactis.

Nisin is a pentacyclic peptide antibiotic produced by some Lactococcus lactis strains. Nisin contains dehydroresidues and thioether rings that are posttranslationally introduced by a membrane-associated enzyme complex, composed of a serine and threonine dehydratase NisB and the cyclase NisC. In addition, the transporter NisT is necessary for export of the modified peptide. We studied the potential of L. lactis expressing NisB and NisT to produce peptides whose serines and threonines are dehydrated. L. lactis containing the nisBT genes and a plasmid coding for a specific leader peptide fusion construct efficiently produced peptides with a series of non-naturally occurring multiple flanking dehydrobutyrines. We demonstrated NisB-mediated dehydration of serines and threonines in a C-terminal nisin(1-14) extension of nisin, which implies that also residues more distant from the leader peptide than those occurring in prenisin or any other lantibiotic can be modified. Furthermore, the feasibility and efficiency of generating a library of peptides containing dehydroresidues were demonstrated. In view of the particular shape and reactivity of dehydroamino acids, such a library provides a novel source for screening for peptides with desired biological and physicochemical properties.

Sec-mediated transport of posttranslationally dehydrated peptides in Lactococcus lactis.

Nisin is a lanthionine-containing antimicrobial peptide produced by Lactococcus lactis. Its (methyl)lanthionines are introduced by two posttranslational enzymatic steps involving the dehydratase NisB, which dehydrates serine and threonine residues, and the cyclase NisC, which couples these dehydrated residues to cysteines, yielding thioether-bridged amino acids called lanthionines. The prenisin is subsequently exported by the ABC transporter NisT and extracellularly processed by the peptidase NisP. L. lactis expressing the nisBTC genes can modify and secrete a wide range of nonlantibiotic peptides. Here we demonstrate that in the absence of NisT and NisC, the Sec pathway of L. lactis can be exploited for the secretion of dehydrated variants of therapeutic peptides. Furthermore, posttranslational modifications by NisB and NisC still occur even when the nisin leader is preceded by a Sec signal peptide or a Tat signal peptide 27 or 44 amino acids long, respectively. However, transport of fully modified prenisin via the Sec pathway is impaired. The extent of NisB-mediated dehydration could be improved by raising the intracellular concentration NisB or by modulating the export efficiency through altering the signal sequence. These data demonstrate that besides the traditional lantibiotic transporter NisT, the Sec pathway with an established broad substrate range can be utilized for the improved export of lantibiotic enzyme-modified (poly)peptides.

Post-translational modification of therapeutic peptides by NisB, the dehydratase of the lantibiotic nisin.

Post-translationally introduced dehydroamino acids often play an important role in the activity and receptor specificity of biologically active peptides. In addition, a dehydroamino acid can be coupled to a cysteine to yield a cyclized peptide with increased biostability and resistance against proteolytic degradation and/or modified specificity. The lantibiotic nisin is an antimicrobial peptide produced by Lactococcus lactis. Its post-translational enzymatic modification involves NisB-mediated dehydration of serines and threonines and NisC-catalyzed coupling of cysteines to dehydroresidues, followed by NisT-mediated secretion. Here, we demonstrate that a L. lactis strain containing the nisBTC genes effectively dehydrates and secretes a wide range of medically relevant nonlantibiotic peptides among which variants of adrenocorticotropic hormone, vasopressin, an inhibitor of tripeptidyl peptidase II, enkephalin, luteinizing hormone-releasing hormone, angiotensin, and erythropoietin. For most of these peptides, ring formation was demonstrated. These data show that lantibiotic enzymes can be applied for the modification of peptides, thereby enabling the biotechnological production of dehydroresidue-containing and/or thioether-bridged therapeutic peptides with enhanced stability and/or modulated activities.

NisT, the transporter of the lantibiotic nisin, can transport fully modified, dehydrated, and unmodified prenisin and fusions of the leader peptide with non-lantibiotic peptides.

Lantibiotics are lanthionine-containing peptide antibiotics. Nisin, encoded by nisA, is a pentacyclic lantibiotic produced by some Lactococcus lactis strains. Its thioether rings are posttranslationally introduced by a membrane-bound enzyme complex. This complex is composed of three enzymes: NisB, which dehydrates serines and threonines; NisC, which couples these dehydrated residues to cysteines, thus forming thioether rings; and the transporter NisT. We followed the activity of various combinations of the nisin enzymes by measuring export of secreted peptides using antibodies against the leader peptide and mass spectroscopy for detection. L. lactis expressing the nisABTC genes efficiently produced fully posttranslationally modified prenisin. Strikingly, L. lactis expressing the nisBT genes could produce dehydrated prenisin without thioether rings and a dehydrated form of a non-lantibiotic peptide. In the absence of the biosynthetic NisBC enzymes, the NisT transporter was capable of excreting unmodified prenisin and fusions of the leader peptide with non-lantibiotic peptides. Our data show that NisT specifies a broad spectrum (poly)peptide transporter that can function either in conjunction with or independently from the biosynthetic genes. NisT secretes both unmodified and partially or fully posttranslationally modified forms of prenisin and non-lantibiotic peptides. These results open the way for efficient production of a wide range of peptides with increased stability or novel bioactivities.