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Lactate dehydrogenase 5 (LDH5) is overexpressed in metastatic tumors and is an attractive target for anticancer therapy. Small-molecule drugs have been developed to target the substrate/cofactor sites of LDH5, but none has reached the clinic to date, and alternative strategies remain almost unexplored. Combining rational and computer-based approaches, we identified peptidic sequences with high affinity toward a β-sheet region that is involved in protein–protein interactions (PPIs) required for the activity of LDH5. To improve stability and potency, these sequences were grafted into a cyclic cell-penetrating β-hairpin peptide scaffold. The lead grafted peptide, cGmC9, inhibited LDH5 activity in vitroin low micromolar range and more efficiently than the small-molecule inhibitor GNE-140. cGmC9 inhibits LDH5 by targeting an interface unlikely to be inhibited by small-molecule drugs. This lead will guide the development of new LDH5 inhibitors and challenges the landscape of drug discovery programs exclusively dedicated to small molecules.

Gomesin, a disulfide-rich antimicrobial peptide produced by the Brazilian spider Acanthoscurria gomesiana, has been shown to be potent against Gram-negative bacteria and to possess selective anticancer properties against melanoma cells. In a recent study, a backbone cyclized analogue of gomesin was shown to be as active but more stable than its native form. In the current study, we were interested in improving the antimicrobial properties of the cyclic gomesin, understanding its selectivity toward melanoma cells and elucidating its antimicrobial and anticancer mode of action. Rationally designed analogues of cyclic gomesin were examined for their antimicrobial potency, selectivity toward cancer cells, membrane-binding affinity, and ability to disrupt cell and model membranes. We improved the activity of cyclic gomesin by ∼10-fold against tested Gram-negative and Gram-positive bacteria without increasing toxicity to human red blood cells. In addition, we showed that gomesin and its analogues are more toxic toward melanoma and leukemia cells than toward red blood cells and act by selectively targeting and disrupting cancer cell membranes. Preference toward some cancer types is likely dependent on their different cell membrane properties. Our findings highlight the potential of peptides as antimicrobial and anticancer leads and the importance of selectively targeting cancer cell membranes for drug development.

Momordicatrypsin inhibitors (TIs) such as those isolated from the seeds of the gấc fruit, Momordica cochinchinensis(MCoTI-I and MCoTI-II), are widely used as scaffolds for drug design studies. To more effectively exploit these molecules in the development of therapeutics, there is a need for wider discovery of the natural sequence diversity among TIs from other species in the Momordicasubfamily. Here we report the discovery of the encoding gene and six TIs from the seeds of the spiny gourd, Momordica dioica, four of which possess novel sequences (Modi 1, 3, 5, and 6) and two (Modi 2 and 4) of which are known peptides (TI-14, TI-17) previously identified in Momordica subangulata. Modi 6 is an acyclic peptide featuring a pyrrolidone carboxylic acid modification, whereas the remaining five TIs are cyclic. All Modi peptides display similar overall structures and trypsin inhibitory activities. No toxicity was observed for these peptides when tested against cancer and insect cells. All Modi peptides were exceptionally stable over 24 h in human serum, indicating a dual strategy to stabilize the peptides in nature, either head-to-tail cyclization or N-pyrolation, which suggests these peptides might be excellent candidates as scaffolds for epitope stabilization in drug design studies.

Cyclotides are macrocyclic cystine-knotted peptides most commonly found in the Violaceae plant family. Although Rinoreais the second-largest genera within the Violaceae family, few studies have examined whether or not they contain cyclotides. To further our understanding of cyclotide diversity and evolution, we examined the cyclotide content of two Rinoreaspecies found in Southeast Asia: R. virgataand R. bengalensis. Seven cyclotides were isolated from R. virgata(named Rivi1–7), and a known cyclotide (cT10) was found in R. bengalensis. Loops 2, 5, and 6 of Rivi1–4 contained sequences not previously seen in corresponding loops of known cyclotides, thereby expanding our understanding of the diversity of cyclotides. In addition, the sequence of loop 2 of Rivi3 and Rivi4 were identical to some related noncyclic “acyclotides” from the Poaceae plant family. As only acyclotides, but not cyclotides, have been reported in monocotyledons thus far, our findings support an evolutionary link between monocotyledon-derived ancestral cyclotide precursors and dicotyledon-derived cyclotides. Furthermore, Rivi2 and Rivi3 had comparable cytotoxic activities to the most cytotoxic cyclotide known to date: cycloviolacin O2 from Viola odorata; yet, unlike cycloviolacin O2, they did not show hemolytic activity. Therefore, these cyclotides represent novel scaffolds for use in future anticancer drug design.

In contrast to Möbius and trypsin inhibitor cyclotides, members of the bracelet subfamily are typically intractable to chemical synthesis and folding. In a significant advance in the field, the bracelet cyclotides ribe 33 and Cter 27 were successfully produced synthetically in moderate yield in a recent study. That synthetic method was a breakthrough as members of the bracelet subfamily of cyclotides had hitherto eluded attempts to be synthetically produced, apart from one report of cyO2 production in which a complicated folding strategy was used. In the current study the successful in vitrofolding of three mutants of bracelet cyclotide Cter 27 is reported. This study broadens our understanding of the folding of bracelet cyclotides and elucidates the three dimensional structure of synthetic Cter 27, providing a new class of cyclotide molecular grafting scaffold for drug design applications.

Melanocortin receptors are pharmaceutically important receptors that are involved in complex physiological functions. They have been associated with various diseases including obesity, erectile dysfunction, acne, and skin cancer. It has been challenging to transform nonselective endogenous agonist and antagonist ligands into selective and potent ligands. In this study, we investigated naturally occurring peptides derived from frog skin secretions for selectivity and activity toward melanocortin receptors. Three peptides (ORB, ORB2K and ranacyclin‐T) were found to have selectivity towards the melanocortin receptor 5 (MC5R). ORB and ORB2K had partial binding affinity at nanomolar concentrations, whereas ranacyclin‐T had 57% binding efficiency at 1.6 μM. Backbone cyclization of ORB and ORB2K altered the binding efficiency to melanocortin receptors. Our results suggest that these frog‐skin peptides could be modified for developing melanocortin‐specific ligands and potentially future therapeutics.

Angiogenesis is important for tumor growth, and accordingly, targeting angiogenesis has become an important pathway for antitumor therapy. A novel proapoptotic peptide, CIGB-300 (P15-Tat), has been shown to be involved in the casein kinase II phosphorylation pathway, conferring it with antiangiogenic activity. Cyclic peptides have been widely used as scaffolds in drug design studies due to their high stability and favorable biopharmaceutical properties. Here, we chose two very stable cyclic trypsin inhibitors, MCoTI-II and SFTI-1, as frameworks to incorporate the bioactive epitope P15 into various backbone loops. NMR studies revealed that all re-engineered analogs had similar secondary structures to their native cyclic frameworks. One key analog, MCoP15, displayed significant improvement for inhibiting human umbilical vein endothelial cell migration, was nontoxic, and had higher stability than the P15 epitope alone. Overall, the results show the value of P15 being engineered into cyclic trypsin inhibitor scaffolds for improving antiangiogenic activity and stability. More broadly, the study highlights the versatility of cyclic peptide frameworks in drug design for antiangiogenic therapies.

Cyclotides are plant-derived peptides found within five families of flowering plants (Violaceae, Rubiaceae, Fabaceae, Solanaceae, and Poaceae) that have a cyclic backbone and six conserved cysteine residues linked by disulfide bonds. Their presence within the Violaceae species seems ubiquitous, yet not all members of other families produce these macrocyclic peptides. The genus PalicoureaAubl. (Rubiaceae) contains hundreds of neotropical species of shrubs and small trees; however, only a few cyclotides have been discovered hitherto. Herein, five previously uncharacterized Möbius cyclotides within Palicourea sessilisand their pharmacological activities are described. Cyclotides were isolated from leaves and stems of this plant and identified as pase A–E, as well as the known peptide kalata S. Cyclotides were de novo sequenced by MALDI-TOF/TOF mass spectrometry, and their structures were solved by NMR spectroscopy. Because some cyclotides have been reported to modulate immune cells, pase A–D were assayed for cell proliferation of human primary activated T lymphocytes, and the results showed a dose-dependent antiproliferative function. The toxicity on other nonimmune cells was also assessed. This study reveals that pase cyclotides have potential for applications as immunosuppressants and in immune-related disorders.