Linker optimization and activity validation of a cell surface vimentin targeted homo-dimeric peptoid antagonist for lung cancer stem cells.

Epithelial-to-mesenchymal transition (EMT) endows epithelia-derived cancer cells with properties of stem cells that govern cancer invasion and metastasis. Vimentin is one of the best studied EMT markers and recent reports indicate that vimentin interestingly translocated onto cell surface under various tumor conditions. We recently reported a cell surface vimentin (CSV) specific peptoid antagonist named JM3A. We now investigated the selective antagonist activity of the optimized homo-dimeric version of JM3A, JM3A-L2D on stem-like cancer cells or cancer stem cells (CSCs) over normal cells in non-small cell lung cancer (NSCLC). Homo-dimerization of JM3A provided the avidity effect and improved the biological activity compared to the monomeric version. We first optimized the central linker length of the dimer by designing seven JM3A derivatives with varying linker lengths/types and evaluated the anti-cancer activity using the standard MTS cell viability assay. The most optimized derivative contains a central lysine linker and two glycines, named JM3A-L2D, which displayed 100 nM vimentin binding affinity (Kd) with an anti-cancer activity (IC50) of 6.7 µM on H1299 NSCLC cells. This is a 190-fold improvement in binding over the original JM3A. JM3A-L2D exhibited better potency on high vimentin-expressing NSCLC cells (H1299 and H460) compared to low vimentin-expressing NSCLC cells (H2122). No activity was observed on normal bronchial HBEC3-KT cells. The anti-CSC activity of JM3A-L2D was evaluated using the standard colony formation assay and JM3A-L2D disrupted the colony formation with IC50 âˆ¼ 400 nM. In addition, JM3A-L2D inhibited cell migration activity at IC50 âˆ¼ 2 µM, assessed via wound healing assay. The underlying mechanism of action seems to be the induction of apoptosis by JM3A-L2D on high-vimentin expressing H1229 and H460 NSCLC cells. Our optimized highly CSV selective peptoid has the potential to be developed as an anti-cancer drug candidate, especially considering the high serum stability and economical synthesis of peptoids.

N-substituting perturbation on the interaction affinity and recognition specificity between rheumatic immune-related Abl SH3 domain and its peptoid ligands.

Abl is a nonreceptor tyrosine kinase involved in a variety of disease pathways such as rheumatic immune. Full-length Abl protein consists of a catalytic tyrosine kinase (TK) domain as well as two regulatory Src homology domains 2 and 3 (SH2 and SH3, respectively); the latter recognizes and binds to those natural proline-rich peptide segments containing a PxxP motif on the protein surface of its interacting partners. However, natural peptides cannot bind effectively to the modular domain in high affinity and strong selectivity due to their small size and broad specificity. Here, a synthetic proline-rich peptide p41 was used as template; its structural diversity was extended by combinationally replacing the Pro0 and Pro+3 residues with a number of N-substituted amino acids. Consequently, peptide affinity change upon the replacement was derived to create a systematic N-substituting perturbation profile, from which we identified several N-substitution combinations at the Pro0 and Pro+3 residues of p41 PxxP motif that may moderately or significantly improve the peptide binding potency to Abl; they represent potent peptoid binders of Abl SH3 domain, with affinity improved considerably relative to p41. More significantly, the designed potent peptoids were also found to exhibit a good SH3-selectivity for their cognate Abl over other noncognate nonreceptor tyrosine kinases, with S = 9.7-fold.

Real-Time Monitoring of Multitarget Antimicrobial Mechanisms of Peptoids Using Label-Free Imaging with Optical Diffraction Tomography.

Antimicrobial peptides (AMPs) are promising therapeutics in the fight against multidrug-resistant bacteria. As a mimic of AMPs, peptoids with N-substituted glycine backbone have been utilized for antimicrobials with resistance against proteolytic degradation. Antimicrobial peptoids are known to kill bacteria by membrane disruption; however, the nonspecific aggregation of intracellular contents is also suggested as an important bactericidal mechanism. Here,structure-activity relationship (SAR) of a library of indole side chain-containing peptoids resulting in peptoid 29 as a hit compound is investigated. Then, quantitative morphological analyses of live bacteria treated with AMPs and peptoid 29 in a label-free manner using optical diffraction tomography (ODT) are performed. It is unambiguously demonstrated that both membrane disruption and intracellular biomass flocculation are primary mechanisms of bacterial killing by monitoring real-time morphological changes of bacteria. These multitarget mechanisms and rapid action can be a merit for the discovery of a resistance-breaking novel antibiotic drug.

Characterization of PGua4, a Guanidinium-Rich Peptoid that Delivers IgGs to the Cytosol via Macropinocytosis.

To investigate the structure-cellular penetration relationship of guanidinium-rich transporters (GRTs), we previously designed PGua4, a five-amino acid peptoid containing a conformationally restricted pattern of eight guanidines, which showed high cell-penetrating abilities and low cell toxicity. Herein, we characterized the cellular uptake selectivity, internalization pathway, and intracellular distribution of PGua4, as well as its capacity to deliver cargo. PGua4 exhibits higher penetration efficiency in HeLa cells than in six other cell lines (A549, Caco-2, fibroblast, HEK293, Mia-PaCa2, and MCF7) and is mainly internalized by clathrin-mediated endocytosis and macropinocytosis. Confocal microscopy showed that it remained trapped in endosomes at low concentrations but induced pH-dependent endosomal membrane destabilization at concentrations ≥10 µM, allowing its diffusion into the cytoplasm. Importantly, PGua4 significantly enhanced macropinocytosis and the cellular uptake and cytosolic delivery of large IgGs following noncovalent complexation. Therefore, in addition to its peptoid nature conferring high resistance to proteolysis, PGua4 presents characteristics of a promising tool for IgG delivery and therapeutic applications.

Short Peptoid Evolved from the Key Hydrophobic Stretch of Amyloid-ß42 Peptide Serves as a Potent Therapeutic Lead of Alzheimer's Disease.

Amyloid-ß 42(Aß42), an enzymatically cleaved (1-42 amino acid long) toxic peptide remnant, has long been reported to play the key role in Alzheimer's disease (AD). Aß42 also plays the key role in the onset of other AD-related factors including hyperphosphorylation of tau protein that forms intracellular neurofibrillary tangles, imbalances in the function of the neurotransmitter acetylcholine, and even generation of reactive oxygen species (ROS), disrupting the cytoskeleton and homeostasis of the cell. To address these issues, researchers have tried to construct several strategies to target multiple aspects of the disease but failed to produce any clinically successful therapeutic molecules. In this article, we report a new peptoid called RA-1 that was designed and constructed from the hydrophobic stretch of the Aß42 peptide, 16KLVFFA21. This hydrophobic stretch is primarily responsible for the Aß42 peptide aggregation. Experimental study showed that the RA-1 peptoid is stable under proteolytic conditions, can stabilize the microtubule, and can inhibit the formation of toxic Aß42 aggregates by attenuating hydrophobic interactions between Aß42 monomers. Furthermore, results from various intracellular assays showed that RA-1 inhibits Aß42 fibril formation caused by the imbalance in AchE activity, reduces the production of cytotoxic reactive oxygen species (ROS), and promotes neurite outgrowth even in the toxic environment. Remarkably, we have also demonstrated that our peptoid has significant ability to improve the cognitive ability and memory impairment in in vivo rats exposed to AlCl3 and d-galactose (d-gal) dementia model. These findings are also validated with histological studies. Overall, our newly developed peptoid emerges as a multimodal potent therapeutic lead molecule against AD.

Evaluation of the cell permeability of bicyclic peptoids and bicyclic peptide-peptoid hybrids.

Bicyclization has proven to be an effective strategy for significantly restricting conformational flexibility in peptides and peptidomimetics such as peptoids. Such constrained bicyclic peptoids would have far higher conformational rigidity than monocyclic and linear ones, allowing them to have enhanced binding affinity and selectivity for their biological targets. Herein, we show that bicyclic peptoids have superior cellular uptake efficiency than their linear counterparts regardless of their side chains and ring sizes. As a representative example, an 8-mer bicyclic peptoid achieves a CP50 value of 1.2 µM, which is > 5-times superior to the corresponding linear peptoid. Additionally, we also demonstrate that bicyclic peptide-peptoid hybrids are much more cell-permeable than native peptides. Due to their favorable properties including improved cellular uptake, resistance to proteolytic degradation, relatively large sizes, and enormous structural diversity, constrained bicyclic peptoids and peptide-peptoid hybrids will play an important role as potential drug leads, especially in targeting intracellular protein-protein interactions, which are traditionally considered undruggable.

Entry inhibition of hepatitis B virus using cyclosporin O derivatives with peptoid side chain incorporation.

Hepatitis B virus (HBV) infection is a serious worldwide health problem causing liver cirrhosis and hepatocellular carcinoma. The development of novel therapeutics targeting distinct steps of the HBV life cycle and combination therapy with approved drugs (i.e., nucleot(s)ides, interferon-α) are considered effective strategies for curing HBV. Among these strategies is the development of entry inhibitors that interfere with the host entry step of HBV to prevent viral infection and transmission. Herein, we generated a novel library of cyclosporin O (CsO) derivatives that incorporate peptoid side chains. Twenty-two CsO derivatives were evaluated for membrane permeability, cytotoxicity, and in vitro HBV entry inhibitory activity. The lead compound (i.e., compound 21) showed the greatest potency in the in vitro HBV entry inhibition assay (IC50 = 0.36 ± 0.01 µM) with minimal cytotoxicity. Our peptide-peptoid hybrid CsO scaffold can readily expand chemical diversity and is applicable for screening various targets requiring macrocyclic chemical entities.

Unbiased peptoid cell screen identifies a peptoid targeting newly appeared cell surface vimentin on tumor transformed early lung cancer cells.

To identify potential new reagents and biomarkers for early lung cancer detection we combined the use of a novel preclinical isogenic model of human lung epithelial cells comparing non-malignant cells with those transformed to full malignancy using defined oncogenic changes and our on-bead two color (red and green stained cells) (OBTC) peptoid combinatorial screening methodology. The preclinical model used normal parent lung epithelial cells (HBEC3-KT, labeled with green dye) and isogenic fully malignant transformed derivatives (labeled with a red dye) via the sequential introduction of key genetic alterations of p53 knockdown, oncogenic KRAS and overexpression of cMYC (HBEC3p53, KRAS, cMYC). Using the unbiased OBTC screening approach, we tested 100,000 different peptoids and identified only one (named JM3A) that bound to the surface of the HBEC3p53, KRAS, cMYC cells (red cells) but not HBEC3-KT cells (green cells). Using the JM3A peptoid and proteomics, we identified the protein bound as vimentin using multiple validation approaches. These all confirmed the cell surface expression of vimentin (CSV) on transformed (HBEC3p53, KRAS, cMYC) but not on untransformed (HBEC3-KT) cells. JM3A coupled with fluorophores was able to detect and stain cell surface vimentin on very early stage lung cancers but not normal lung epithelial cells in a fashion comparable to that using anti-vimentin antibodies. We conclude: using a combined isogenic preclinical model of lung cancer and two color screening of a large peptoid library, we have identified differential expression of cell surface vimentin (CSV) after malignant transformation of lung epithelial cells, and developed a new peptoid reagent (JM3A) for detection of CSV which works well in staining of early stage NSCLCs. This new, highly specific, easy to prepare, CSV detecting JM3A peptoid provides an important new reagent for identifying cancer cells in early stage tumors as well as a resource for detection and isolating of CSV expressing circulating tumor cells.

"Molecular Masks" for ACE2 to Effectively and Safely Block SARS-CoV-2 Virus Entry.

Coronavirus Disease 2019 (COVID-19) remains a global health crisis, despite the development and success of vaccines in certain countries. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the virus that causes COVID-19, uses its spike protein to bind to the human cell surface receptor angiotensin-converting enzyme 2 (ACE2), which allows the virus to enter the human body. Using our unique cell screening technology, we identified two ACE2-binding peptoid compounds and developed dimeric derivatives (ACE2P1D1 and ACE2P2D1) that effectively blocked spike protein-ACE2 interaction, resulting in the inhibition of SARS-CoV-2 pseudovirus entry into human cells. ACE2P1D1 and ACE2P2D1 also blocked infection by a D614G mutant pseudovirus. More importantly, these compounds do not decrease ACE2 expression nor its enzyme activity (which is important in normal blood pressure regulation), suggesting safe applicability in humans.

Cell-penetrating, amphipathic cyclic peptoids as molecular transporters for cargo delivery.

Here we describe the design, synthesis, and biological evaluation of cell-penetrating, amphipathic cyclic peptoids as a novel class of molecular transporters. We demonstrated that macrocyclization, along with the introduction of hydrophobic residues, greatly enhanced cellular uptake of polyguanidine linear peptoids. The amphipathic cyclic peptoids showed an order of magnitude more efficient intracellular delivery ability, compared to a commonly used polyarginine cell-penetrating peptide, representing one of the best molecular transporters reported to date. Given the excellent cell-permeability, proteolytic stability, and ease of synthesis, the amphipathic cyclic peptoids would be broadly applicable to a wide range of clinical and biological applications.

Approaches to Evaluate the Impact of a Small-Molecule Binder to a Noncatalytic Site of the Proteasome.

Proteasome activity is crucial for cell survival and proliferation. In recent years, small molecules have been discovered that can affect the catalytic activity of the proteasome. Rather than targeting the active sites of the proteasome, it might be possible to affect ubiquitin-dependent degradation of proteins by limiting the association of the 19S regulatory particle (19S RP) with the 20S core particle (20S CP) of the proteasome. We recently described the discovery of TXS-8, a peptoid that binds to Rpn-6. Rpn-6 is a proteasome-associated protein that makes critical contacts with the 19S RP and the 20S CP. Herein, we present a general workflow to evaluate the impact of a small-molecule binder on proteasome activity by using TXS-8 as an example. This workflow contains three steps in which specific probes or overexpressed proteins in cells are used to determine whether the hydrolysis activity of the proteasome is affected. Although, in our case, TXS-8 did not affect proteasome activity, our workflow is highly amenable to studying a variety of small-molecule-proteasome subunit interactions.

Sequence-function relationship within water-soluble Peptoid Chelators for Cu2.

Chelation of Cu2+ by synthetic molecules is an emerging therapeutic approach for treating several illnesses in human body such as Wilson disease, cancer and more. Among synthetic metal chelators, those based on peptoids - N-substituted glycine oligomers - are advantageous due to their structural similarity to peptides, ease of synthesis on solid support and versatile controlled sequences. Tuning peptoid sequences, via systematically changing at least one side chain, can facilitate and control their function. Along these lines, this work aims to explore the role of the non-coordinating side chain within peptoid chelators in order to understand the factors that control the selectivity of these chelators to Cu2+ in water medium. To this aim, a set of peptoid trimers having a pyridine group at the acetylated N-terminal, a 2,2'-bipyridine group at the second position and a non-coordinating group at the C-terminus, where the latter is systematically varied between aromatic, aliphatic, chiral or non-chiral, were investigated as selective chelators for Cu2+. The effect of the position of the non-coordinating group on the selectivity of the peptoid to Cu2+ was also tested. Based on extensive spectroscopic data, we found that the choice of the non-coordinating group along with its position dramatically influences the selectivity of the peptoids to Cu2+. We showed that peptoids having bulky chiral groups at the C-terminus enable high selectivity to Cu2+. We further demonstrated the ability of one of the selective chelators to remove Cu2+ from the natural copper binding protein metallothionein in HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) buffer medium.

Structural characterization of a peptoid-inspired conformationally constrained oligomer (PICCO) bound to streptavidin.

A high affinity Streptavidin ligand was mined from a DNA-encoded library of non-peptidic oligimers and characterized structurally.

Helicity of Peptoid Ions in the Gas Phase.

Peptoids are attractive substitutes for peptides in several research areas, especially when they adopt a helical structure. The chain-size evolution of the secondary structure of the widely studied (S)-N-1-phenylethyl peptoids is here analyzed by means of the ion mobility mass spectrometry technique increasingly used as a powerful analytical tool and is further supported by theoretical modeling. We conclude that the helical shape of the peptoids prevailing in solution is lost in the gas phase by the need to screen the positive charge borne by the peptoid even though the collisional cross sections are close to the values expected for helical systems. We further illustrate that trend line analyses predicting molecular shapes from fits of the size evolution of cross sections can be very misleading since they critically depend on the range of polymerization degrees under study.

Novel peptoid-based adsorbents for purifying IgM and IgG from polyclonal and recombinant sources.

Polyclonal immunoglobulin therapeutics comprising dosed IgG and IgM combinations are powerful tools in fighting cancer and severe infections. The inability of protein ligands to produce polyclonal IgG- and IgM-enriched formulations and recover monoclonal IgM calls for novel ligands with superior biorecognition activity. In this study, a peptoid ligand discovered by our group, and integrated into affinity adsorbents LigaTrap Technologies' "Human IgG" and "Human IgM", were utilized to purify IgG and IgM from complex fluids. IgG purification from human serum using LigaTrap IgG afforded 94.6% purity and 62.9% yield, on par with Protein A/G resins. When challenged with CHO and HEK cell culture harvests with low IgG titer (<1 mg/mL), LigaTrap IgG returned values of yield and purity well above 60% and 90%. LigaTrap IgM was evaluated for purifying IgM in comparison with commercial adsorbents, and afforded a product purity of 93% from a CHO harvest (IgM titer of 1 mg/mL) and 75.1% yield from a HEK harvest (0.5 mg/mL). LigaTrap-M provided IgM enrichment up to 11-fold higher than HiTrap resin. The peptoid adsorbents separated IgG-depleted human serum into IgM- and IgA-enriched fractions. These results demonstrate the potential of the peptoid ligand for manufacturing polyclonal Ig formulations and monoclonal IgM therapeutics.

Synthesis and Application of an Activity-Based Peptide-Peptoid Hybrid Probe for the Immunoproteasome.

The immunoproteasome (iCP), a specific isoform of the proteasome's catalytic particle, is becoming an important protein complex of interest in various diseases. However, there is still much left to be learned about its activity in cells and how this can be altered by various endogenous conditions or with treatment with small molecules. Current strategies to investigate the iCP lack in their ability to be used in live, intact cells, limiting them to use in endpoint experiments. The iCP-selective probe presented here has been shown to be compatible with various live-cell assays, including monitoring iCP activity kinetically in a plate reader-based assay and observing single cells with confocal microscopy. A well-studied iCP-selective inhibitor, ONX-0914, has also been demonstrated to decrease the fluorescence signal of the iCP probe in both of these assays, showing its potential function in investigating small-molecule modulators of the iCP. © 2019 by John Wiley & Sons, Inc. Basic Protocol 1: Synthesis of an immunoproteasome-selective peptide-peptoid hybrid probe Basic Protocol 2: Expression of the immunoproteasome in A549 cells Basic Protocol 3: Using the immunoproteasome probe to monitor activity in live cells with a fluorescence plate reader Basic Protocol 4: Using the immunoproteasome probe to monitor activity in live cells with confocal microscopy.

DNA-Encoded Combinatorial Library of Macrocyclic Peptoids.

We report the design and synthesis of a DNA-encoded one-bead one-compound library of cyclic peptoids composed of more than 11 million molecules. We show that affinity-based screening of this large library can identify cyclic peptoid ligands for a target protein. In this work, we developed a simple method for amplifying the PCR product from DNA tags on a single bead, thereby enabling determination of the structures of hit cyclic peptoids with no need for high-throughput sequencing and complicated data analysis. We also developed a sublibrary screening strategy to minimize false positives caused by the interference of coding DNA tags before starting laborious and impractical hit confirmation. Given the simplicity and robustness of the synthesis and screening, along with the desirable features of macrocyclic peptoids including improved conformational rigidity, our method will be highly useful for discovering biologically active molecules modulating challenging targets such as protein-protein interactions that are not easily targeted by typical peptidomimetics and small-molecules.

Fluorescent analogs of peptoid-based HDAC inhibitors: Synthesis, biological activity and cellular uptake kinetics.

Fluorescent tagging of bioactive molecules is a powerful tool to study cellular uptake kinetics and is considered as an attractive alternative to radioligands. In this study, we developed fluorescent histone deacetylase (HDAC) inhibitors and investigated their biological activity and cellular uptake kinetics. Our approach was to introduce a dansyl group as a fluorophore in the solvent-exposed cap region of the HDAC inhibitor pharmacophore model. Three novel fluorescent HDAC inhibitors were synthesized utilizing efficient submonomer protocols followed by the introduction of a hydroxamic acid or 2-aminoanilide moiety as zinc-binding group. All compounds were tested for their inhibition of selected HDAC isoforms, and docking studies were subsequently performed to rationalize the observed selectivity profiles. All HDAC inhibitors were further screened in proliferation assays in the esophageal adenocarcinoma cell lines OE33 and OE19. Compound 2, 6-((N-(2-(benzylamino)-2-oxoethyl)-5-(dimethylamino)naphthalene)-1-sulfonamido)-N-hydroxyhexanamide, displayed the highest HDAC inhibitory capacity as well as the strongest anti-proliferative activity. Fluorescence microscopy studies revealed that compound 2 showed the fastest uptake kinetic and reached the highest absolute fluorescence intensity of all compounds. Hence, the rapid and increased cellular uptake of 2 might contribute to its potent anti-proliferative properties.

Oligomers of α-ABpeptoid/ß3 -peptide hybrid.

Peptoids, oligomers of N-substituted glycines, have been attracting increasing interest due to their advantageous properties as peptidomimetics. However, due to the lack of chiral centers and amide hydrogen atoms, peptoids, in general, do not form folding structures except that they have α-chiral side chains. We have recently developed "peptoids with backbone chirality" as a new class of peptoid foldamers called α-ABpeptoids and demonstrated that they could have folding conformations owing to the methyl groups on chiral α-carbons in the backbone structure. Here we report α-ABpeptoid/ß3 -peptide oligomers as a unique peptidomimetic structure with a heterogeneous backbone. This hybrid structure contains a mixed α-ABpeptoid and ß3 -peptide residues arranged in an alternate manner. These α-ABpeptoid/ß3 -peptide oligomers could form intramolecular hydrogen bonding and have better cell permeability relative to pure peptide sequences. These oligomers were shown to adopt ordered folding structures based on circular dichroism studies. Overall, α-ABpeptoid/ß3 -peptide oligomers may represent a novel class of peptidomimetic foldamers and will find a wide range of applications in biomedical and material sciences.

Toward a clinical antifungal peptoid: Investigations into the therapeutic potential of AEC5.

Cryptococcus neoformans is a fungal pathogen that causes cryptococcal meningitis in immunocompromised individuals. Existing antifungal treatment plans have high mammalian toxicity and increasing drug resistance, demonstrating the dire need for new, nontoxic therapeutics. Antimicrobial peptoids are one alternative to combat this issue. Our lab has recently identified a tripeptoid, AEC5, with promising efficacy and selectivity against C. neoformans. Here, we report studies into the broad-spectrum efficacy, killing kinetics, mechanism of action, in vivo half-life, and subchronic toxicity of this compound. Most notably, these studies have demonstrated that AEC5 rapidly reduces fungal burden, killing all viable fungi within 3 hours. Additionally, AEC5 has an in vivo half-life of 20+ hours and no observable in vivo toxicity following 28 days of daily injections. This research represents an important step in the characterization of AEC5 as a practical treatment option against C. neoformans infections.