A novel peptidomimetic therapeutic for selective suppression of lung cancer stem cells over non-stem cancer cells.

Cancers are highly heterogeneous and typically contain a small subset of drug-resisting cells called tumor initiating cells or cancer stem cells (CSCs). CSCs can self-renew, divide asymmetrically, and often cause tumor invasion and metastasis. Therefore, treatments specifically targeting CSCs are critical to improve patient survival. Recently, we identified a highly specific peptidomimetic (peptoid - PCS2) that selectively binds to the CSC subpopulation of lung cancer over the remaining cancer cells (non-CSCs). Subsequently, we identified plectin as the target of PCS2. Plectin is an intracellular structural protein, which is involved in tumor invasion and metastasis when it appears on cell surface. While PCS2 monomer did not display any anti-cancer activity, we designed a series of homo-dimeric versions of PCS2, and identified PCS2D1.2 optimized homo-dimer that displayed highly specific cytotoxicity towards CSCs over non-CSCs. PCS2D1.2 effectively blocked the in vitro colony formation and cell migration, hallmarks of CSCs. Furthermore, PCS2D1.2 reduced the in vivo tumor formation. In both in vitro and in vivo studies, PCS2D1.2 effectively reduced plectin expression and/or plectin-rich CSCs, but had no effect on non-CSCs. Therefore, PCS2D1.2 has the potential to be developed as a highly CSC specific drug candidate, which can be used in combination with current anti-cancer drugs.

"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.

Design of a dual ERK5 kinase activation and autophosphorylation inhibitor to block cancer stem cell activity.

The importance of ERK5 kinase signaling in tumorigenicity, metastasis, and drug resistance of cancer stem cells (CSCs) has been recognized recently, and we report a unique dual inhibitor that blocks binding of the ERK5 activator and ERK5 autophosphorylation simultaneously. The conventional ATP-binding site inhibitors have not yet yielded expected level of anti-cancer effects, due to complexities in converting ERK5 activation into CSC biological effects. We designed the first ERK5-targeted anti-CSC dual active hetero-bivalent inhibitor that blocks the regulatory peptide interaction involved in ERK5 kinase activation and that simultaneously inhibits the conventional ATP-binding pocket as well. We utilized two assay systems to independently prove disruption of these two ERK5 activities via a single compound. We also showed that this compound inhibited CSC activities, such as colony formation, cell proliferation, and migration.

Identification of side arm-modified DOTA scaffolds as multi-site binding ligands for cancer cells over normal cells.

The metal-chelated 1,4,7,10-tetraazacyclododecan-1,4,7,10-tetraacetic acid-tetraamide (DOTA) scaffold has been widely used as a contrast agent for diagnostic purposes in positron emission tomography (PET) and magnetic resonance imaging (MRI), but not as a biomarker targetable ligand. While the oxygen atoms at the stem of the four arms of the DOTA scaffold are needed for metal chelation, we previously introduced various physiochemical properties to extend these arms in a chemical library fashion to enhance the imaging contrast mechanism. We developed two such on-bead libraries, with 80 and 76 DOTA derivatives, where one arm was used to attach the DOTA scaffold onto resin beads and the other three arms were chemically modified. We now hypothesized that the chemical moieties used to modify these three arms can also recognize biomarkers on a cell surface. Therefore in this current study, we used such 76 derivatives of DOTA library to screen against HeLa cervical cancer cells. We found that two of the four 'hits' identified displayed higher binding towards HeLa cells than the unmodified parent DOTA. Furthermore, one of those 'hits' displayed better binding towards cervical and prostate cancer cells than lung and breast cancer cells and normal HBEC-3KT and RWPE1 cells. This indicates that this derivative can recognize a biomarker specific for certain types of cancer cells. If the compound has intrinsic activity, this can be used as a theranostic agent for real time therapy monitoring applications in the future. We believe that our DOTA derivative-based library approach can be applied to other types of cell and protein screens on various disease types in the future.

Converting a weaker ATP-binding site inhibitor into a potent hetero-bivalent ligand by tethering to a unique peptide sequence derived from the same kinase.

Attaching an additional binding site directed moiety or a ligand to an ATP-binding site inhibitor has been used as a strategy to increase kinase binding affinity and specificity. The moieties typically used here as the second binding partner are varied from simple organic groups to ligands such as peptides derived from substrate binding site sequences. So far these hetero-bivalent ligands were developed targeting additional binding sites closer to the ATP-binding pocket. Here we report a unique expansion of this hetero-bivalent idea by: (I) targeting a new binding site much farther away from ATP-binding site, (II) using a peptide uniquely derived from a portion of the same kinase sequence that has been reported to turn and bind to the above distance binding pocket (used as the second binding ligand), and (III) optimizing a much longer and flexible linker (to connect ATP-binding site inhibitor and above mentioned second peptide sequence) with multistep, yet complete on-bead synthesis approach. We converted a very weak EphA3-kinase ATP-binding site inhibitor-PP2 into a potent hetero-bivalent ligand by tethering to a unique 5-mer peptide sequence that derived from the linker region of EphA3 that connects kinase and sterile alpha motif (SAM) domains. Our design highlight the use of distance binding pockets to ATP-binding site as the second targeted site, while introducing the idea of extracting natural peptide sequences that already exist within the same kinase sequence, by a careful screening of available crystal structures.

A comprehensive lipid binding and activity validation of a cancer-specific peptide-peptoid hybrid PPS1.

We recently identified a peptide-peptoid hybrid, PPS1, which recognizes lipids that have an overall negative charge, such as phosphatidylserine (PS), phosphatidylglycerol (PG), phosphatidic acid (PA), and phosphatidylinositol (PI), but that does not bind to neutral lipids, such as phosphatidylcholine (PC), phosphatidylethanolamine (PE), and sphingomyelin (SM). The simple dimeric version of PPS1, PPS1D1, displayed strong cytotoxicity to cancer cells over normal cells in vitro and tumor burden in vivo. In this study, we comprehensively characterized the direct binding and activity of PPS1 on PS, PG, and PA using liposome-based assays and lung cancer cell lines that express these negatively charged lipids. First, the fluorescence polarization (FP) binding studies of fluoresceinated-PPS1 (PPS1-FITC) to PS-, PG-, and PA-containing PC-liposomes showed that the binding of PPS1 to PC-liposomes increased as concentrations of these lipids increased. In terms of activity, PPS1D1 induced the release of calcein from large, unilamellar PC-liposomes containing 15-30% PS, PG, and PA. PPS1D1 had no activity when the liposomes were composed of 100% PC. This effect was higher at 30% lipids than 15%, and the EC50 for PG and PA were higher than that of PS, indicating that PPS1D1 is more specific towards PS. PPS1D1 binds to and induces significant cytotoxicity in lung cancer cell lines H1693, HCC95, and H1395, which express negatively charged lipids, but had no effect on normal HBEC30KT cells, which has mostly PC in the outer layer. In addition, a series of previously developed PPS1D1 derivatives, which retain or lose activity, were tested with these liposome-based assays, and the data were equivalent to previous observations. This study provides comprehensive binding and activity validations of a unique peptide-peptoid hybrid, PPS1, on negatively charged lipids PS, PA, and PG that are elevated on cancer cell surfaces relative to normal human cell surfaces.

On-bead combinatorial synthesis and imaging of europium(III)-based paraCEST agents aids in identification of chemical features that enhance CEST sensitivity.

The rate of water exchange between the inner sphere of a paramagnetic ion and bulk water is an important parameter in determining the magnitude of the chemical exchange saturation transfer signal from paramagnetic CEST agents (paraCEST). This is governed by various geometric, steric and ligand field factors created by macrocyclic ligands surrounding the paramagnetic metal ion. Our previous on-bead combinatorial studies of di-peptoid-europium(III)-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA)-tetraamide complexes revealed that negatively charged groups in the immediate vicinity of the metal center strongly enhances the CEST signal. Here, we report a solid phase synthesis and on-bead imaging of 76 new DOTA derivatives that are developed by coupling with a single residue onto each of the three arms of a DOTA-tetraamide scaffold attached to resin beads. This single residue predominantly carries negatively charged groups blended with various physico-chemical characteristics. We found that non-bulky negatively charged groups are best suited at the immediate vicinity of the metal ion, while positive, bulky and halogen containing moieties suppress the CEST signal. Copyright © 2017 John Wiley & Sons, Ltd.

Identification of the minimum pharmacophore of lipid-phosphatidylserine (PS) binding peptide-peptoid hybrid PPS1D1.

We previously reported a unique peptide-peptoid hybrid, PPS1 that specifically recognizes lipid-phosphatidylserine (PS) and a few other negatively charged phospholipids, but not neutral phospholipids, on the cell membrane. The dimeric version of PPS1, i.e., PPS1D1 triggers strong cancer cell cytotoxicity and has been validated in lung cancer models both in vitro and in vivo. Given that PS and other negatively charged phospholipids are abundant in almost all tumor microenvironments, PPS1D1 is an attractive drug lead that can be developed into a globally applicable anti-cancer agent. Therefore, it is extremely important to identify the minimum pharmacophore of PPS1D1. In this study, we have synthesized alanine/sarcosine derivatives as well as truncated derivatives of PPS1D1. We performed ELISA-like competitive binding assay to evaluate the PS-recognition potential and standard MTS cell viability assay on HCC4017 lung cancer cells to validate the cell cytotoxicity effects of these derivatives. Our studies indicate that positively charged residues at the second and third positions, as well as four hydrophobic residues at the fifth through eighth positions, are imperative for the binding and activity of PPS1D1. Methionine at the first position was not essential, whereas the positively charged Nlys at the fourth position was minimally needed, as two derivatives that were synthesized replacing this residue were almost as active as PPS1D1.

Potent and selective photo-inactivation of proteins with peptoid-ruthenium conjugates.

Advances in high-throughput screening now enable the rapid discovery of bioactive small molecules, but these primary hits almost always exhibit modest potency. We report a strategy for the transformation of these hits into much more potent inhibitors without compound optimization. Appending a derivative of Ru(II)(tris-bipyridyl)(2+), an efficient photosensitizer of singlet oxygen production, to synthetic protein-binding compounds results in highly potent and specific target protein inactivation upon irradiation with visible light.

A peptoid interleukin-15 receptor antagonist suppresses inflammation and arthritis in mice.

Objective: To discover a novel peptoid antagonist that targets the interleukin-15 (IL-15) receptor and to evaluate its therapeutic efficacy in the treatment of inflammation and arthritis. Methods: A new compound (IFRA3, interleukin-15 receptor antagonist 3) was discovered using a unique on-bead two-colour combinatorial cell screening of a peptoid library. The interaction of IFRA3 with IL-15 receptor was assessed by in vitro pull-down and thermal shift assays. The efficacy of IFRA3 in treating inflammation and arthritis was evaluated in mouse models. Results: IFRA3Q1 (a tetrameric derivative of IFRA3) inhibited the activity of IL-15 and suppressed CTLL-2 cell proliferation (which depends on IL-15 activity). IFRA3Q1 exhibited strong in vivo anti-inflammatory activity in carrageenan-induced inflammation in mice. Furthermore, IFRA3Q1 inhibited collagen-induced arthritis in DBA/1J mice. Conclusion: By binding to and inhibiting the function of IL-15 receptor, IFRA3Q1 exhibited significant anti-arthritis activity. Our findings suggest that IFRA3Q1 represents a new paradigm for arthritis therapy by targeting IL-15 signalling.

On-bead combinatorial synthesis and imaging of chemical exchange saturation transfer magnetic resonance imaging agents to identify factors that influence water exchange.

The sensitivity of magnetic resonance imaging (MRI) contrast agents is highly dependent on the rate of water exchange between the inner sphere of a paramagnetic ion and bulk water. Normally, identifying a paramagnetic complex that has optimal water exchange kinetics is done by synthesizing and testing one compound at a time. We report here a rapid, economical on-bead combinatorial synthesis of a library of imaging agents. Eighty different 1,4,7,10-tetraazacyclododecan-1,4,7,10-tetraacetic acid (DOTA)-tetraamide peptoid derivatives were prepared on beads using a variety of charged, uncharged but polar, hydrophobic, and variably sized primary amines. A single chemical exchange saturation transfer image of the on-bead library easily distinguished those compounds having the most favorable water exchange kinetics. This combinatorial approach will allow rapid screening of libraries of imaging agents to identify the chemical characteristics of a ligand that yield the most sensitive imaging agents. This technique could be automated and readily adapted to other types of MRI or magnetic resonance/positron emission tomography agents as well.

MRI detection of VEGFR2 in vivo using a low molecular weight peptoid-(Gd)8-dendron for targeting.

The synthesis of a polylysine dendron containing eight GdDOTA units conjugated to a peptoid dimer known to have a high affinity for the vascular endothelial growth factor receptor 2 (VEGFR2) is described. This simple low molecular weight system with a molecular r(1) relaxivity of ∼48 mM(-1) s(-1) is shown to enhance MR images of tumors grown in mice in vivo.

GU81, a VEGFR2 antagonist peptoid, enhances the anti-tumor activity of doxorubicin in the murine MMTV-PyMT transgenic model of breast cancer.

BACKGROUND: Vascular endothelial growth factor (VEGF) is a primary stimulant of angiogenesis under physiological and pathological conditions. Anti-VEGF therapy is a clinically proven strategy for the treatment of a variety of cancers including colon, breast, lung, and renal cell carcinoma. Since VEGFR2 is the dominant angiogenic signaling receptor, it has become an important target in the development of novel anti-angiogenic therapies. We have reported previously the development of an antagonistic VEGFR2 peptoid (GU40C4) that has promising anti-angiogenic activity in vitro and in vivo. METHODS: In the current study, we utilize a derivative of GU40C4, termed GU81 in therapy studies. GU81 was tested alone or in combination with doxorubicin for in vivo efficacy in the MMTV-PyMT transgenic model of breast cancer. RESULTS: The derivative GU81 has increased in vitro efficacy compared to GU40C4. Single agent therapy (doxorubicin or GU81 alone) had no effect on tumor weight, histology, tumor fat content, or tumor growth index. However, GU81 is able to significantly to reduce total vascular area as a single agent. GU81 used in combination with doxorubicin significantly reduced tumor weight and growth index compared to all other treatment groups. Furthermore, treatment with combination therapy significantly arrested tumor progression at the premalignant stage, resulting in increased tumor fat content. Interestingly, treatment with GU81 alone increased tumor-VEGF levels and macrophage infiltration, an effect that was abrogated when used in combination with doxorubicin. CONCLUSION: This study demonstrates the VEGFR2 antagonist peptoid, GU81, enhances the anti-tumor activity of doxorubicin in spontaneous murine MMTV-PyMT breast tumors.

A peptoid "antibody surrogate" that antagonizes VEGF receptor 2 activity.

We report a two-color, cell-based screen to identify specific receptor-binding compounds in a combinatorial library of peptoids displayed on beads. We apply this strategy to the isolation of vascular endothelial growth factor receptor 2 (VEGFR2)-binding peptoids. A dimeric derivative of one of these lead compounds is shown to be an antagonist of VEGFR2 activity both in vitro and in vivo. This methodology provides a potentially general route to synthetic molecules that bind integral membrane receptors with affinities and specificities similar to those of antibodies, but which are far smaller and easier to make and manipulate.

The pharmacophore of a peptoid VEGF receptor 2 antagonist includes both side chain and main chain residues.

Here we identify the pharmacophore in a peptoid that antagonizes Vascular Endothelial Growth Factor Receptor-2 (VEGFR2) in vitro and in vivo. Only three of the side chains in the peptoid are required for activity. Surprisingly, however, main chain atoms also form critical interactions with the receptor.

A peptoid antagonist of VEGF receptor 2 recognizes a 'hotspot' in the extracellular domain distinct from the hormone-binding site.

Antagonists of VEGF-mediated angiogenesis are of great interest clinically for the treatment of solid tumors and certain forms of macular degeneration. We recently described a novel peptoid antagonist of VEGF Receptor 2 (VEGFR2) that binds to the extracellular domain of the receptor and inhibits VEGF-mediated autophosphorylation and subsequent downstream signaling. Given the structural similarities between peptides and peptoids, an obvious model for the mode of action of the peptoid is that it competes with VEGF for binding to VEGFR2. However, we present evidence here that this is not the case and that VEGF and the peptoid antagonist recognize non-overlapping surfaces located within the first three immunoglobulin-like subdomains of the receptor. These data argue that the peptoid inhibits receptor-mediated autophosphorylation by a novel allosteric mechanism that may prevent the receptor from acquiring the conformation necessary to propagate downstream signals.

A mini-library system to investigate non-essential residues of lipid-phosphatidylserine (PS) binding peptide-peptoid hybrid PPS1.

We recently identified a peptide-peptoid hybrid, PPS1, which specifically recognized lipid-phosphatidylserine (PS). PPS1 consists of distinct positively charged and hydrophobic residue-containing regions. PPS1 monomer was inactive, but the dimeric form, PPS1D1, displayed strong cytotoxicity for lung cancer cells compared to normal cells in vitro, and reduced the tumor growth in vivo. The minimum pharmacophore of PPS1D1 showed that the first (methionine) and fourth (N-lysine) residues were not important for PPS1D1 cytotoxic activity. In this study, we further investigated these two residues, in particular the fourth residue that lies between the most important four residue hydrophobic region and two positively charged residues, to determine whether replacements of these moieties could gain activity improvements, or render PPS1D1 totally insensitive for binding recognition. The positively charged fourth residue N-lysine was replaced with the substituents having varied physiochemical properties, such as aromatic-hydrophobic, aliphatic-alicyclic, heterocyclic, and negatively charged residues, developing a mini-library of 39 derivatives. The standard 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) colorimetric and/or the calcein AM cell viability assays performed on HCC4017 lung cancer cells indicated that the fourth position of PPS1D1 was insensitive to most changes, except reversal to the negative charge significantly affected the activity. This observation may be due to the neutralization of the nearby positively charged residue that is essential for binding. In addition, shortening each monomeric sequence by eliminating the methionine at the first position did not affect the activity.

A unique mid-sequence linker used to multimerize the lipid-phosphatidylserine (PS) binding peptide-peptoid hybrid PPS1.

Ligand multimerizations enhance the binding affinity towards cell surface biomarkers through their avidity effects. Typical linkers connect individual monomeric ligand moieties from one end (e.g., C- or N-terminus of a peptide) and exclusively target protein receptors. The lipid phosphatidylserine (PS) is normally present on the cytoplasmic side of the eukaryotic cell membrane, but in tumors and tumor endothelial cells, this negatively charged PS flips to the outer layer. We recently reported a PS binding peptide-peptoid hybrid (PPS1) that has distinct positively charged and hydrophobic residue-containing regions. The PPS1 monomer is inactive, and upon C-terminal dimerization (PPS1D1), it triggers cytotoxicity. In the current study, a unique series of PPS1 multimeric derivatives were synthesized by switching the linker from the C-terminus to an internal position. The unimportant fourth residue (N-lys) from the C-terminus was utilized to build the linker. The synthesis strategy was developed employing variations of (I) the linker size, (II) the number of positively charged residues, and (III) the number of hydrophobic regions. Cytotoxicity of these new derivatives on HCC4017 lung cancer cells showed that a minimum of two hydrophobic regions was important to retain the activity and that the shortest linker length was optimal for activity.

Unbiased Selection of Peptide-Peptoid Hybrids Specific for Lung Cancer Compared to Normal Lung Epithelial Cells.

To develop widely applicable diagnostic and potentially therapeutic approaches overcoming protein heterogeneity in human cancer, we have developed a technology to unbiasedly select high specificity compound(s) that bind any biomolecule (e.g., proteins, lipids, carbohydrates) presented on the cancer cell surface but not on normal cells. We utilized a peptidomimetic based on-bead two-color (OBTC) combinatorial cell screen that can detect differences between two cell surfaces at high accuracy by looking for beads (where each bead in the library had one peptide-peptoid hybrid on the surface) that only bound cancer but not normal cells. We screened a library of 393 216 compounds targeting HCC4017 lung adenocarcinoma cells (labeled in red) in the presence of HBEC30KT normal bronchial epithelial cells (labeled in green) derived from the same tissue of the same patient. This screen identified a peptide-peptoid hybrid called PPS1 which displayed high specific binding for HCC4017 cancer cells over HBEC30KT cells. Specificity was validated through on-bead, ELISA-like and magnetic bead pulldown studies, while a scrambled version of PPS1 did not show any binding. Of interest, the simple dimeric version (PPS1D1) displayed cytotoxic activity on HCC4017 cells, but not on normal HBEC30KT cells. PPS1D1 also strongly accumulated in HCC4017 lung cancer xenografts in mice over control constructs. We conclude that such combinatorial screens using tumor and normal cells from the same patient have significant potential to develop new reagents for cancer biology, diagnosis, and potentially therapy.