Incorporating a ß-hairpin sequence motif to increase intracellular stability of a peptide-based PROTAC.

Proteolysis targeting chimeras (PROTACs) have emerged as a new class of therapeutics that utilize the ubiquitin-proteasome system (UPS) to facilitate proteasomal degradation of "undruggable" targets. Peptide-based PROTACs contain three essential components: a binding motif for the target protein, a short amino acid sequence recognized by an E3 ligase called a degron, and a cell penetrating peptide to facilitate uptake into intact cells. While peptide-based PROTACs have been shown to successfully degrade numerous targets, they have often been found to exhibit low cell permeability and high protease susceptibility. Prior work identified peptides containing a ß-hairpin sequence motif that function not only as protecting elements, but also as CPPs and degrons. The goal of this study was to investigate if a ß-hairpin sequence could replace commonly used unstructured peptides sequences as the degron and the CPP needed for PROTAC uptake and function. The degradation of the protein Tau was selected as a model system as several published works have identified a Tau binding element that could easily be conjugated to the ß-hairpin sequence. A series of time- and concentration-dependent studies confirmed that the ßhairpin sequence was an adequate alternative CPP and degron to facilitate the proteasomemediated degradation of Tau. Microscopy studies confirmed the time-dependent uptake of the PROTAC and a degradation assay confirmed that the ß-hairpin conjugated PROTAC had a greater lifetime in cells.

Characterization of PMI-5011 on the Regulation of Deubiquitinating Enzyme Activity in Multiple Myeloma Cell Extracts.

Deubiquitinating enzyme (DUB)-targeted therapeutics have shown promise in recent years as alternative cancer therapeutics, especially when coupled with proteasome-based inhibitors. While a majority of DUB-based therapeutics function by inhibiting DUB enzymes, studies show that positive regulation of these enzymes can stabilize levels of protein degradation. Unfortunately, there are currently no clinically available therapeutics for this purpose. The goal of this work was to understand the effect of a botanical extract from Artemisia dracunculus L called PMI-5011 on DUB activity in cancer cells. Through a series of kinetic analyses and mathematical modeling, it was found that PMI-5011 positively regulated DUB activity in two model multiple myeloma cells line (OPM2 and MM.1S). This suggests that PMI-5011 interacts with the active domains of DUBs to enhance their activity directly or indirectly, without apparently affecting cellular viability. Similar kinetic profiles of DUB activity were observed with three bioactive compounds in PMI-5011 (DMC-1, DMC-2, davidigenin). Interestingly, a differential cell line-independent trend was observed at higher concentrations which suggested variances in inherent gene expressions of UCHL1, UCHL5, USP7, USP15, USP14, and Rpn11 in OPM2 and MM.1S cell lines. These findings highlight the therapeutic potential of PMI-5011 and its selected bioactive compounds in cancer.

Direct measurement of deubiquitinating enzyme activity in intact cells using a protease-resistant, cell-permeable, peptide-based reporter.

Deubiquitinating enzymes (DUBs) regulate the removal of the polyubiquitin chain from proteins targeted for degradation. Current approaches to quantify DUB activity are limited to test tube-based assays that incorporate enzymes or cell lysates, but not intact cells. The goal of this work was to develop a novel peptide-based biosensor of DUB activity that is cell permeable, protease-resilient, fluorescent, and specific to DUBs. The biosensor consists of an N-terminal ß-hairpin motif that acts as both a 'protectide' to increase intracellular stability and a cell penetrating peptide (CPP) to facilitate the uptake into intact cells. The ß-hairpin was conjugated to a C-terminal substrate consisting of the last four amino acids in ubiquitin (LRGG) to facilitate DUB mediated cleavage of a C-terminal fluorophore (AFC). The kinetics of the peptide reporter were characterized in cell lysates by dose response and inhibition enzymology studies. Inhibition studies with an established DUB inhibitor (PR-619) confirmed the specificity of both reporters to DUBs. Fluorometry and fluorescent microscopy experiments followed by mathematical modeling established the capability of the biosensor to measure DUB activity in intact cells while maintaining cellular integrity. The novel reporter introduced here is compatible with high-throughput single cell analysis platforms such as FACS and droplet microfluidics facilitating direct quantification of DUB activity in single intact cells with direct application in point-of-care cancer diagnostics and drug discovery.

Design of novel lipidated peptidomimetic conjugates for targeting EGFR heterodimerization in HER2 + cancer.

The human epidermal growth factor receptor (EGFR) family is known to be involved in cell signaling pathways. The extracellular domain of EGFR consists of four domains, of which domain II and domain IV are known to be involved in the dimerization process. Overexpression of these receptors is known to play a significant role in heterodimerization of these receptors leading to the development of cancer. We have designed peptidomimetic molecules to inhibit the EGFR heterodimerization interaction that have shown antiproliferative activity and specificity for HER2-positive cancer cell lines. Among these, a peptidomimetic, compound 5, exhibited antiproliferative activity at low nanomolar concentrations in HER2-overexpressing cancer cell lines. To improve the stability of this peptidomimetic, we have designed and synthesized a novel conjugate of peptidomimetic compound 5 with a lipid, stearic acid. The antiproliferative activity of this conjugate was evaluated in HER2-positive cancer cell lines. Results suggested that the conjugate exhibited selective antiproliferative activity in HER2-overexpressing breast and lung cancer cell lines and was able to block HER2:HER3 heterodimerization. Also, the conjugate showed improved stability with a half-life of 5 h in human serum compared to the half-life of 2 h for parent compound 5. The binding affinity of the conjugate to HER2 protein was evaluated by SPR analysis, and the mode of binding of the lipid conjugate to domain IV of HER2 protein was demonstrated by docking analysis. Thus, this novel lipid conjugate can be used to target HER2-overexpressing cancers.

Design of cyclic and d-amino acids containing peptidomimetics for inhibition of protein-protein interactions of HER2-HER3.

HER2 receptors are surface proteins belonging to the epidermal growth factor family of receptors. Their numbers are elevated in breast, lung, and ovarian cancers. HER2-positive cancers are aggressive, have higher mortality rate, and have a poor prognosis. We have designed peptidomimetics that bind to HER2 and block the HER2-mediated dimerization of epidermal growth factor family of receptors. Among these, a symmetrical cyclic peptidomimetic (compound 18) exhibited antiproliferative activity in HER2-overexpressing lung cancer cell lines with IC50 values in the nanomolar concentration range. To improve the stability of the peptidomimetic, d-amino acids were introduced into the peptidomimetic, and several analogs of compound 18 were designed. Among the analogs of compound 18, compound 32, a cyclic, d-amino acid-containing peptidomimetic, was found to have an IC50 value in the nanomolar range in HER2-overexpressing cancer cell lines. The antiproliferative activity of compound 32 was also measured by using a 3D cell culture model that mimics the in vivo conditions. The binding of compound 32 to the HER2 protein was studied by surface plasmon resonance. In vitro stability studies indicated that compound 32 was stable in serum for 48 hours and intact peptide was detectable in vivo for 12 hours. Results from our studies indicated that 1 of the d-amino acid analogs of 18, compound 32, binds to the HER2 extracellular domain, inhibiting the phosphorylation of kinase of HER2.

Structure-activity relationships of peptidomimetics that inhibit PPI of HER2-HER3.

Human epidermal growth factor receptor-2 (HER2) is a tyrosine kinase family protein receptor that is known to undergo heterodimerization with other members of the family of epidermal growth factor receptors (EGFR) for cell signaling. Overexpression of HER2 and deregulation of signaling has implications in breast, ovarian, and lung cancers. We have designed several peptidomimetics to block the HER2-mediated dimerization, resulting in antiproliferative activity for cancer cells. In this work, we have investigated the structure-activity relationships of peptidomimetic analogs of Compound 5. Compound 5 was conformationally constrained by N- and C-terminal modification and cyclization as well as by substitution with d-amino acids at the N-and C-termini. Among the compounds studied in this work, a peptidomimetic Compound 21 with d-amino acid substitution and its N- and C-termini capped with acetyl and amide functional groups and a reversed sequence compared to that of Compound 5 exhibited better antiproliferative activity in HER2-overexpressed breast, ovarian, and lung cancer cell lines. Compound 21 was further evaluated for its protein-protein interaction (PPI) inhibition ability using enzyme fragment complementation assay, proximity ligation assay, and Western blot analysis. Results suggested that Compound 21 is able to block HER2:HER3 interaction and inhibit phosphorylation of the kinase domain of HER2. The mode of binding of Compound 21 to HER2 protein was modeled using a docking method. Compound 21 seems to bind to domain IV of HER2 near the PPI site of EGFR:HER2, and HER:HER3 and inhibit PPI.

Monitoring a nuclear factor-κB signature of drug resistance in multiple myeloma.

The emergence of acquired drug resistance results from multiple compensatory mechanisms acting to prevent cell death. Simultaneous monitoring of proteins involved in drug resistance is a major challenge for both elucidation of the underlying biology and development of candidate biomarkers for assessment of personalized cancer therapy. Here, we have utilized an integrated analytical platform based on SDS-PAGE protein fractionation prior to liquid chromatography coupled to multiple reaction monitoring mass spectrometry, a versatile and powerful tool for targeted quantification of proteins in complex matrices, to evaluate a well-characterized model system of melphalan resistance in multiple myeloma (MM). Quantitative assays were developed to measure protein expression related to signaling events and biological processes relevant to melphalan resistance in multiple myeloma, specifically: nuclear factor-κB subunits, members of the Bcl-2 family of apoptosis-regulating proteins, and Fanconi Anemia DNA repair components. SDS-PAGE protein fractionation prior to liquid chromatography coupled to multiple reaction monitoring methods were developed for quantification of these selected target proteins in amounts of material compatible with direct translation to clinical specimens (i.e. less than 50,000 cells). As proof of principle, both relative and absolute quantification were performed on cell line models of MM to compare protein expression before and after drug treatment in naïve cells and in drug resistant cells; these liquid chromatography-multiple reaction monitoring results are compared with existing literature and Western blots. The initial stage of a systems biology platform for examining drug resistance in MM has been implemented in cell line models and has been translated to MM cells isolated from a patient. The ultimate application of this platform could assist in clinical decision-making for individualized patient treatment. Although these specific assays have been developed to monitor MM, these techniques are expected to have broad applicability in cancer and other types of disease.

Conformationally constrained cyclic grafted peptidomimetics targeting protein-protein interactions.

Sunflower trypsin inhibitor-1 (SFTI-1) structure is used for designing grafted peptides as a possible therapeutic agent. The grafted peptide exhibits multiple conformations in solution due to the presence of proline in the structure of the peptide. To lock the grafted peptide into a major conformation in solution, a dibenzofuran moiety (DBF) was incorporated in the peptide backbone structure, replacing the Pro-Pro sequence. NMR studies indicated a major conformation of the grafted peptide in solution. Detailed structural studies suggested that SFTI-DBF adopts a twisted beta-strand structure in solution. The surface plasmon resonance analysis showed that SFTI-DBF binds to CD58 protein. A model for the protein-SFTI-DBF complex was proposed based on docking studies. These studies suggested that SFTI-1 grafted peptide can be used to design stable peptides for therapeutic purposes by grafting organic functional groups and amino acids. However, when a similar strategy was used with another grafted peptide, the resulting peptide did not produce a single major conformation, and its biological activity was lost. Thus, conformational constraints depend on the sequence of amino acids used for SFTI-1 grafting.

How Cargo Identity Alters the Uptake of Cell-Penetrating Peptide (CPP)/Cargo Complexes: A Study on the Effect of Net Cargo Charge and Length.

Cell-penetrating peptides (CPPs) have emerged as a powerful tool for the delivery of otherwise impermeable cargoes into intact cells. Recent efforts to improve the delivery capability of peptides have mainly focused on the identity of the CPP; however, there is evidence that the identity of the cargo itself affects the uptake. The goal of this work was to investigate how the characteristics of a peptide cargo, including net charge and length, either enhance or diminish the internalization efficiency of the CPP/cargo complex. A small library of CPP/cargo complexes were synthesized consisting of structured and unstructured CPPs with cargoes of net positive, negative, or neutral charge and lengths of 4 or 8 amino acids. Cargoes with a net positive charge were found to enhance the overall uptake of the complexes while net neutral and negatively charged cargoes diminished uptake. Conversely, the net length of the cargo had no significant effect on uptake of the CPP/cargo complexes. Microcopy images confirmed the increased uptake of the positively charged cargoes; however, an increase in punctate regions with the addition of a cargo was also observed. The effects of the net positively charged cargoes were confirmed with both structured and unstructured CPPs, which demonstrated similar trends of an increase in uptake with the addition of positively charged residues. These findings demonstrate that the net charge of cargoes impacts the uptake of the complex, which can be considered in the future when designing peptide-based reporters or therapeutics.

Modulation of co-stimulatory signal from CD2-CD58 proteins by a grafted peptide.

Peptides were designed to inhibit the protein-protein interaction of CD2 and CD58 to modulate the immune response. This work involved the design and synthesis of eight different peptides by replacing each amino acid residue in peptide 6 with alanine as well as grafting the peptide to the sunflower trypsin-inhibitor framework. From the alanine scanning studies, mutation at position 2 of the peptide was shown to result in increased potency to inhibit cell adhesion interactions. The most potent peptide from the alanine scanning was further studied for its detailed three-dimensional structure and binding to CD58 protein using surface plasmon resonance and flow cytometry. This peptide was used to graft to the sunflower trypsin inhibitor to improve the stability of the peptide. The grafted peptide, SFTI-a1, was further studied for its potency as well as its thermal, chemical, and enzymatic stability. The grafted peptide exhibited improved activity compared to our previously grafted peptide and was stable against thermal and enzymatic degradation.

Lipidated Peptidomimetic Ligand-Functionalized HER2 Targeted Liposome as Nano-Carrier Designed for Doxorubicin Delivery in Cancer Therapy.

The therapeutic index of chemotherapeutic agents can be improved by the use of nano-carrier-mediated chemotherapeutic delivery. Ligand-targeted drug delivery can be used to achieve selective and specific delivery of chemotherapeutic agents to cancer cells. In this study, we prepared a peptidomimetic conjugate (SA-5)-tagged doxorubicin (Dox) incorporated liposome (LP) formulation (SA-5-Dox-LP) to evaluate the targeted delivery potential of SA-5 in human epidermal growth factor receptor-2 (HER2) overexpressed non-small-cell lung cancer (NSCLC) and breast cancer cell lines. The liposome was prepared using thin lipid film hydration and was characterized for particle size, encapsulation efficiency, cell viability, and targeted cellular uptake. In vivo evaluation of the liposomal formulation was performed in a mice model of NSCLC. The cell viability studies revealed that targeted SA-5-Dox-LP showed better antiproliferative activity than non-targeted Dox liposomes (Dox-LP). HER2-targeted liposome delivery showed selective cellular uptake compared to non-targeted liposomes on cancer cells. In vitro drug release studies indicated that Dox was released slowly from the formulations over 24 h, and there was no difference in Dox release between Dox-LP formulation and SA-5-Dox-LP formulation. In vivo studies in an NSCLC model of mice indicated that SA-5-Dox-LP could reduce the lung tumors significantly compared to vehicle control and Dox. In conclusion, this study demonstrated that the SA-5-Dox-LP liposome has the potential to increase therapeutic efficiency and targeted delivery of Dox in HER2 overexpressing cancer.

Bioactivity improvement via display of the hydrophobic core of HYD1 in a cyclic ß-hairpin-like scaffold, MTI-101.

HYD1 is an all D-amino acid linear 10-mer peptide that was discovered by one-bead-one-compound screening. HYD1 has five hydrophobic amino acids flanked by polar amino acids. Alanine scanning studies showed that alternating hydrophobic amino acid residues and N- and C-terminal lysine side chains were contributors to the biological activity of the linear 10-mer analogs. This observation led us to hypothesize that display of the hydrophobic pentapeptide sequence of HYD1 in a cyclic beta-hairpin-like scaffold could lead to better bioavailability and biological activity. An amphipathic pentapeptide sequence was used to form an antiparallel strand and those strands were linked via dipeptide-like sequences selected to promote ß-turns. Early cyclic analogs were more active but otherwise mimicked the biological activity of the linear HYD1 peptide. The cyclic peptidomimetics were synthesized using standard Fmoc solid phase synthesis to form linear peptides, followed by solution phase or on-resin cyclization. SAR studies were carried out with an aim to increase the potency of these drug candidates for the killing of multiple myeloma cells in vitro. The solution structures of 1, 5, and 10 were elucidated using NMR spectroscopy. 1H NMR and 2D TOCSY studies of these peptides revealed a downfield Hα proton chemical shift and 2D NOE spectral analysis consistent with a ß-hairpin-like structure.

CPProtectides: Rapid uptake of well-folded ß-hairpin peptides with enhanced resistance to intracellular degradation.

Cell penetrating peptides (CPPs) have emerged as powerful tools for delivering bioactive cargoes, such as biosensors or drugs to intact cells. One limitation of CPPs is their rapid degradation by intracellular proteases. ß-hairpin "protectides" have previously been demonstrated to be long-lived under cytosolic conditions due to their secondary structure. The goal of this work was to demonstrate that arginine-rich ß-hairpin peptides function as both protectides and as CPPs. Peptides exhibiting a ß-hairpin motif were found to be rapidly internalized into cells with their uptake efficiency dependent on the number of arginine residues in the sequence. Cellular internalization of the ß-hairpin peptides was compared to unstructured, scrambled sequences and to commercially available, arginine-rich CPPs. The unstructured peptides displayed greater uptake kinetics compared to the structured ß-hairpin sequences; however, intracellular stability studies revealed that the ß-hairpin peptides exhibited superior stability under cytosolic conditions with a 16-fold increase in peptide half-life. This study identifies a new class of long-lived CPPs that can overcome the stability limitations of peptide-based reporters or bioactive delivery mechanisms in intact cells.

Design of a doxorubicin-peptidomimetic conjugate that targets HER2-positive cancer cells.

Doxorubicin (DOX) belongs to the anthracycline class of drugs that are used in the treatment of various cancers. It has limited cystostatic effects in therapeutic doses, but higher doses can cause cardiotoxicity. In the current approach, we conjugated a peptidomimetic (Arg-aminonaphthylpropionic acid-Phe, compound 5) known to bind to HER2 protein to DOX via a glutaric anhydride linker. Antiproliferative assays suggest that the DOX-peptidomimetic conjugate has activity in the lower micromolar range. The conjugate exhibited higher toxicity in HER2-overexpressed cells than in MCF-7 and MCF-10A cells that do not overexpress HER2 protein. Cellular uptake studies using confocal microscope experiments showed that the conjugate binds to HER2-overexpressed cells and DOX is taken up into the cells in 4 h compared to conjugate in MCF-7 cells. Binding studies using surface plasmon resonance indicated that the conjugate binds to the HER2 extracellular domain with high affinity compared to compound 5 or DOX alone. The conjugate was stable in the presence of cells with a half-life of nearly 4 h and 1 h in human serum. DOX is released from the conjugate and internalized into the cells in 4 h, causing cellular toxicity. These results suggest that this conjugate can be used to target DOX to HER2-overexpressing cells and can improve the therapeutic index of DOX for HER2-positive cancer.

Simultaneous delivery of antibiotics neomycin and ampicillin in drinking water inhibits fermentation of resistant starch in rats.

SCOPE: Antibiotics ampicillin 1 g/L and neomycin 0.5 g/L were added to drinking water before or during feeding of resistant starch (RS) to rats to inhibit fermentation. METHODS AND RESULTS: In a preliminary study, antibiotics and no RS were given prior to rats receiving a transplant of cecal contents via gavage from donor rats fed RS (without antibiotics) or a water gavage before feeding resistant starch to both groups. Antibiotics given prior to feeding RS did not prevent later fermentation of RS regardless of either type of gavage. In the second study, antibiotics were given simultaneously with feeding of RS. This resulted in inhibition of fermentation of RS with cecal contents pH >8 and low amounts of acetate and butyrate. Rats treated with antibiotics had reduced Bifidobacteria spp., but similar Bacteroides spp. to control groups to reduce acetate and butyrate and preserve the production of propionate. Despite reduced fermentation, rats given antibiotics had increased glucagon-like peptide 1 (GLP-1) and cecum size, measures that are usually associated with fermentation. CONCLUSIONS: A simultaneous delivery of antibiotics inhibited fermentation of RS. However, increased GLP-1 and cecum size would be confounding effects in assessing the mechanism for beneficial effects of dietary RS by knocking out fermentation.

Constrained Cyclic Peptides as Immunomodulatory Inhibitors of the CD2:CD58 Protein-Protein Interaction.

The interaction between the cell-cell adhesion proteins CD2 and CD58 plays a crucial role in lymphocyte recruitment to inflammatory sites, and inhibitors of this interaction have potential as immunomodulatory drugs in autoimmune diseases. Peptides from the CD2 adhesion domain were designed to inhibit CD2:CD58 interactions. To improve the stability of the peptides, ß-sheet epitopes from the CD2 region implicated in CD58 recognition were grafted into the cyclic peptide frameworks of sunflower trypsin inhibitor and rhesus theta defensin. The designed multicyclic peptides were evaluated for their ability to modulate cell-cell interactions in three different cell adhesion assays, with one candidate, SFTI-a, showing potent activity in the nanomolar range (IC50: 51 nM). This peptide also suppresses the immune responses in T cells obtained from mice that exhibit the autoimmune disease rheumatoid arthritis. SFTI-a was resistant to thermal denaturation, as judged by circular dichroism spectroscopy and mass spectrometry, and had a half-life of ∼24 h in human serum. Binding of this peptide to CD58 was predicted by molecular docking studies and experimentally confirmed by surface plasmon resonance experiments. Our results suggest that cyclic peptides from natural sources are promising scaffolds for modulating protein-protein interactions that are typically difficult to target with small-molecule compounds.

Asymmetric Co(II)-catalyzed cyclopropanation with succinimidyl diazoacetate: general synthesis of chiral cyclopropyl carboxamides.

[Co(P1)] is an effective catalyst for asymmetric cyclopropanation with succinimidyl diazoacetate. The Co(II)-catalyzed reaction is suitable for various olefins, providing the desired cyclopropane succinimidyl esters in high yields and excellent diastereo- and enantioselectivity. The resulting enantioenriched cyclopropane succinimidyl esters can serve as convenient synthons for the general synthesis of optically active cyclopropyl carboxamides.