Designing Fluorescent Nuclear Permeable Peptidomimetics to Target Proliferating Cell Nuclear Antigen.

Human proliferating cell nuclear antigen (PCNA) is a critical mediator of DNA replication and repair, acting as a docking platform for replication proteins. Disrupting these interactions with a peptidomimetic agent presents as a promising avenue to limit proliferation of cancerous cells. Here, a p21-derived peptide was employed as a starting scaffold to design a modular peptidomimetic that interacts with PCNA and is cellular and nuclear permeable. Ultimately, a peptidomimetic was produced which met these criteria, consisting of a fluorescein tag and SV40 nuclear localization signal conjugated to the N-terminus of a p21 macrocycle derivative. Attachment of the fluorescein tag was found to directly affect cellular uptake of the peptidomimetic, with fluorescein being requisite for nuclear permeability. This work provides an important step forward in the development of PCNA targeting peptidomimetics for use as anti-cancer agents or as cancer diagnostics.

PPARγ Corepression Involves Alternate Ligand Conformation and Inflation of H12 Ensembles.

Inverse agonists of peroxisome proliferator activated receptor γ (PPARγ) have emerged as safer alternatives to full agonists for their reduced side effects while still maintaining impressive insulin-sensitizing properties. To shed light on their molecular mechanism, we characterized the interaction of the PPARγ ligand binding domain with SR10221. X-ray crystallography revealed a novel binding mode of SR10221 in the presence of a transcriptionally repressing corepressor peptide, resulting in much greater destabilization of the activation helix, H12, than without corepressor peptide. Electron paramagnetic resonance provided in-solution complementary protein dynamic data, which revealed that for SR10221-bound PPARγ, H12 adopts a plethora of conformations in the presence of corepressor peptide. Together, this provides the first direct evidence for corepressor-driven ligand conformation for PPARγ and will allow the development of safer and more effective insulin sensitizers suitable for clinical use.

Unlocking the PIP-box: A peptide library reveals interactions that drive high-affinity binding to human PCNA.

The human sliding clamp, Proliferating Cell Nuclear Antigen (hPCNA), interacts with over 200 proteins through a conserved binding motif, the PIP-box, to orchestrate DNA replication and repair. It is not clear how changes to the features of a PIP-box modulate protein binding and thus how they fine-tune downstream processes. Here, we present a systematic study of each position within the PIP-box to reveal how hPCNA-interacting peptides bind with drastically varied affinities. We synthesized a series of 27 peptides derived from the native protein p21 with small PIP-box modifications and another series of 19 peptides containing PIP-box binding motifs from other proteins. The hPCNA-binding affinity of all peptides, characterized as KD values determined by surface plasmon resonance, spanned a 4000-fold range, from 1.83 nM to 7.59 µM. The hPCNA-bound peptide structures determined by X-ray crystallography and modeled computationally revealed intermolecular and intramolecular interaction networks that correlate with high hPCNA affinity. These data informed rational design of three new PIP-box sequences, testing of which revealed the highest affinity hPCNA-binding partner to date, with a KD value of 1.12 nM, from a peptide with PIP-box QTRITEYF. This work showcases the sequence-specific nuances within the PIP-box that are responsible for high-affinity hPCNA binding, which underpins our understanding of how nature tunes hPCNA affinity to regulate DNA replication and repair processes. In addition, these insights will be useful to future design of hPCNA inhibitors.

Ultra-low fouling photocrosslinked coatings for the selective capture of cells expressing CD44.

The effective control of biointerfacial interactions is of outstanding interest in a broad range of biomedical applications, ranging from cell culture tools to biosensors and implantable medical devices. For many of these applications, highly specific interactions between cells and material surfaces are desired. Sophisticated control over these interactions requires reducing or preventing non-specific interactions on the one hand and displaying highly specific signals that can be recognized by extracellular receptors on the other. We have recently developed ultra-low fouling coatings that can be applied in a single step using photoreactive copolymers of 2-hydroxypropyl acrylamide and N-benzophenone acrylamide. Here, we have expanded this approach by incorporating polymerizable peptide monomers into these copolymers. The monomers QQGWFGAGK(acrylamide) and acrylamide-GAGQQGWF were synthesized after identifying the QQGWF sequence as a binding motif for CD44 by phage display for the first time. Our results demonstrate that UV-crosslinked coatings fabricated using the QQGWFGAGK(acrylamide) monomer are effective at selectively binding hMSC in the presence of HepG2 and HEK293 cells due to the difference in CD44 expression. Our results also demonstrate that the peptide modified coatings retain their low biofouling character using a BCA protein binding assay as well as an E. coli bacterial attachment assay over a 24 h period. Our approach provides an alternative to traditional integrin-mediated selective cell binding on surfaces and opens the door to new diagnostic applications, exploiting the fact that the transmembrane protein CD44 is highly expressed in multiple diseases.

The binding of boronated peptides to low affinity mammalian saccharides.

A 54-member library of boronated octapeptides, with all but the boronated residue being proteinogenic, was tested for affinity to a set of saccharides commonly found on the terminus of mammalian glycans. After experimentation with a high-throughput dye-displacement assay, attention was focused on isothermal titration calorimetry as a tool to provide reliable affinity data, including enthalpy and entropy of binding. A small number of boronated peptides showed higher affinity and significant selectivity for N-acetylneuraminic acid over methyl-α-d-galactopyranoside, methyl-α/ß-l-fucopyranoside and N-acetyl-d-glucosamine. Thermodynamic data showed that for most of the boronated peptides studied, saccharide binding was associated with a significant increase in entropy, presumably resulting from the displacement of semiordered water molecules from around the sugar and/or peptide.

A thrombopoietin receptor antagonist is capable of depleting myelofibrosis hematopoietic stem and progenitor cells.

Recently, interactions between thrombopoietin (TPO) and its receptor, the myeloproliferative leukemia (MPL) virus oncogene, have been shown to play a role in the development and progression of myeloproliferative neoplasms including myelofibrosis (MF). These observations have led to the development of strategies to disrupt the association of TPO with its receptor as a means of targeting MF hematopoietic stem cells (HSCs) and hematopoietic progenitor cells (HPCs). In this report, we show that although both splenic and peripheral blood MF CD34(+) cells expressed lower levels of MPL than normal CD34(+) cells, TPO promoted the proliferation of MF CD34(+) cells and HPCs in a dose-dependent fashion. Furthermore, the treatment of MF but not normal CD34(+) cells with a synthesized MPL antagonist, LCP4, decreased the number of CD34(+)Lin(-) cells and all classes of assayable HPCs (colony-forming unit-megakaryocyte [CFU-MK], CFU-granulocyte/macrophage, burst-forming unit-erythroid/CFU-erythroid, and CFU-granulocyte/erythroid/macrophage/MK) irrespective of their mutational status. In addition, LCP4 treatment resulted in the depletion of the number of MF HPCs that were JAK2V617F(+) Moreover, the degree of human cell chimerism and the proportion of malignant donor cells were significantly reduced in immunodeficient mice transplanted with MF CD34(+) cell grafts treated with LCP4. These effects of LCP4 on MF HSCs/HPCs were associated with inhibition of JAK-STAT activity, leading to the induction of apoptosis. These findings demonstrate that such specific anti-cytokine receptor antagonists represent a new class of drugs that are capable of targeting MF HSCs.

Intracellular nucleic acid delivery by the supercharged dengue virus capsid protein.

Supercharged proteins are a recently identified class of proteins that have the ability to efficiently deliver functional macromolecules into mammalian cells. They were first developed as bioengineering products, but were later found in the human proteome. In this work, we show that this class of proteins with unusually high net positive charge is frequently found among viral structural proteins, more specifically among capsid proteins. In particular, the capsid proteins of viruses from the Flaviviridae family have all a very high net charge to molecular weight ratio (> +1.07/kDa), thus qualifying as supercharged proteins. This ubiquity raises the hypothesis that supercharged viral capsid proteins may have biological roles that arise from an intrinsic ability to penetrate cells. Dengue virus capsid protein was selected for a detailed experimental analysis. We showed that this protein is able to deliver functional nucleic acids into mammalian cells. The same result was obtained with two isolated domains of this protein, one of them being able to translocate lipid bilayers independently of endocytic routes. Nucleic acids such as siRNA and plasmids were delivered fully functional into cells. The results raise the possibility that the ability to penetrate cells is part of the native biological functions of some viral capsid proteins.

Reverse thioether ligation route to multimeric peptide antigens.

Multimeric presentation, a rather effective way of enhancing peptide immunogenicity, is best exemplified by MAP (multiple antigenic peptide) dendrimers consisting of a branched Lys core on which several copies of the peptide epitope are displayed. While accessible by solid-phase synthesis, MAPs can also be conveniently made in solution, e.g., by linking the epitope (N-acetylated and with a C-terminal Cys) through a thioether bond onto the α and ε (haloacetyl-activated) positions of the Lys core. We now report the reverse version of this approach, whereby a chloroacetyl-derivatised epitope is tethered to a thiol-functionalised form of a Lys dendron core. This convergent approach can be carried out either in solution or in the solid phase and is advantageous because (i) in situ tris(2-carboxyethyl)phosphine (TCEP)-mediated reduction of disulfide bonds maintains the thiol platform reactive throughout the ligation process; (ii) the low amounts of TCEP used pose minimal risk to chloroacetyl groups in the peptide, resulting in (iii) significantly reduced byproduct formation, hence cleaner products. For the solid phase version of the method, an optimised procedure has been devised to convert the Lys core into a tetrathiol dendron.

Chimeric infectious bursal disease virus-like particles as potent vaccines for eradication of established HPV-16 E7-dependent tumors.

Cervical cancer is caused by persistent high-risk human papillomavirus (HR-HPV) infection and represents the second most frequent gynecological malignancy in the world. The HPV-16 type accounts for up to 55% of all cervical cancers. The HPV-16 oncoproteins E6 and E7 are necessary for induction and maintenance of malignant transformation and represent tumor-specific antigens for targeted cytotoxic T lymphocyte-mediated immunotherapy. Therapeutic cancer vaccines have become a challenging area of oncology research in recent decades. Among current cancer immunotherapy strategies, virus-like particle (VLP)-based vaccines have emerged as a potent and safe approach. We generated a vaccine (VLP-E7) incorporating a long C-terminal fragment of HPV-16 E7 protein into the infectious bursal disease virus VLP and tested its therapeutic potential in HLA-A2 humanized transgenic mice grafted with TC1/A2 tumor cells. We performed a series of tumor challenge experiments demonstrating a strong immune response against already-formed tumors (complete eradication). Remarkably, therapeutic efficacy was obtained with a single dose without adjuvant and against two injections of tumor cells, indicating a potent and long-lasting immune response.

Selenomethionine incorporation into amyloid sequences regulates fibrillogenesis and toxicity.

BACKGROUND: The capacity of a polypeptide chain to engage in an amyloid formation process and cause a conformational disease is contained in its sequence. Some of the sequences undergoing fibrillation contain critical methionine (Met) residues which in vivo can be synthetically substituted by selenomethionine (SeM) and alter their properties. METHODOLOGY/PRINCIPAL FINDINGS: Using peptide synthesis, biophysical techniques and cell viability determinations we have studied the effect of the substitution of methionine (Met) by selenomethionine (SeM) on the fibrillogenesis and toxic properties of Aß40 and HuPrP(106-140). We have found that the effects display site-specificity and vary from inhibition of fibrillation and decreased toxicity ([SeM(35)]Aß40, [SeM(129)]HuPrP(106-140) and [SeM(134)]HuPrP(106-140)), retarded assembly, modulation of polymer shape and retention of toxicity ([SeM(112)]HuPrP(106-140) to absence of effects ([SeM(109)]HuPrP(106-140)). CONCLUSIONS/SIGNIFICANCE: This work provides direct evidence that the substitution of Met by SeM in proamyloid sequences has a major impact on their self-assembly and toxic properties, suggesting that the SeM pool can play a major role in dictating the allowance and efficiency of a polypeptide chain to undergo toxic polymerization.

Synthesis of multiple antigenic peptides (MAPs)-strategies and limitations.

Dendrimeric platforms such as MAPs can be synthesized either entirely by solid-phase methods (SPPS, direct approach) or by conjugation in solution of preformed, SPPS-made building blocks (indirect approach). Although MAPs and MAP-like constructs have been extensively and successfully used for various biological (mainly immunological) applications, experimental reports are most often lacking in chemical detail about their preparation and characterization. Here, we provide complete accounts of the synthesis and analytical documentation of MAPs and similar dendrimers by either all-SPPS (direct) or chemoselective thioether ligation (indirect) methods. We have chosen as model epitopes a 24-residue sequence of the ectodomain of protein M2 from influenza virus (M2e), which is found to be a rather challenging peptide epitope, and a far more manageable, shortened (12-residue) version of the same peptide. The advantages and shortcomings of both direct and indirect methods are discussed.

Escherichia coli cell surface perturbation and disruption induced by antimicrobial peptides BP100 and pepR.

The potential of antimicrobial peptides (AMPs) as an alternative to conventional therapies is well recognized. Insights into the biological and biophysical properties of AMPs are thus key to understanding their mode of action. In this study, the mechanisms adopted by two AMPs in disrupting the gram-negative Escherichia coli bacterial envelope were explored. BP100 is a short cecropin A-melittin hybrid peptide known to inhibit the growth of phytopathogenic gram-negative bacteria. pepR, on the other hand, is a novel AMP derived from the dengue virus capsid protein. Both BP100 and pepR were found to inhibit the growth of E. coli at micromolar concentrations. Zeta potential measurements of E. coli incubated with increasing peptide concentrations allowed for the establishment of a correlation between the minimal inhibitory concentration (MIC) of each AMP and membrane surface charge neutralization. While a neutralization-mediated killing mechanism adopted by either AMP is not necessarily implied, the hypothesis that surface neutralization occurs close to MIC values was confirmed. Atomic force microscopy (AFM) was then employed to visualize the structural effect of the interaction of each AMP with the E. coli cell envelope. At their MICs, BP100 and pepR progressively destroyed the bacterial envelope, with extensive damage already occurring 2 h after peptide addition to the bacteria. A similar effect was observed for each AMP in the concentration-dependent studies. At peptide concentrations below MIC values, only minor disruptions of the bacterial surface occurred.

Strategies and limitations in dendrimeric immunogen synthesis. The influenza virus M2e epitope as a case study.

Dendrimeric platforms such as multiple antigen peptides (MAPs) are regarded as one of the most efficacious approaches for antigenic presentation. Originally described as available by stepwise solid-phase peptide synthesis (SPPS), MAPs have also been prepared by chemical (thioether, oxime, hydrazone) ligation of appropriately functionalized tetra- or octavalent polylysine scaffolds with the peptide antigen to be multiply displayed. In this work, the advantages and limitations of two of the most frequent methods of MAP preparation, namely, chemoselective thioether ligation in solution, and all-solid-phase synthesis, have been tested in the case of a particularly troublesome epitope model, the ectodomain of protein M2 from influenza virus (M2e). The strong tendency of M2e to self-associate is a serious inconvenient for conjugation in solution, which as a result fails to produce the target MAPs with the specified number of M2e copies. In contrast, the fully stepwise SPPS approach is shown to be quite practical, especially when 6-aminohexanoic acid spacer units providing increased internal flexibility are inserted at each branching point.

Structure-activity relationship of CART (cocaine- and amphetamine-regulated transcript) peptide fragments.

CART (cocaine- and amphetamine-regulated transcript) peptides are neuropeptides abundant in the central nervous system and periphery found to be involved in the regulation of food intake behavior and other physiological processes. Recently, we reported specific binding of (125)I-CART(61-102) to the rat adrenal pheochromocytoma cell line PC12, both intact cells and cell membranes. In this study, several fragments of CART(61-102) corresponding to its structural loops were synthesized and tested for their potency in binding experiments using PC12 intact cells and cell membranes and in feeding test with fasted mice. From all shorter peptides tested, only CART(74-86) and CART(62-86) containing disulfide bridges kept partial binding potency of the original molecule with K(i) in 10(-5) and 10(-4)M range. However, these fragments were not able to inhibit food intake after their central administration up to a dose of 4 nmol/mouse. The results showed that a compact structure containing three disulfide bridges is necessary for preservation of full biological activity of CART peptides.

The effects of N-terminal part modification of arginine vasopressin analogues with 2-aminoindane-2-carboxylic acid: a highly potent V2 agonist.

In this study we present the synthesis and some pharmacological properties of nine new analogues of arginine vasopressin modified in the N-terminal part of the molecule with 2-aminoindane-2-carboxylic acid (Aic). The peptides were tested for their in vitro uterotonic and in vivo pressor and antidiuretic activities. One of the new peptides, [Mpa1,Aic2,Val4,D-Arg8]VP, exhibited an antidiuretic activity similar to that of [Mpa1,D-Arg8]VP, thus being one of the most potent antidiuretic vasopressin analogues reported to date.

New bradykinin analogues substituted in positions 7 and 8 with sterically restricted 1-aminocyclopentane-1-carboxylic acid.

A sterically constrained non-coded amino acid, 1-aminocyclopentane-1-carboxylic acid (Apc), was introduced in position 7 or 8 of the bradykinin (BK) B(2) receptor antagonist, [D-Arg(0), Hyp(3), Thi(5, 8), D-Phe(7)]BK, previously synthesized by Stewart's group. This modification is believed to reduce the flexibility of the peptides, thereby forcing the peptide backbone and side chains to adopt specific orientations. Apc substitution was combined with acylation of the N-terminus with 1-adamantaneacetic acid (Aaa). The activity of four new analogues was assayed in isolated rat uterus and in rat blood pressure tests. The results clearly demonstrated that the Apc residue inserted in position 7 led to a reduction of antagonistic properties in the rat uterus assay or even restored the agonism in the blood pressure test, whereas Apc at position 8 enhanced antagonistic potency in both the tests. In both cases, acylation of the N-terminus led to the enhancement of the antagonistic potency. On the basis of these findings, new potent and selective B(2) blockers might be designed.

Analogues of neurohypophyseal hormones, oxytocin and arginine vasopressin, conformationally restricted in the N-terminal part of the molecule.

It is generally accepted that the conformation of the N-terminal part of neurohypophyseal hormones analogues is important for their pharmacological activity. In this work, we decided to investigate how the substitution of positions 2 and 3 with the ethylene-bridged dipeptide would alter the pharmacological properties of OT, [Mpa1]OT, and [Cpa1]OT (OT=oxytocin; Mpa=3-mercaptopropionic acid; Cpa=1-mercaptocyclohexaneacetic acid) and to investigate how a bulky 3,3-diphenyl-L-alanine residue incorporated in position 2 of AVP, [Mpa1]AVP, and [Cpa1]AVP (AVP=arginine vasopressin) would change the pharmacological profile of the compounds. The next analogues, [Val4]AVP, [Mpa1,Val4]AVP, and [Cpa1,Val4]AVP, had N-benzyl-L-alanine introduced at position 3. The last peptide was designed by Cys1 substitution in AVP by its sterically restricted bulky counterpart, alpha-hydroxymethylcysteine. All the peptides were tested for their in vitro uterotonic, pressor, and antidiuretic activities in the rat. The results of these assays showed that the reduction of conformational freedom of the N-terminal part of the molecule had a significant impact on pharmacological activities.

Investigation of cis/trans ratios of peptide bonds in AVP analogues containing N-methylphenylalanine enantiomers.

The solution conformation of vasopressin analogues, modified at positions 2 and 3 with N-methylphenylalanine or its enantiomer, [D-MePhe2,MePhe3]AVP and [MePhe2,D-MePhe3]AVP, were studied by 2D NMR spectroscopy in H2O/D2O and theoretical calculations (EDMC/ANALYZE). In the case of [MePhe2,D-MePhe3]AVP, the synthesis afforded two products, A and B, which had identical molecular ions and similar retention times on HPLC. This finding was explained by racemization of Cys1, which gave an additional analogue, [D-Cys1,MePhe2,D-MePhe3]AVP (B). The possibility is not excluded of racemization of Cys1 in the remaining analogues of this series. However, only in the case of [MePhe2,D-MePhe3]AVP was this process so distinct that two strong peaks in the HPLC chromatogram were observed. The NMR spectra of all the analogues showed several distinct sets of residual proton resonances. This suggests that the peptides adopt more than two groups of conformations in H2O/D2O. This fact is due to cis/trans isomerization. Two more populated isomers arise from the cis/trans isomerization across the 2-3 peptide bond in [D-MePhe2,MePhe3]AVP and [MePhe2,D-MePhe3]AVP (A) and across the 1-2 peptide bond in [D-Cys1,MePhe2,D-MePhe3]AVP (B).

New bradykinin analogues modified in the C-terminal part with sterically restricted 1-aminocyclohexane-1-carboxylic acid.

In the present work, a sterically constrained noncoded amino acid, 1-aminocyclohexane-1-carboxylic acid (Acc), was substituted in position 8 of the peptide chain of bradykinin (BK) and position 6, 7, or 8 of its B2 receptor antagonist [D-Arg0,Hyp3,Thi,(5,8)D-Phe7]BK, previously synthesized by Stewart's group, to reduce the flexibility of the peptides, thus forcing the peptide backbone and side chains to adopt specific orientations. Knowing that acylation of the N-terminus of several known B2 blockers with a variety of bulky groups has consistently improved their antagonistic potency in the rat blood pressure assay, the Acc substituted analogues were also synthesized in the N-acylated form with 1-adamantaneacetic acid (Aaa). The activity of eight new analogues was assayed in isolated rat uterus and in rat blood pressure tests. The results clearly demonstrated the importance of the position in the peptide chain into which the sterically restricted Acc residue was inserted. Meanwhile, Acc at positions 6 and 7 led to reduction of antagonistic qualities or even restored the agonism, respectively. Acc at position 8 enhanced antagonistic qualities in both tests. The Acc at position 8 of BK strongly reduced the agonistic potency. In most cases acylation of the N-terminus led either to enhancement of antagonistic potencies or to further decrease of agonistic potency. Our findings offer new possibilities for designing new potent and selective B2 blockers.

The influence of 1-aminocyclopentane-1-carboxylic acid at position 2 or 3 of AVP and its analogues on their pharmacological properties.

This study describes the synthesis and some pharmacological properties of eight new analogues of arginine vasopressin (AVP) substituted at position 2 or 3 with cycloleucine (1-aminocyclopentane-1-carboxylic acid, Apc). All new peptides were tested for their pressor, antidiuretic and uterotonic in vitro potency. The Apc3 modification resulted in an almost complete loss of potency in all three tests, which is interpreted as a loss of interaction with all three neurohypophyseal hormone receptors. On the other hand, the Apc2 modification resulted in compounds having differently modified activities (high antidiuretic potency, low and graded pressor activity and either no activity or low oxytocin antagonizing activity in the uterotonic in vitro test) thus selectively altering the interaction with the receptors similar to that of 1-aminocyclohexane-1-carboxylic acid (Acc). The results obtained may be helpful for designing new analogues of arginine vasopressin.