Alternating Cationic-Hydrophobic Peptide/Peptoid Hybrids: Influence of Hydrophobicity on Antibacterial Activity and Cell Selectivity.

The influence of hydrophobicity on antibacterial activity versus the effect on the viability of mammalian cells for peptide/peptoid hybrids was examined for oligomers based on the cationic Lys-like peptoid residue combined with each of 28 hydrophobic amino acids in an alternating sequence. Their relative hydrophobicity was correlated to activity against both Gram-negative and Gram-positive species, human red blood cells, and HepG2 cells. This identified hydrophobic side chains that confer potent antibacterial activity (e. g., MICs of 2-8 µg/mL against E. coli) and low toxicity toward mammalian cells (<10 % hemolysis at 400 µg/mL and IC50 >800 µg/mL for HepG2 viability). Most peptidomimetics retained activity against drug-resistant strains. These findings corroborate the hypothesis that for related peptidomimetics two hydrophobicity thresholds may be identified: i) it should exceed a certain level in order to confer antibacterial activity, and ii) there is an upper limit, beyond which cell selectivity is lost. It is envisioned that once identified for a given subclass of peptide-like antibacterials such thresholds can guide further optimisation.

Design, Synthesis and Biological Evaluation of Biphenylglyoxamide-Based Small Molecular Antimicrobial Peptide Mimics as Antibacterial Agents.

There has been an increasing interest in the development of antimicrobial peptides (AMPs) and their synthetic mimics as a novel class of antibiotics to overcome the rapid emergence of antibiotic resistance. Recently, phenylglyoxamide-based small molecular AMP mimics have been identified as potential leads to treat bacterial infections. In this study, a new series of biphenylglyoxamide-based small molecular AMP mimics were synthesised from the ring-opening reaction of N-sulfonylisatin bearing a biphenyl backbone with a diamine, followed by the conversion into tertiary ammonium chloride, quaternary ammonium iodide and guanidinium hydrochloride salts. Structure-activity relationship studies of the analogues identified the octanesulfonyl group as being essential for both Gram-positive and Gram-negative antibacterial activity, while the biphenyl backbone was important for Gram-negative antibacterial activity. The most potent analogue was identified to be chloro-substituted quaternary ammonium iodide salt 15c, which possesses antibacterial activity against both Gram-positive (MIC against Staphylococcus aureus = 8 µM) and Gram-negative bacteria (MIC against Escherichia coli = 16 µM, Pseudomonas aeruginosa = 63 µM) and disrupted 35% of pre-established S. aureus biofilms at 32 µM. Cytoplasmic membrane permeability and tethered bilayer lipid membranes (tBLMs) studies suggested that 15c acts as a bacterial membrane disruptor. In addition, in vitro toxicity studies showed that the potent compounds are non-toxic against human cells at therapeutic dosages.

Discovery of a Small Molecule Probe of Rpn-6, an Essential Subunit of the 26S Proteasome.

A considerable number of essential cellular proteins have no catalytic activity and serve instead as structural components to aid in assembling protein complexes. For example, the assembly and function of the 26S proteasome, the major enzymatic complex necessary for ubiquitin-dependent protein degradation, require a number of essential protein contacts to associate the 19S regulatory particle with the 20S core particle. Previously, small molecule inhibitors of the active sites of the 20S core particle have been developed, but the activity of the 26S proteasome could also be altered via the disruption of its assembly. We were interested in discovering a small molecule binder of Rpn-6, as it is among several essential proteins that facilitate 26S assembly, which could be used to further our understanding of the association of the 19S regulatory particle with the 20S core particle. Additionally, we were interested in whether a small molecule-Rpn-6 interaction could potentially be cytotoxic to cancer cells that rely heavily on proteasome activity for survival. A workflow for utilizing a one-bead, one-compound library and a thermal shift assay was developed to discover such a molecule. TXS-8, our lead hit, was discovered to have a low micromolar binding affinity for Rpn-6 as well as very limited binding to other proteins. The cytotoxicity of TXS-8 was evaluated in several cell lines, revealing increased cytotoxicity to hematological cancers. Discovery of this peptoid binder of Rpn-6 provides the initial evidence that Rpn-6 could be a druggable target to affect protein degradation and serves as a primary scaffold from which to design more potent binders. We suspect that Rpn-6 could have additional essential roles beyond that of a molecular clamp of the proteasome to help hematological cancer cells survive and that TXS-8 can serve as a useful tool for further elucidating its roles.

Host defense peptide mimicking poly-ß-peptides with fast, potent and broad spectrum antibacterial activities.

Microbial infections have always been serious challenges to human health considering that antibiotics almost inevitably induce microbial resistance. Therefore, it is urgent to develop a new antibacterial agent that is active against drug-resistant bacteria and is less susceptible to microbial resistance. In this work, a series of host defense peptide (HDP) mimicking antibacterial poly-ß-peptides were synthesized, characterized and evaluated for their biological activities. The best poly-ß-peptide within this study (20 : 80 Bu : DM) displays potent and broad spectrum antibacterial activity against antibiotic-resistant super bugs and low toxicity toward mammalian cells. Moreover, these poly-ß-peptides are bactericidal and kill bacteria very fast within 5 min. An antimicrobial resistance test demonstrated that bacteria develop no resistance toward the selected poly-ß-peptides even over 1000 generations. Our studies demonstrate that random copolymers of heterochiral poly-ß-peptides, without the need for defined secondary structures, can mimic the antimicrobial HDP. These results imply the potential application of these poly-ß-peptides as new antimicrobial agents to tackle drug resistant antimicrobial infections.

Searching for improved mimetic peptides inhibitors preventing conformational transition of amyloid-ß42 monomer.

A series of novel mimetic peptides were designed, synthesised and biologically evaluated as inhibitors of Aß42 aggregation. One of the synthesised peptidic compounds, termed compound 7 modulated Aß42 aggregation as demonstrated by thioflavin T fluorescence, acting also as an inhibitor of the cytotoxicity exerted by Aß42 aggregates. The early stage interaction between compound 7 and the Aß42 monomer was investigated by replica exchange molecular dynamics (REMD) simulations and docking studies. Our theoretical results revealed that compound 7 can elongate the helical conformation state of an early stage Aß42 monomer and it helps preventing the formation of ß-sheet structures by interacting with key residues in the central hydrophobic cluster (CHC). This strategy where early "on-pathway" events are monitored by small molecules will help the development of new therapeutic strategies for Alzheimer's disease.

Selectively targeting bacteria by tuning the molecular design of membrane-active peptidomimetic amphiphiles.

Here we report the design of membrane-active peptidomimetic molecules with a tunable arrangement of hydrophobic and polar groups. In spite of having the same chemical composition, the effective hydrophobicities of the compounds were different as a consequence of their chemical structure and conformational properties. The compound with lower effective hydrophobicity demonstrated antibacterial activity that was highly selective towards bacteria over mammalian cells. This study, highlighting the role in membrane selectivity of the specific arrangement of the different moieties in the molecular structure, provides useful indications for developing non-toxic antibacterial agents.

Anticancer Gold(III) Peptidomimetics: From Synthesis to in vitro and ex vivo Biological Evaluations.

Five new AuIII -peptidodithiocarbamato complexes of the type [AuIII Br2 (dtc-AA1 -AA2 -OR] (in which AA1 =N-methylglycine (Sar), l/d-Pro; AA2 =l/d-Ala, α-aminoisobutyric acid (Aib); R=OtBu, triethylene glycol methyl ether), differing with regard to the amino acid sequence and/or the chiral amino acid configuration, were designed to enhance tumor selectivity and bioavailability. The gold(III)-based moiety was functionalized to exploit the targeting properties of the peptidomimetic ligand toward two peptide transporters (namely PEPT1 and PEPT2), which are upregulated in several tumor cells. The compounds were synthesized and fully characterized, mainly by means of elemental analysis, one- and two-dimensional NMR spectroscopy, FT-IR, and UV/Vis spectrophotometry. The crystal structures of three compounds were also solved by X-ray diffraction. In vitro cytotoxicity studies using a panel of human tumor cell lines (A549 [non-small-cell lung carcinoma], MCF-7 [breast cancer], A2780 [ovarian carcinoma], H1975 [non-small-cell lung carcinoma], H460 [large-cell lung carcinoma], and A431 [human epidermoid carcinoma]) showed the dtc-Pro-Aib-OtBu derivative to be very effective, with GI50 values much lower than those of cisplatin. This complex was thus selected for evaluating stability under physiological conditions and possible interactions with serum albumin, as well in PARP-1 enzyme inhibition assays and preliminary ex vivo toxicity experiments on healthy rat tissues.

Indoloazepinone-Constrained Oligomers as Cell-Penetrating and Blood-Brain-Barrier-Permeating Compounds.

Non-cationic and amphipathic indoloazepinone-constrained (Aia) oligomers have been synthesized as new vectors for intracellular delivery. The conformational preferences of the [l-Aia-Xxx]n oligomers were investigated by circular dichroism (CD) and NMR spectroscopy. Whereas Boc-[l-Aia-Gly]2,4 -OBn oligomers 12 and 13 and Boc-[l-Aia-ß3 -h-l-Ala]2,4 -OBn oligomers 16 and 17 were totally or partially disordered, Boc-[l-Aia-l-Ala]2 -OBn (14) induced a typical turn stabilized by C5 - and C7 -membered H-bond pseudo-cycles and aromatic interactions. Boc-[l-Aia-l-Ala]4 -OBn (15) exhibited a unique structure with remarkable T-shaped π-stacking interactions involving the indole rings of the four l-Aia residues forming a dense hydrophobic cluster. All of the proposed FITC-6-Ahx-[l-Aia-Xxx]4 -NH2 oligomers 19-23, with the exception of FITC-6-Ahx-[l-Aia-Gly]4 -NH2 (18), were internalized by MDA-MB-231 cells with higher efficiency than the positive references penetratin and Arg8 . In parallel, the compounds of this series were successfully explored in an in vitro blood-brain barrier (BBB) permeation assay. Although no passive diffusion permeability was observed for any of the tested Ac-[l-Aia-Xxx]4 -NH2 oligomers in the PAMPA model, Ac-[l-Aia-l-Arg]4 -NH2 (26) showed significant permeation in the in vitro cell-based human model of the BBB, suggesting an active mechanism of cell penetration.

Design and synthesis of cell selective α/ß-diastereomeric peptidomimetic with potent in vivo antibacterial activity against methicillin resistant S. Aureus.

Design of therapeutically viable antimicrobial peptides with cell selectivity against microorganisms is an important step towards the development of new antimicrobial agents. Here, we report four de novo designed, short amphipathic sequences based on a α-helical template comprising of Lys, Trp and Leu or their corresponding D-and/or ß-amino acids. Sequence A-12 was protease susceptible whereas its α/ß-diastereomeric analogue UNA-12 was resistant to trypsin and proteinase K up to 24 h. A-12 and UNA-12 exhibited broad-spectrum antibacterial activity (MIC: 2-32 µg/mL) against pathogens including methicillin resistant S. aureus (MRSA) and methicillin-resistant S. epidermidis (MRSE). Interestingly, A-12 was found to be most toxic (>50% haemolytic at 250 µg/mL) whereas UNA-12 was found to be non cytotoxic among the all analogues against hRBCs and human keratinocytes. Interaction studies with artificial membranes by tryptophan fluorescence and acrylamide quenching assay demonstrated A-12 interacted equally in bacterial as well as mammalian mimic membrane whereas UNA-12 was found to be more selective towards bacterial mimic membrane. Further microscopic tool has revealed membrane damaging ability of A-12 and UNA-12 with bactericidal mode of action against MRSA. Encouragingly, peptidomimetics analogue UNA-12 showed remarkable safety and efficacy against MRSA in in-vivo neutropenic mice thigh infection model. In summary, simultaneous replacement of the natural amino acids with D-/ß-congeners is a promising strategy for designing of potent, cell selective and protease stable peptide based antibiotics.

Tumor Targeting with an isoDGR-Drug Conjugate.

Herein we report the first example of an isoDGR-drug conjugate (2), designed to release paclitaxel selectively within cancer cells expressing integrin αV ß3 . Conjugate 2 was synthesized by connecting the isoDGR peptidomimetic 5 with paclitaxel via the lysosomally cleavable Val-Ala dipeptide linker. Conjugate 2 displayed a low nanomolar affinity for the purified integrin αV ß3 receptor (IC50 =11.0 nm). The tumor targeting ability of conjugate 2 was assessed in vitro in anti-proliferative assays on two isogenic cancer cell lines characterized by different integrin αV ß3 expression: human glioblastoma U87 (αV ß3 +) and U87 ß3 -KO (αV ß3 -). The isoDGR-PTX conjugate 2 displayed a remarkable targeting index (TI=9.9), especially when compared to the strictly related RGD-PTX conjugate 4 (TI=2.4).

Structural and Physico-Chemical Interpretation (SPCI) of QSAR Models and Its Comparison with Matched Molecular Pair Analysis.

This paper describes the Structural and Physico-Chemical Interpretation (SPCI) approach, which is an extension of a recently reported method for interpretation of quantitative structure-activity relationship (QSAR) models. This approach can efficiently be used to reveal structural motifs and the major physicochemical factors affecting the investigated properties. Its efficacy was demonstrated both on the classical Free-Wilson data set and on several data sets with different end points (permeability of the blood-brain barrier, fibrinogen receptor antagonists, acute oral toxicity). Structure-activity patterns extracted from QSAR models with SPCI were in good correspondence with experimentally observed relationships and molecular docking, regardless of the machine learning method used. Comparison of SPCI with the matched molecular pair (MMP) method clearly shows an advantage of our approach over MMP, especially for small or structurally diverse data sets. The developed approach has been implemented in the SPCI software tool with a graphical user interface, which is publicly available at http://qsar4u.com/pages/sirms_qsar.php .

Antifungal properties of peptidomimetics with an arginine-[ß-(2,5,7-tri-tert-butylindol-3-yl)alanine]-arginine motif against Saccharomyces cerevisiae and Zygosaccharomyces bailii.

Due to increased occurrence of infections and food spoilage caused by yeast, there is an unmet need for new antifungal agents. The arginine-ß-(2,5,7-tri-tert-butylindol-3-yl) alanine-arginine (R-Tbt-R) motif was previously proved useful in the design of an antifungal tripeptide. Here, an array of peptidomimetics based on this motif was investigated for antifungal and hemolytic activity. The five most promising modified tetrapeptide analogues ( 6: and 9-12: contain an additional C-terminal hydrophobic residue, and these were found to exhibit antifungal activity against Saccharomyces cerevisiae (MIC 6 and 12 µg mL(-1)) and Zygosaccharomyces bailii (MIC 6-25 µg mL(-1)). Four compounds ( 6: and 9-11: , had limited hemolytic activity (<10% hemolysis at 8 × MIC). Determination of their killing kinetics revealed that compound 9: displayed fungicidal effect. Testing against cells from an S. cerevisiae deletion mutant library indicated that interaction with yeast-specific fungal sphingolipids, most likely constitutes a crucial step in the mode of action. Interestingly, a lack of activity of peptidomimetics 6: and 9-11: towards Candida spp. was shown to be due to degradation or sequestering by the yeast. Due to their ultrashort nature, antifungal activity and low toxicity, the four compounds may have potential as leads for novel preservatives.

Antibacterial evaluation of structurally amphipathic, membrane active small cationic peptidomimetics: synthesized by incorporating 3-amino benzoic acid as peptidomimetic element.

A new series of small cationic peptidomimetics were synthesized by incorporating 3-amino benzoic acid (3-ABA) in a small structural framework with the objective to mimic essential properties of natural antimicrobial peptides (AMPs). The new design approach resulted into improvement of activity and selectivity in comparison to linear peptides and allowed us to better understand the influence of structural amphipathicity on biological activity. Lead peptidomimetics displayed antibacterial activities against resistant pathogens (MRSA & MRSE). A calcein dye leakage experiment revealed a membranolytic effect of 4g and 4l which was further confirmed by fluorescence microscopy. In addition, proteolytic stability and no sign of resistance development against Staphylococcus aureus and MRSA demonstrate their potential for further development as novel antimicrobial therapeutics.

Preclinical pharmacokinetic and toxicological evaluation of MIF-1 peptidomimetic, PAOPA: examining the pharmacology of a selective dopamine D2 receptor allosteric modulator for the treatment of schizophrenia.

Schizophrenia is a mental illness characterized by a breakdown in cognition and emotion. Over the years, drug treatment for this disorder has mainly been compromised of orthosteric ligands that antagonize the active site of the dopamine D2 receptor. However, these drugs are limited in their use and often lead to the development of adverse movement and metabolic side effects. Allosteric modulators are an emerging class of therapeutics with significant advantages over orthosteric ligands, including an improved therapeutic and safety profile. This study investigates our newly developed allosteric modulator, PAOPA, which is a specific modulator of the dopamine D2 receptor. Previous studies have shown PAOPA to attenuate schizophrenia-like behavioral abnormalities in preclinical models. To advance this newly developed allosteric drug from the preclinical to clinical stage, this study examines the pharmacokinetic behavior and toxicological profile of PAOPA. Results from this study prove the effectiveness of PAOPA in reaching the implicated regions of the brain for therapeutic action, particularly the striatum. Pharmacokinetic parameters of PAOPA were found to be comparable to current market antipsychotic drugs. Necropsy and histopathological analyses showed no abnormalities in all examined organs. Acute and chronic treatment of PAOPA indicated no movement abnormalities commonly found with the use of current typical antipsychotic drugs. Moreover, acute and chronic PAOPA treatment revealed no hematological or metabolic abnormalities classically found with the use of atypical antipsychotic drugs. Findings from this study demonstrate a better safety profile of PAOPA, and necessitates the progression of this newly developed therapeutic for the treatment of schizophrenia.

Peptidomimetic small-molecule compounds promoting cardiogenesis of stem cells.

Embryonic stem (ES) cells may be used as an alternative source of functionally intact cardiomyocytes for ischemic heart disease. Several natural and synthetic small molecules have been identified as useful tools for controlling and manipulating stem cell renewal and differentiation. Currently, there is an urgent requirement for novel small molecules that specifically induce differentiation of stem cells into cardiomyocytes. To identify compounds that promote cardiomyogenesis of stem cells, cell-based screening of a peptidomimetic small-molecule library was carried out. A series of ß-turn peptidomimetic compounds, including CW209E, increased the expression of α-MHC promoter-driven enhanced green fluorescent protein (EGFP) and ratio of beating embryoid bodies (EBs) without inducing cytotoxicity in mouse embryonic stem cells. CW209E also increased the number of beating EBs in human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs). Thus, this chemical compound should be useful for elucidation of the molecular pathway of cardiogenesis and generation of cardiomyocytes ex vivo, which can be further applied for experimental or clinical cell therapy for ischemic heart diseases.

Peptides that anneal to natural collagen in vitro and ex vivo.

Collagen comprises » of the protein in humans and ¾ of the dry weight of human skin. Here, we implement recent discoveries about the structure and stability of the collagen triple helix to design new chemical modalities that anchor to natural collagen. The key components are collagen mimetic peptides (CMPs) that are incapable of self-assembly into homotrimeric triple helices, but are able to anneal spontaneously to natural collagen. We show that such CMPs containing 4-fluoroproline residues, in particular, bind tightly to mammalian collagen in vitro and to a mouse wound ex vivo. These synthetic peptides, coupled to dyes or growth factors, could herald a new era in assessing or treating wounds.

Jointly handling potency and toxicity of antimicrobial peptidomimetics by simple rules from desirability theory and chemoinformatics.

Today, emerging and increasing resistance to antibiotics has become a threat to public health worldwide. Antimicrobial peptides have unique action mechanisms making them an attractive therapeutic prospect to be applied against resistant bacteria. However, the major drawback is related with their high hemolytic activity which cancels out the safety requirements for a human antibiotic. Therefore, additional efforts are needed to develop new antimicrobial peptides that possess a greater potency for bacterial cells and less or no toxicity over erythrocytes. In this paper, we introduce a practical approach to simultaneously deal with these two conflicting properties. The convergence of machine learning techniques and desirability theory allowed us to derive a simple, predictive, and interpretable multicriteria classification rule for simultaneously handling the antibacterial and hemolytic properties of a set of cyclic ß-hairpin cationic peptidomimetics (Cß-HCPs). The multicriteria classification rule exhibited a prediction accuracy of about 80% on training and external validation sets. Results from an additional concordance test have shown an excellent agreement between the multicriteria classification rule predictions and the predictions from independent classifiers for complementary antibacterial and hemolytic activities, respectively, evidencing the reliability of the multicriteria classification rule. The rule was also consistent with the general mode of action of cationic peptides pointing out its biophysical relevance. We also propose a multicriteria virtual screening strategy based on the joint use of the multicriteria classification rule, desirability, similarity, and chemometrics concepts. The ability of such a virtual screening strategy to prioritize selective (nonhemolytic) antibacterial Cß-HCPs was assessed and challenged for their predictivity regarding the training, validation, and overall data. In doing so, we were able to rank a selective antibacterial Cß-HCP earlier than a biologically inactive or nonselective antibacterial Cß-HCP with a probability of ca. 0.9. Our results thus indicate that promising chemoinformatics tools were obtained by considering both the multicriteria classification rule and the virtual screening strategy, which could, for instance, be used to aid the discovery and development of potent and nontoxic antimicrobial peptides.