ABSTRACT
In recent decades, bioactive peptides have been gaining recognition in various biomedical areas, such as intracellular drug delivery (cell-penetrating peptides, CPPs) or anti-infective action (antimicrobial peptides, AMPs), closely associated to their distinct mode of interaction with biological membranes. Exploiting the interaction of membrane-active peptides with diverse targets (healthy, tumoral, bacterial or parasitic cell membranes) is opening encouraging prospects for peptides in therapeutics. However, ordinary peptides formed by L-amino acids are easily decomposed by proteases in biological fluids. One way to sidestep this limitation is to use topoisomers, namely versions of the peptide made up of D-amino acids in either canonic (enantio) or inverted (retroenantio) sequence. Rearranging peptide sequences in this fashion provides a certain degree of native structure mimicry that, in appropriate contexts, may deliver desirable biological activity while avoiding protease degradation. In this review, we will focus on recent accounts of membrane-active topoisomeric peptides with therapeutic applications as CPP drug delivery vectors, or as antimicrobial and anticancer candidates. We will also discuss the most common modes of interaction of these peptides with their membrane targets.
ABSTRACT
Tumour suppressor p53 plays a key role in the development of cancer and has therefore been widely studied in recent decades. While it is well known that p53 is biologically active as a tetramer, the tetramerisation mechanism is still not completely understood. p53 is mutated in nearly 50% of cancers, and mutations can alter the oligomeric state of the protein, having an impact on the biological function of the protein and on cell fate decisions. Here, we describe the effects of a number of representative cancer-related mutations on tetramerisation domain (TD) oligomerisation defining a peptide length that permits having a folded and structured domain, thus avoiding the effect of the flanking regions and the net charges at the N- and C-terminus. These peptides have been studied under different experimental conditions. We have applied a variety of techniques, including circular dichroism (CD), native mass spectrometry (MS) and high-field solution NMR. Native MS allows us to detect the native state of complexes maintaining the peptide complexes intact in the gas phase; the secondary and quaternary structures were analysed in solution by NMR, and the oligomeric forms were assigned by diffusion NMR experiments. A significant destabilising effect and a variable monomer population were observed for all the mutants studied.
ABSTRACT
Infectious diseases caused by microbial pathogens (bacteria, virus, fungi, parasites) claim millions of deaths per year worldwide and have become a serious challenge to global human health in our century. Viral infections are particularly notable in this regard, not only because humankind is facing some of the deadliest viral pandemics in recent history, but also because the arsenal of drugs to combat the high levels of mutation, and hence the antigenic variability of (mostly RNA) viruses, is disturbingly scarce. Therefore, the search for new antivirals able to successfully fight infection with minimal or no adverse effects on the host is a pressing task. Traditionally, antiviral therapies have relied on relatively small-sized drugs acting as proteases, polymerases, integrase inhibitors, etc. In recent decades, novel approaches involving targeted delivery such as that achieved by peptide-drug conjugates (PDCs) have gained attention as alternative (pro)drugs for tackling viral diseases. Antiviral PDC therapeutics typically involve one or more small drug molecules conjugated to a cell-penetrating peptide (CPP) carrier either directly or through a linker. Such integration of two bioactive elements into a single molecular entity is primarily aimed at achieving improved bioavailability in conditions where conventional drugs are challenged, but may also turn up novel unexpected functionalities and applications. Advances in peptide medicinal chemistry have eased the way to antiviral PDCs, but challenges remain on the way to therapeutic success. In this paper, we review current antiviral CPP-drug conjugates (antiviral PDCs), with emphasis on the types of CPP and antiviral cargo. We integrate the conjugate and the chemical approaches most often applied to combine both entities. Additionally, we comment on various obstacles faced in the design of antiviral PDCs and on the future outlooks for this class of antiviral therapeutics.
ABSTRACT
Viral disease outbreaks affect hundreds of millions of people worldwide and remain a serious threat to global health. The current SARS-CoV-2 pandemic and other recent geographically- confined viral outbreaks (severe acute respiratory syndrome (SARS), Ebola, dengue, zika and ever-recurring seasonal influenza), also with devastating tolls at sanitary and socio-economic levels, are sobering reminders in this respect. Among the respective pathogenic agents, Zika virus (ZIKV), transmitted by Aedes mosquito vectors and causing the eponymous fever, is particularly insidious in that infection during pregnancy results in complications such as foetal loss, preterm birth or irreversible brain abnormalities, including microcephaly. So far, there is no effective remedy for ZIKV infection, mainly due to the limited ability of antiviral drugs to cross blood-placental and/or blood-brain barriers (BPB and BBB, respectively). Despite its restricted permeability, the BBB is penetrable by a variety of molecules, mainly peptide-based, and named BBB peptide shuttles (BBBpS), able to ferry various payloads (e.g., drugs, antibodies, etc.) into the brain. Recently, we have described peptide-porphyrin conjugates (PPCs) as successful BBBpS-associated drug leads for HIV, an enveloped virus in which group ZIKV also belongs. Herein, we report on several brain-directed, low-toxicity PPCs capable of targeting ZIKV. One of the conjugates, PP-P1, crossing both BPB and BBB, has shown to be effective against ZIKV (IC50 1.08 µM) and has high serum stability (t1/2 ca. 22 h) without altering cell viability at all tested concentrations. Peptide-porphyrin conjugation stands out as a promising strategy to fill the ZIKV treatment gap.
ABSTRACT
Passing through the blood-brain barrier (BBB) to treat neurological conditions is one of the main hurdles in modern medicine. Many drugs with promising in vitro profiles become ineffective in vivo due to BBB restrictive permeability. In particular, this includes drugs such as antiviral porphyrins, with the ability to fight brain-resident viruses causing diseases such as HIV-associated neurocognitive disorders (HAND). In the last two decades, BBB shuttles, particularly peptide-based ones, have shown promise in carrying various payloads across the BBB. Thus, peptide-drug conjugates (PDCs) formed by covalent attachment of a BBB peptide shuttle and an antiviral drug may become key therapeutic tools in treating neurological disorders of viral origin. In this study, we have used various approaches (guanidinium, phosphonium, and carbodiimide-based couplings) for on-resin synthesis of new peptide-porphyrin conjugates (PPCs) with BBB-crossing and potential antiviral activity. After careful fine-tuning of the synthetic chemistry, DIC/oxyma has emerged as a preferred method, by which 14 different PPCs have been made and satisfactorily characterized. The PPCs are prepared by coupling a porphyrin carboxyl group to an amino group (either N-terminal or a Lys side chain) of the peptide shuttle and show effective in vitro BBB translocation ability, low cytotoxicity toward mouse brain endothelial cells, and low hemolytic activity. Three of the PPCs, MP-P5, P4-MP, and P4-L-MP, effectively inhibiting HIV infectivity in vitro, stand out as most promising. Their efficacy against other brain-targeting viruses (Dengue, Zika, and SARS-CoV-2) is currently under evaluation, with preliminary results confirming that PPCs are a promising strategy to treat viral brain infections.
Subject(s)
Anti-HIV Agents/pharmacokinetics , Blood-Brain Barrier/metabolism , Peptides/pharmacokinetics , Porphyrins/pharmacokinetics , Animals , Anti-HIV Agents/chemistry , Anti-HIV Agents/pharmacology , Biological Transport , Cell Line , Drug Discovery , HEK293 Cells , HIV/drug effects , HIV Infections/drug therapy , Humans , Mice , Peptides/chemistry , Peptides/pharmacology , Porphyrins/chemistry , Porphyrins/pharmacologyABSTRACT
Ulcerative colitis and Crohn's disease are forms of inflammatory bowel disease whose incidence and prevalence are increasing worldwide. These diseases lead to chronic inflammation of the gastrointestinal tract as a result of an abnormal response of the immune system. Recent studies positioned Cortistatin, which shows low stability in plasma, as a candidate for IBD treatment. Here, using NMR structural information, we design five Cortistatin analogues adopting selected native Cortistatin conformations in solution. One of them, A5, preserves the anti-inflammatory and immunomodulatory activities of Cortistatin in vitro and in mouse models of the disease. Additionally, A5 displays an increased half-life in serum and a unique receptor binding profile, thereby overcoming the limitations of the native Cortistatin as a therapeutic agent. This study provides an efficient approach to the rational design of Cortistatin analogues and opens up new possibilities for the treatment of patients that fail to respond to other therapies.
Subject(s)
Anti-Inflammatory Agents, Non-Steroidal/therapeutic use , Colitis, Ulcerative/drug therapy , Immunologic Factors/therapeutic use , Immunomodulation/drug effects , Neuropeptides/therapeutic use , Animals , Cells, Cultured , Colitis, Ulcerative/chemically induced , Colitis, Ulcerative/pathology , Dextran Sulfate/toxicity , Disease Models, Animal , Gastrointestinal Tract/pathology , Humans , Inflammation/drug therapy , Male , Mice , Mice, Inbred C57BL , Molecular Conformation , Trinitrobenzenesulfonic Acid/toxicityABSTRACT
We have developed a nanocarrier consisting of large unilamellar vesicles (LUVs) for combined delivery of two human immunodeficiency virus type 1 (HIV-1) entry inhibitors, enfuvirtide (ENF) and protoporphyrin IX (PPIX). The intrinsic lipophilicity of ENF and PPIX, a fusion inhibitor and an attachment inhibitor, respectively, leads to their spontaneous incorporation into the lipid bilayer of the LUVs nanocarrier. Both entry inhibitors partition significantly toward LUVs composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and a 9:1 mixture of POPC:1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (DPPE-PEG2000), representative of conventional and immune-evasive drug delivery formulations, respectively. These colocalize in the core of lipid membranes. Dual-loaded nanocarriers are monodispersed and retain the size distribution, thermotropic behavior, and surface charge of the unloaded form. Combination of the two entry inhibitors in the nanocarrier resulted in improved synergy against HIV-1 entry compared to combination in free form, strongly when immune-evasive formulations are used. We propose that the improved action of the entry inhibitors when loaded into the nanocarriers results from their slow release at the site of viral entry. Overall, liposomes remain largely unexplored platforms for combination of viral entry inhibitors, with potential for improvement of current antiretroviral therapy drug safety and application. Our work calls for a reappraisal of the potential of entry inhibitor combinations and delivery for clinical use in antiretroviral therapy.
Subject(s)
Enfuvirtide/pharmacology , HIV-1/drug effects , HIV-1/physiology , Protoporphyrins/pharmacology , Virus Internalization/drug effects , Cell Line , Drug Synergism , Humans , Inhibitory Concentration 50 , Liposomes/chemistry , Nanoparticles/chemistry , Polyethylene GlycolsABSTRACT
Random peptide libraries that cover large search spaces are often used for the discovery of new binders, even when the target is unknown. To ensure an accurate population representation, there is a tendency to use large libraries. However, parameters such as the synthesis scale, the number of library members, the sequence deconvolution and peptide structure elucidation, are challenging when increasing the library size. To tackle these challenges, we propose an algorithm-supported approach to peptide library design based on molecular mass and amino acid diversity. The aim is to simplify the tedious permutation identification in complex mixtures, when mass spectrometry is used, by avoiding mass redundancy. For this purpose, we applied multi (two- and three-)-objective genetic algorithms to discriminate between library members based on defined parameters. The optimizations led to diverse random libraries by maximizing the number of amino acid permutations and minimizing the mass and/or sequence overlapping. The algorithm-suggested designs offer to the user a choice of appropriate compromise solutions depending on the experimental needs. This implies that diversity rather than library size is the key element when designing peptide libraries for the discovery of potential novel biologically active peptides.
ABSTRACT
Somatostatin is a 14-residue peptide hormone that regulates the endocrine system by binding to five G-protein-coupled receptors (SSTR1-5). We have designed six new Somatostatin analogs with L-3-(3',5'-difluorophenyl)-alanine (Dfp) as a substitute of Phe and studied the effect of an electron-poor aromatic ring in the network of aromatic interactions present in Somatostatin. Replacement of each of the Phe residues (positions 6, 7 and 11) by Dfp and use of a D-Trp8 yielded peptides whose main conformations could be characterized in aqueous solution by NMR. Receptor binding studies revealed that the analog with Dfp at position 7 displayed a remarkable affinity to SSTR2 and SSTR3. Analogs with Dfp at positions 6 or 11 displayed a π-π interaction with the Phe present at 11 or 6, respectively. Interestingly, these analogs, particularly [D-Trp8,L-Dfp11]-SRIF, showed high selectivity towards SSTR2, with a higher value than that of Octreotide and a similar one to that of native Somatostatin.
Subject(s)
Peptides, Cyclic/chemical synthesis , Peptides, Cyclic/pharmacology , Somatostatin/analogs & derivatives , Alanine/chemistry , Amino Acid Sequence , Binding Sites , Halogenation , Magnetic Resonance Spectroscopy , Models, Molecular , Molecular Structure , Peptides, Cyclic/chemistry , Somatostatin/chemistry , Structure-Activity RelationshipABSTRACT
Fingermark autofluorescence changes with time, both spectrally and in total intensity. In this study we investigate which components in the aged fingermarks cause this change in autofluorescent signal. Thin layer chromatography combined with fluorescence spectroscopy was used to identify fluorescent aging products. Based on our results, tryptophan derivatives, including indoleacetic acid, (nor)harman and xanthurenic acid are indicated as important contributors to the autofluorescence of aged fingermarks. Knowledge about which fluorescent aging products are present in fingermarks might be useful in the development of fingermark age estimation methods. This work is part of a larger project of which the major goal is to develop a method to estimate the time of deposition of fingermarks. Additionally, by selective targeting of aging products the development of aged fingermarks might be improved.
Subject(s)
Dermatoglyphics , Fluorescence , Benzaldehydes/chemistry , Carbolines/chemistry , Chloroform/chemistry , Chromatography, Thin Layer , Humans , Indicators and Reagents , Indoleacetic Acids/chemistry , Methanol/chemistry , Solvents/chemistry , Spectrometry, Fluorescence , Time Factors , Tryptophan/chemistry , Xanthurenates/chemistryABSTRACT
The syntheses of large peptides and of those containing non-natural amino acids can be facilitated by the application of convergent approaches, dissecting the native sequence into segments connected through a ligation reaction. We describe an improvement of the ligation protocol used to prepare peptides and proteins without cysteine residues at the ligation junction. We have found that the addition of HOBt to the ligation, improves the conversion of the ligation reaction without affecting the epimerization rate or chemoselectivity, and it can be efficiently used with peptides containing phosphorylated amino acids.
Subject(s)
Amines/chemistry , Esters/chemistry , Sulfhydryl Compounds/chemistry , Triazoles/chemistry , Amino Acid Sequence , Amino Acids/chemistry , Chromatography, High Pressure Liquid , Chromatography, Reverse-Phase , Molecular Sequence Data , Peptides/chemistryABSTRACT
To demonstrate the utility of the coarse-grained united-residue (UNRES) force field to compare experimental and computed kinetic data for folding proteins, we have performed long-time millisecond-timescale canonical Langevin molecular dynamics simulations of the triple ß-strand from the Formin binding protein 28 WW domain and six nonnatural variants, using UNRES. The results have been compared with available experimental data in both a qualitative and a quantitative manner. Complexities of the folding pathways, which cannot be determined experimentally, were revealed. The folding mechanisms obtained from the simulated folding kinetics are in agreement with experimental results, with a few discrepancies for which we have accounted. The origins of single- and double-exponential kinetics and their correlations with two- and three-state folding scenarios are shown to be related to the relative barrier heights between the various states. The rate constants obtained from time profiles of the fractions of the native, intermediate, and unfolded structures, and the kinetic equations fitted to them, correlate with the experimental values; however, they are about three orders of magnitude larger than the experimental ones for most of the systems. These differences are in agreement with the timescale extension derived by scaling down the friction of water and averaging out the fast degrees of freedom when passing from all-atom to a coarse-grained representation. Our results indicate that the UNRES force field can provide accurate predictions of folding kinetics of these WW domains, often used as models for the study of the mechanisms of proein folding.
Subject(s)
Microscopy/methods , Protein Folding , Protein Structure, Tertiary , KineticsABSTRACT
Cells continuously produce reactive oxidative species that can modify all cellular components. In proteins, for example, cysteine, methionine, tryptophan (Trp), and tyrosine residues are particularly prone to oxidation. Here, we report two new approaches to distinguish two isomeric oxidation products of Trp residues, i.e. 5-hydroxytryptophan (5-HTP) and oxindolylalanine (Oia) residues, in peptides. First, 2-nitrobenzenesulfenyl chloride, known to derivatize Trp residues in position 2 of the indole ring, was used to label 5-HTP residues. The mass shift of 152.98 m/z units allowed identifying 5-HTP- besides Trp-containing peptides by mass spectrometry, whereas Oia residues were not labeled. Second, fragmentation of the Oia- and 5-HTP-derived immonium ions at m/z 175.08 produced ions characteristic for each residue that allowed their identification even in the presence of y(1) ions at m/z 175.12 derived from peptides with C-terminal arginine residues. The pseudo MS(3) spectra acquired on a quadrupole time-of-flight hybrid mass spectrometer displayed two signals at m/z 130.05 and m/z 132.05 characteristic for Oia-containing peptides and a group of six signals (m/z 103.04, 120.04, 130.04, 133.03, 146.04, and 148.04) for 5-HTP-cointaining peptides. In both cases, the relative signal intensities appeared to be independent of the sequence providing a specific fingerprint of each oxidative modification.
Subject(s)
5-Hydroxytryptophan/chemistry , Alanine/analogs & derivatives , Peptides/chemistry , Spectrometry, Mass, Electrospray Ionization/methods , Alanine/chemistry , Amino Acid Sequence , Arginine/chemistry , Ions/chemistry , Isomerism , Molecular Sequence Data , Oxindoles , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization/methodsABSTRACT
The term reactive oxygen species refers to small molecules that can oxidize, for example, nearby proteins, especially cysteine, methionine, tryptophan, and tyrosine residues. Tryptophan oxidation is always irreversible in the cell and can yield several oxidation products, such as 5-hydroxy-tryptophan (5-HTP), oxindolylalanine (Oia), kynurenine (Kyn), and N-formyl-kynurenine (NFK). Because of the severe effects that oxidized tryptophan residues can have on proteins, there is a great need to develop generally applicable and highly sensitive techniques to identify the oxidized residue and the oxidation product. Here, the fragmentation behavior of synthetic peptides corresponding to sequences recently identified in three skeletal muscle proteins as containing oxidized tryptophan residues were studied using postsource decay and collision-induced dissociation (CID) in matrix-assisted laser desorption/ionization (MALDI) time-of-flight (TOF)/TOF mass spectrometry (MS) and CID in an electrospray ionization (ESI) double quadrupole TOF-MS. For each sequence, a panel of five different peptides containing Trp, 5-HTP, Kyn, NFK, or Oia residue was studied. It was always possible to identify the modified positions by the y-series and also to distinguish the different oxidation products by characteristic fragment ions in the lower mass range by tandem MS. NFK- and Kyn-containing peptides displayed an intense signal at m/z 174.1, which could be useful in identifying accordingly modified peptides by a sensitive precursor ion scan. Most importantly, it was always possible to distinguish isomeric 5-HTP and Oia residues. In ESI- and MALDI-MS/MS, this was achieved by the signal intensity ratios of two signals obtained at m/z 130.1 and 146.1. In addition, high collision energy CID in the MALDI-TOF/TOF-MS also permitted the identification of these two isomeric residues by their v- and w-ions, respectively.
Subject(s)
Muscle Proteins/chemistry , Peptides/chemistry , Spectrometry, Mass, Electrospray Ionization , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization , Tryptophan/analysis , 5-Hydroxytryptophan/analysis , Alanine/analogs & derivatives , Alanine/analysis , Amino Acid Sequence , Animals , Kynurenine/analogs & derivatives , Kynurenine/analysis , Molecular Sequence Data , Oxidation-Reduction , OxindolesABSTRACT
ROS, continuously produced in cells, can reversibly or irreversibly oxidize proteins, lipids, and DNA. At the protein level, cysteine, methionine, tryptophan, and tyrosine residues are particularly prone to oxidation. Here, we describe the solid phase synthesis of peptides containing four different oxidation products of tryptophan residues that can be formed by oxidation in proteins in vitro and in vivo: 5-HTP, Oia, Kyn, and NFK. First, we synthesized Oia and NFK by selective oxidation of tryptophan and then protected the α-amino group of both amino acids, and the commercially available 5-HTP, with Fmoc-succinimide. High yields of Fmoc-Kyn were obtained by acid hydrolysis of Fmoc-NFK. All four Fmoc derivatives were successfully incorporated, at high yields, into three different peptide sequences from skeletal muscle actin, creatin kinase (M-type), and ß-enolase. The correct structure of all modified peptides was confirmed by tandem mass spectrometry. Interestingly, isobaric peptides containing 5-HTP and Oia were always well separated in an acetonitrile gradient with TFA as the ion-pair reagent on a C18-phase. Such synthetic peptides should prove useful in future studies to distinguish isobaric oxidation products of tryptophan.