Amino Acid Analogues Research Articles

The synthesis of an EDTA-like chelating peptidomimetic building block suitable for solid-phase peptide synthesis.

A novo trifunctional EDTA-like peptidomimetic amino acid is described. This unique building block, which is prepared in a straightforward manner from commercialized starting materials, contains three moieties: a hexadentate chelating unit similar to that present in EDTA, and amino and carboxylic groups, which facilitate its introduction into the backbone of peptides using conventional SPPS. As a proof of concept, this building block is introduced into a cyclic peptide inspired from the family of Gratisin analogues. The designed peptide contains the amino acid analogue in one of the turns, and chelates Ca2+ with nanomolar affinity at physiological pH.

Low-molecular-weight components of the metabolome control the proliferative activity in cellular and bacterial cultures.

The effects of blood metabolites and model cell culture exometabolites found in the human and animal blood metabolomes have been assessed. Specifically, the influence of carboxylic acids that are structural analogues of amino acids and the drug Aktoflor-C have been studied. Methods of organotypic culturing of rat tissues and Escherichia coli bacterial culturing have been used. It has been found that all the tested compounds either stimulate or inhibit cell proliferation in tissue cultures and proliferation in bacterial cultures. The metabolites that are present in blood and interstitial fluids can exert regulation effects on the tissues of the body and intestinal microbiota.

Methoxinine - an alternative stable amino acid substitute for oxidation-sensitive methionine in radiolabelled peptide conjugates

Radiolabelled peptides with high specificity and affinity towards receptors that are overexpressed by tumour cells are used in nuclear medicine for the diagnosis (imaging) and therapy of cancer. In some cases, the sequences of peptides under investigations contain methionine (Met), an amino acid prone to oxidation during radiolabelling procedures. The formation of oxidative side products can affect the purity of the final radiopharmaceutical product and/or impair its specificity and affinity towards the corresponding receptor. The replacement of Met with oxidation resistant amino acid analogues, for example, norleucine (Nle), can provide a solution. While this approach has been applied successfully to different radiolabelled peptides, a Met → Nle switch only preserves the length of the amino acid side chain important for hydrophobic interactions but not its hydrogen-bonding properties. We report here the use of methoxinine (Mox), a non-canonical amino acid that resembles more closely the electronic properties of Met in comparison to Nle. Specifically, we replaced Met15 by Mox15 and Nle15 in the binding sequence of a radiometal-labelled human gastrin derivative [d-Glu10 ]HG(10-17), named MG11 (d-Glu-Ala-Tyr-Gly-Trp-Met-Asp-Phe-NH2 ). A comparison of the physicochemical properties of 177 Lu-DOTA[X15 ]MG11 (X = Met, Nle, Mox) in vitro (cell internalization/externalization properties, receptor affinity (IC50 ), blood plasma stability and logD) showed that Mox indeed represents a suitable, oxidation-stable amino acid substitute of Met in radiolabelled peptide conjugates. Copyright © 2017 European Peptide Society and John Wiley & Sons, Ltd.

Diagnostic performance and safety of NMK36 (trans-1-amino-3-[18F] fluorocyclobutanecarboxylic acid)-PET/CT in primary prostate pancer: multicenter Phase IIb clinical trial.

OBJECTIVE We performed a multicenter Phase IIb clinical trial of NMK36, a novel amino acid analog for positron emission tomography containing trans-1-amino-3-[(18)F]fluorocyclobutanecarboxylic acid, to evaluate its safety and diagnostic performance for primary prostate cancer. METHODS Sixty-eight subjects with primary prostate cancer scheduled for radical prostatectomy or hormone therapy underwent whole-body positron emission tomography/computed tomography after injection of NMK36. The diagnostic performances of NMK36-positron emission tomography/computed tomography were evaluated for (i) regional lymph node metastasis: comparison with contrast-enhanced computed tomography under setting reference standard (histopathology or 6-month follow-up), (ii) bone metastasis: concordance rate with conventional imaging (combination of bone scintigraphy and contrast-enhanced computed tomography) and (iii) primary lesion: comparison with histopathological findings. RESULTS The accuracy of NMK36-positron emission tomography/computed tomography and contrast-enhanced computed tomography for regional lymph node metastasis were 85.5 and 87.3%, respectively. NMK36-positron emission tomography/computed tomography showed positive findings for regional lymph nodes with short-axis diameters of 5-9 mm at 23 regions in 13 patients of hormone therapy cohort, but they were not confirmed with reference standard in this study. The concordance rate of NMK36-positron emission tomography/computed tomography with conventional imaging for bone metastases was 83.3%, and seven patients had positive findings only by NMK36-positron emission tomography/computed tomography. The sensitivity and specificity of NMK36-positron emission tomography/computed tomography for primary lesion in six-segment analysis was 92.5 and 90.1%, respectively. Seven of non-serious adverse events were observed in six patients. CONCLUSIONS This study showed the comparable diagnostic performance of NMK36-positron emission tomography/computed tomography compared with conventional imaging. Some lesions of lymph node and bone were positive solely by NMK36-positron emission tomography/computed tomography, which needs to be confirmed with reference standard in future study to evaluate the usefulness of NMK36-positron emission tomography/computed tomography in staging prostate cancer.

Batch and in-flow kinetic resolution of racemic 1-(N-acylamino)alkylphosphonic and 1-(N-acylamino)alkylphosphinic acids and their esters using immobilized penicillin G acylase

The kinetic resolution of racemic 1-(N-acylamino)alkylphosphonic acids 3 (R3=OH) and their dimethyl esters 1, as well as 1-(N-acylamino)alkylphosphinic acids 4 (R3=H or Ph) using penicillin G acylase (PGA) immobilized on three types of mesoporous silicas in both a batch slurry system and in a continuous-flow reactor was studied. The initial hydrolytic deacylation rates in the presence of those catalysts were measured and the relationships between the substrate structure and the enzyme efficiency are discussed. The stereospecific hydrolysis of the N-acyl group of both racemic N-acylated phosphorus analogues of amino acids and their esters catalyzed by the immobilized PGA proved to be a highly effective method for the kinetic resolution of all the investigated compounds, with the stereochemical preference of PGA for (R)-substrates.

Optimization and in Vivo Validation of Peptide Vectors Targeting the LDL Receptor

Active targeting and delivery to pathophysiological organs of interest is of paramount importance to increase specific accumulation of therapeutic drugs or imaging agents while avoiding systemic side effects. We recently developed a family of new peptide ligands of the human and rodent LDL receptor (LDLR), an attractive cell-surface receptor with high uptake activity and local enrichment in several normal or pathological tissues (Malcor et al., J. Med. Chem. 2012, 55 (5), 2227). Initial chemical optimization of the 15-mer, all natural amino acid compound 1/VH411 (DSGL[CMPRLRGC]cDPR) and structure-activity relationship (SAR) investigation led to the cyclic 8 amino acid analogue compound 22/VH445 ([cMPRLRGC]c) which specifically binds hLDLR with a KD of 76 nM and has an in vitro blood half-life of ∼3 h. Further introduction of non-natural amino acids led to the identification of compound 60/VH4106 ([(d)-"Pen"M"Thz"RLRGC]c), which showed the highest KD value of 9 nM. However, this latter analogue displayed the lowest in vitro blood half-life (∼1.9 h). In the present study, we designed a new set of peptide analogues, namely, VH4127 to VH4131, with further improved biological properties. Detailed analysis of the hLDLR-binding kinetics of previous and new analogues showed that the latter all displayed very high on-rates, in the 106 s-1.M-1 range, and off-rates varying from the low 10-2 s-1 to the 10-1 s-1 range. Furthermore, all these new analogues showed increased blood half-lives in vitro, reaching ∼7 and 10 h for VH4129 and VH4131, respectively. Interestingly, we demonstrate in cell-based assays using both VH445 and the most balanced optimized analogue VH4127 ([cM"Thz"RLRG"Pen"]c), showing a KD of 18 nM and a blood half-life of ∼4.3 h, that its higher on-rate correlated with a significant increase in both the extent of cell-surface binding to hLDLR and the endocytosis potential. Finally, intravenous injection of tritium-radiolabeled 3H-VH4127 in wild-type or ldlr -/- mice confirmed their active LDLR targeting in vivo. Overall, this study extends our previous work toward a diversified portfolio of LDLR-targeted peptide vectors with validated LDLR-targeting potential in vivo.

Safety and Tolerability of Plecanatide in Patients with Chronic Idiopathic Constipation: Long-term Evidence from an Open-Label Study

Introduction: Plecanatide is an orally administered 16 amino acid analog of the human GI peptide uroguanylin (UG). UG binds to and activates the guanylate cyclase-C (GC-C) receptor in the proximal small intestine in a pH-dependent manner, contributing to fluid secretion and normal bowel function. Plecanatide similarly binds to GC-C in a pH-dependent manner and is being developed for the treatment of CIC. Results are presented from the recently completed long-term, open-label study of plecanatide in CIC patients. Methods: The primary objective of this multicenter study was to assess the long term safety and tolerability of once daily plecanatide for the treatment of CIC. Patients who completed a previous double-blind study (Phase 2b or either of the two Phase 3 studies), select patients who had screen failed for either of the Phase 3 studies, or select patients who had not previously been treated with plecanatide may have been eligible for this study. Enrolled patients received 52 weeks (3mg) or up to 72 weeks (6mg) of plecanatide. Dose adjustments were not permitted. In addition to spontaneous adverse event (AE) reporting, patients completed Patient Global Assessment (PGA) questionnaires which included measures of patient satisfaction and desire to continue treatment on a scale of 1 to 5 (higher scores are better). Results: There were 2370 patient exposures in this study with the majority (90.5%) receiving treatment with the 6 mg dose; 1932 (81.5%) completed or were receiving study drug at the time the study met its enrollment goal and was ended. Treatment emergent adverse events (TEAEs) were qualitatively and quantitatively similar to those observed in prior double blindstudies. The most common AEs were diarrhea (7.1%) and urinary tract infection (2.2%). The remainder of the AEs occurred in < 2% of the patients. AEs leading to discontinuation occurred in 5.3% of patients; diarrhea leading to discontinuation occurred in 3.1%. The majority of TEAEs were mild or moderate in intensity and were generally considered not related to plecanatide treatment. The median score for treatment satisfaction was 4.0 (4=quite satisfied) and for continuation of treatment was 4.0 (4=quite likely). Conclusion: In this long term trial of CIC patients, plecanatide was well tolerated with low AE rates and low discontinuation rates. PGA assessments indicate that the patients were both satisfied with their treatment and likely to continue with plecanatide therapy.

Evaluation of Prostate Cancer with Radiolabeled Amino Acid Analogs.

Conventional imaging of prostate cancer has limitations related to the frequently indolent biology of the disease. PET is a functional imaging method that can exploit various aspects of tumor biology to enable greater detection of prostate cancer than can be provided by morphologic imaging alone. Radiotracers that are in use or under investigation for targeting salient features of prostate cancer include those directed to glucose, choline, acetate, prostate-specific membrane antigen, bombesin, and amino acids. The tumor imaging features of this last class of radiotracers mirror the upregulation of transmembrane amino acid transport that is necessary in carcinomas because of increased amino acid use for energy requirements and protein synthesis. Natural and synthetic amino acids radiolabeled for PET imaging have been investigated in prostate cancer patients. Early work with naturally occurring amino acid-derived radiotracers, such as l-11C-methionine and l-1-11C-5-hydroxytryptophan, demonstrated promising results, including greater sensitivity than 18F-FDG for intraprostatic and extraprostatic cancer detection. However, limitations with naturally occurring amino acid-derived compounds, including metabolism of the radiotracer itself, led to the development of synthetic amino acid radiotracers, which are not metabolized and therefore more accurately reflect transmembrane amino acid transport. Of the synthetic amino acid-derived PET radiotracers, anti-1-amino-3-18F-fluorocyclobutane-1-carboxylic acid (18F-FACBC or 18F-fluciclovine) has undergone the most promising translation to human use, including the availability of simplified radiosynthesis. Several studies have indicated advantageous biodistribution in the abdomen and pelvis with little renal excretion and bladder activity-characteristics beneficial for prostate cancer imaging. Studies have demonstrated improved lesion detection and diagnostic performance of 18F-fluciclovine in comparison with conventional imaging, especially for recurrent prostate cancer, although issues with nonspecific uptake limit the potential role of 18F-fluciclovine in the diagnosis of primary prostate cancer. Although work is ongoing, recently published intrapatient comparisons of 18F-fluciclovine with 11C-choline reported higher overall diagnostic performance of the former, especially for the detection of disease relapse. This review is aimed at providing a detailed overview of amino acid-derived PET compounds that have been studied for use in prostate cancer imaging.

Intermolecular Amination of Unactivated C(sp3 )-H Bonds with Cyclic Alkylamines: Formation of C(sp3 )-N Bonds through Copper/Oxygen-Mediated C(sp3 )-H/N-H Activation.

The first example of intermolecular amination of unactivated C(sp3 )-H bonds by cyclic alkylamines mediated by Cu(OAc)2 /O2 is reported. This method avoids the use of benzoyloxyamines as the aminating reagent, which are normally prepared from alkylamines in extra steps. A variety of unnatural β2, 2 -amino acid analogues are synthesized by this simple and efficient procedure. This approach offers a solution to the previous unmet challenge of C(sp3 )-H/N-H activation for the formation of C(sp3 )-N bonds.

Very low protein diets supplemented with keto-analogues in ESRD predialysis patients and its effect on vascular stiffness and AVF Maturation.

BackgroundNative arteriovenous fistula (AVF) is the most appropriate type of vascular access for chronic dialysis. Its patency rates depend on vascular wall characteristics. Ketoacid analogues of essential amino acids (KA/EAA) are prescribed in end-stage renal disease (ESRD) pre-dialysis patients to lower toxic metabolic products generation and improve nutritional status. We hypothesized that very-low protein diet (VLPD) supplemented with KA/EAA may influence arterial wall stiffness and affect AVF maturation rates and duration in pre-dialysis ESRD patients.MethodsIn a prospective, cohort, 3 years study we enrolled 67 consecutive non-diabetic early referral ESRD patients that underwent AVF creation in our hospital. Patients were divided in two groups based on their regimen 12 months prior to surgery: a VLPD supplemented with KA/EAA study group versus a low protein diet non-KA/EAA-supplemented control group. For each patient we performed serum analysis for the parameters of bone mineral disease, inflammation and nutritional status, one pulse wave velocity (PWV) measurement and one Doppler ultrasound (US) determination prior the surgery, followed by consequent Doppler US assessments at 4, 6, 8 and 12 weeks after it. Rates and duration of mature AVF achievement were noted. We used logistic regression to analyze the association between AVF maturation and KA/EAA administration, by comparing rates and durations between groups, unadjusted and adjusted for systolic blood pressure, C-reactive protein, PWV, phosphorus values. All parameters in the logistic model were transformed in binary variables. A p-value < α = 0.05 was considered significant; data were processed using SPSS 16 software and Excel.ResultsIn the study group (n = 28, aged 57 ± 12.35, 13 females) we registered better serum phosphate (p = 0.022) and C-reactive protein control (p = 0.021), lower PWV (p = 0.007) and a higher percent of AVF creation success (33.3 % versus 17.8 %, p < 0.05). AVF maturation duration was lower in study group (5.91 versus 7.15 weeks, p < 0.001).ConclusionsVLPD supplemented with KA/EAA appear to improve the native AVF primary outcome, decreasing the initial vascular stiffness, possible by preserving vascular wall quality in CKD patients through a better serum phosphate levels control and the limitation of inflammatory response.

Enantiomeric α,β-diaminoethylphosphonic acids as potent inhibitors of aminopeptidases—stereoselective synthesis and biological activity

α,β-Diaminoethylphosphonic acids are efficient transition-state analogue inhibitors of alanyl aminopeptidase and endoplasmic reticulum aminopeptidase 2, biomedically important members of the M01 family of proteases. In this work, the individual enantiomers of selected inhibitors were separated and their inhibitory potency verified. The synthesis involved the use of a chiral α-methylbenzylamine to obtain diastereomeric aziridinephosphonates as key intermediates of the multistep process. The diastereomeric aziridines were resolved chromatographically, and the heterocyclic ring opened with the appropriate amines. The inhibition study revealed an interesting correlation between the absolute configuration and inhibitory activity. Certain enantiomeric phosphonic amino acid analogues exhibited inhibition constants in the nanomolar range.

Evaluating Fmoc-amino acids as selective inhibitors of butyrylcholinesterase.

Cholinesterases are involved in neuronal signal transduction, and perturbation of function has been implicated in diseases, such as Alzheimer's and Huntington's disease. For the two major classes of cholinesterases, such as acetylcholinesterase (AChE) and butyrylcholinesterase (BChE), previous studies reported BChE activity is elevated in patients with Alzheimer's disease, while AChE levels remain the same or decrease. Thus, the development of potent and specific inhibitors of BChE have received much attention as a potential therapeutic in the alleviation of neurodegenerative diseases. In this study, we evaluated amino acid analogs as selective inhibitors of BChE. Amino acid analogs bearing a 9-fluorenylmethyloxycarbonyl (Fmoc) group were tested, as the Fmoc group has structural resemblance to previously described inhibitors. We identified leucine, lysine, and tryptophan analogs bearing the Fmoc group as selective inhibitors of BChE. The Fmoc group contributed to inhibition, as analogs bearing a carboxybenzyl group showed ~tenfold higher values for the inhibition constant (K I value). Inclusion of a t-butoxycarbonyl on the side chain of Fmoc tryptophan led to an eightfold lower K I value compared to Fmoc tryptophan alone suggesting that modifications of the amino acid side chains may be designed to create inhibitors with higher affinity. Our results identify Fmoc-amino acids as a scaffold upon which to design BChE-specific inhibitors and provide the foundation for further experimental and computational studies to dissect the interactions that contribute to inhibitor binding.

Residue-specific Incorporation of Noncanonical Amino Acids into Model Proteins Using an &lt;em&gt;Escherichia coli&lt;/em&gt; Cell-free Transcription-translation System

The canonical set of amino acids leads to an exceptionally wide range of protein functionality. Nevertheless, the set of residues still imposes limitations on potential protein applications. The incorporation of noncanonical amino acids can enlarge this scope. There are two complementary approaches for the incorporation of noncanonical amino acids. For site-specific incorporation, in addition to the endogenous canonical translational machineries, an orthogonal aminoacyl-tRNA-synthetase-tRNA pair must be provided that does not interact with the canonical ones. Consequently, a codon that is not assigned to a canonical amino acid, usually a stop codon, is also required. This genetic code expansion enables the incorporation of a noncanonical amino acid at a single, given site within the protein. The here presented work describes residue-specific incorporation where the genetic code is reassigned within the endogenous translational system. The translation machinery accepts the noncanonical amino acid as a surrogate to incorporate it at canonically prescribed locations, i.e., all occurrences of a canonical amino acid in the protein are replaced by the noncanonical one. The incorporation of noncanonical amino acids can change the protein structure, causing considerably modified physical and chemical properties. Noncanonical amino acid analogs often act as cell growth inhibitors for expression hosts since they modify endogenous proteins, limiting in vivo protein production. In vivo incorporation of toxic noncanonical amino acids into proteins remains particularly challenging. Here, a cell-free approach for a complete replacement of L-arginine by the noncanonical amino acid L-canavanine is presented. It circumvents the inherent difficulties of in vivo expression. Additionally, a protocol to prepare target proteins for mass spectral analysis is included. It is shown that L-lysine can be replaced by L-hydroxy-lysine, albeit with lower efficiency. In principle, any noncanonical amino acid analog can be incorporated using the presented method as long as the endogenous in vitro translation system recognizes it.

Residue-specific Incorporation of Noncanonical Amino Acids into Model Proteins Using an Escherichia coli Cell-free Transcription-translation System.

The canonical set of amino acids leads to an exceptionally wide range of protein functionality. Nevertheless, the set of residues still imposes limitations on potential protein applications. The incorporation of noncanonical amino acids can enlarge this scope. There are two complementary approaches for the incorporation of noncanonical amino acids. For site-specific incorporation, in addition to the endogenous canonical translational machineries, an orthogonal aminoacyl-tRNA-synthetase-tRNA pair must be provided that does not interact with the canonical ones. Consequently, a codon that is not assigned to a canonical amino acid, usually a stop codon, is also required. This genetic code expansion enables the incorporation of a noncanonical amino acid at a single, given site within the protein. The here presented work describes residue-specific incorporation where the genetic code is reassigned within the endogenous translational system. The translation machinery accepts the noncanonical amino acid as a surrogate to incorporate it at canonically prescribed locations, i.e., all occurrences of a canonical amino acid in the protein are replaced by the noncanonical one. The incorporation of noncanonical amino acids can change the protein structure, causing considerably modified physical and chemical properties. Noncanonical amino acid analogs often act as cell growth inhibitors for expression hosts since they modify endogenous proteins, limiting in vivo protein production. In vivo incorporation of toxic noncanonical amino acids into proteins remains particularly challenging. Here, a cell-free approach for a complete replacement of L-arginine by the noncanonical amino acid L-canavanine is presented. It circumvents the inherent difficulties of in vivo expression. Additionally, a protocol to prepare target proteins for mass spectral analysis is included. It is shown that L-lysine can be replaced by L-hydroxy-lysine, albeit with lower efficiency. In principle, any noncanonical amino acid analog can be incorporated using the presented method as long as the endogenous in vitro translation system recognizes it.

Discovery of potent and selective inhibitors of human aminopeptidases ERAP1 and ERAP2 by screening libraries of phosphorus-containing amino acid and dipeptide analogues

A collection of fifty phosphonic and phosphinic acids was screened for inhibition of ERAP1 and ERAP2, the human endoplasmic reticulum aminopeptidases. The cooperative action of these enzymes is manifested by trimming a variety of antigenic precursors to be presented on the cell surface by major histocompatibility class I. The SAR studies revealed several potent compounds, particularly among the phosphinic dipeptide analogues, that were strong inhibitors of ERAP2 (Ki=100–350nM). A wide structural diversity of the applied organophosphorus compounds, predominantly non-proteinogenic analogues, allowed identification of representatives selective toward only one form of ERAP. For example, N′-substituted α,β-diaminophosphonates and phosphinates exhibited potency only toward ERAP2, which is in agreement with the P1 basic substrate-oriented specificity. Such discriminating ligands are invaluable tools for elucidating the precise role of a particular aminopeptidase in the concerted function of antigen processing and in human diseases.

Design and synthesis of N-benzoyl amino acid derivatives as DNA methylation inhibitors.

The inhibition of human DNA Methyl Transferases (DNMT) is a novel promising approach to address the epigenetic dysregulation of gene expression in different diseases. Inspired by the validated virtual screening hit NSC137546, a series of N-benzoyl amino acid analogues was synthesized and obtained compounds were assessed for their ability to inhibit DNMT-dependent DNA methylation in vitro. The biological screening allowed the definition of a set of preliminary structure-activity relationships and the identification of compounds promising for further development. Among the synthesized compounds, L-glutamic acid derivatives 22, 23, and 24 showed the highest ability to prevent DNA methylation in a total cell lysate. Compound 22 inhibited DNMT1 and DNMT3A activity in a concentration-dependent manner in the micromolar range. In addition, compound 22 proved to be stable in human serum and it was thus selected as a starting point for further biological studies.

Incorporation of β-Silicon-β3-Amino Acids in the Antimicrobial Peptide Alamethicin Provides a 20-Fold Increase in Membrane Permeabilization.

Incorporation of silicon-containing amino acids in peptides is known to endow the peptide with desirable properties such as improved proteolytic stability and increased lipophilicity. In the presented study, we demonstrate that incorporation of β-silicon-β3-amino acids into the antimicrobial peptide alamethicin provides the peptide with improved membrane permeabilizing properties. A robust synthetic procedure for the construction of β-silicon-β3-amino acids was developed and the amino acid analogues were incorporated into alamethicin at different positions of the hydrophobic face of the amphipathic helix by using SPPS. The incorporation was shown to provide up to 20-fold increase in calcein release as compared with wild-type alamethicin.

Unnatural amino acid analogues of membrane-active helical peptides with anti-mycobacterial activity and improved stability.

The emergence of MDR-TB, coupled with shrinking antibiotic pipelines, has increased demands for new antimicrobials with novel mechanisms of action. Antimicrobial peptides have increasingly been explored as promising alternatives to antibiotics, but their inherent poor in vivo stability remains an impediment to their clinical utility. We therefore systematically evaluated unnatural amino acid-modified peptides to design analogues with enhanced anti-mycobacterial activities. Anti-mycobacterial activities were evaluated in vitro and intracellularly against drug-susceptible and MDR isolates of Mycobacterium tuberculosis using MIC, killing efficacy and intracellular growth inhibition studies. Toxicity profiles were assessed against mammalian cells to verify cell selectivity. Anti-mycobacterial mechanisms were investigated using microfluidic live-cell imaging with time-lapse fluorescence microscopy and confocal laser-scanning microscopy. Unnatural amino acid incorporation was well tolerated without an appreciable effect on toxicity profiles and secondary conformations of the synthetic peptides. The modified peptides also withstood proteolytic digestion by trypsin. The all d-amino acid peptide, i(llkk)2i (II-D), displayed superior activity against all six mycobacterial strains tested, with a 4-fold increase in selectivity index as compared with the unmodified l-amino acid peptide in broth. II-D effectively reduced the intracellular bacterial burden of both drug-susceptible and MDR clinical isolates of M. tuberculosis after 4 days of treatment. Live-cell imaging studies demonstrated that II-D permeabilizes the mycobacterial membrane, while confocal microscopy revealed that II-D not only permeates the cell membrane, but also accumulates within the cytoplasm. Unnatural amino acid modifications not only decreased the susceptibility of peptides to proteases, but also enhanced mycobacterial selectivity.

Distinct stress conditions result in aggregation of proteins with similar properties.

Protein aggregation is the abnormal association of proteins into larger aggregate structures which tend to be insoluble. This occurs during normal physiological conditions and in response to age or stress-induced protein misfolding and denaturation. In this present study we have defined the range of proteins that aggregate in yeast cells during normal growth and after exposure to stress conditions including an oxidative stress (hydrogen peroxide), a heavy metal stress (arsenite) and an amino acid analogue (azetidine-2-carboxylic acid). Our data indicate that these three stress conditions, which work by distinct mechanisms, promote the aggregation of similar types of proteins probably by lowering the threshold of protein aggregation. The proteins that aggregate during physiological conditions and stress share several features; however, stress conditions shift the criteria for protein aggregation propensity. This suggests that the proteins in aggregates are intrinsically aggregation-prone, rather than being proteins which are affected in a stress-specific manner. We additionally identified significant overlaps between stress aggregating yeast proteins and proteins that aggregate during ageing in yeast and C. elegans. We suggest that similar mechanisms may apply in disease- and non-disease settings and that the factors and components that control protein aggregation may be evolutionary conserved.

NFκB is a central regulator of protein quality control in response to protein aggregation stresses via autophagy modulation.

During cell life, proteins often misfold, depending on particular mutations or environmental changes, which may lead to protein aggregates that are toxic for the cell. Such protein aggregates are the root cause of numerous diseases called "protein conformational diseases," such as myofibrillar myopathy and familial amyotrophic lateral sclerosis. To fight against aggregates, cells are equipped with protein quality control mechanisms. Here we report that NFκB transcription factor is activated by misincorporation of amino acid analogues into proteins, inhibition of proteasomal activity, expression of the R120G mutated form of HspB5 (associated with myofibrillar myopathy), or expression of the G985R and G93A mutated forms of superoxide dismutase 1 (linked to familial amyotrophic lateral sclerosis). This noncanonical stimulation of NFκB triggers the up-regulation of BAG3 and HspB8 expression, two activators of selective autophagy, which relocalize to protein aggregates. Then NFκB-dependent autophagy allows the clearance of protein aggregates. Thus NFκB appears as a central and major regulator of protein aggregate clearance by modulating autophagic activity. In this context, the pharmacological stimulation of this quality control pathway might represent a valuable strategy for therapies against protein conformational diseases.