Brønsted Acid-Lewis Acid (BA-LA) Induced Final Deprotection/Peptide Resin Cleavage in Fmoc/t-Bu Solid-Phase Peptide Synthesis: HCl/FeCl3 and AcOH/FeCl3 as Viable PFAS-Free Alternatives for TFA.

The widely used Fmoc/t-Bu solid-phase peptide synthesis (SPPS) is hampered by relying on corrosive, per/polyfluoroalkyl substance (PFAS) classified trifluoroacetic acid (TFA) as a universal protecting group (PG) removal/resin cleavage reagent. We report that suitable combinations of Brønsted acids (BAs) and Lewis acids (LAs) such as HCl/FeCl3 and AcOH/FeCl3 constitute viable alternatives for TFA as PFAS-free cleavage agents. Using water miscible dimethyl carbonate (DMC) and acetonitrile (MeCN) as solvents enabled diluting cleavage mixtures with suitable aqueous solutions, allowing for direct use in purification in which removal of >99.99% iron from an HCl/FeCl3 induced cleavage was demonstrated.

Unexpected binding modes of inhibitors to the histone chaperone ASF1 revealed by a foldamer scanning approach.

In the search for foldamer inhibitors of the histone chaperone ASF1, we explored the possibility of substituting four α-residues (≈one helix turn) by 3-urea segments and scanned the sequence of a short α-helical peptide known to bind ASF1. By analysing the impact of the different foldamer replacements within the peptide chain, we uncovered new binding modes of the peptide-urea chimeras to ASF1.

Iterative Structure-Based Optimization of Short Peptides Targeting the Bacterial Sliding Clamp.

The bacterial DNA sliding clamp (SC), or replication processivity factor, is a promising target for the development of novel antibiotics. We report a structure-activity relationship study of a new series of peptides interacting within the Escherichia coli SC (EcSC) binding pocket. Various modifications were explored including N-alkylation of the peptide bonds, extension of the N-terminal moiety, and introduction of hydrophobic and constrained residues at the C-terminus. In each category, single modifications were identified that increased affinity to EcSC. A combination of such modifications yielded in several cases to a substantially increased affinity compared to the parent peptides with Kd in the range of 30-80 nM. X-ray structure analysis of 11 peptide/EcSC co-crystals revealed new interactions at the peptide-protein interface (i.e., stacking interactions, hydrogen bonds, and hydrophobic contacts) that can account for the improved binding. Several compounds among the best binders were also found to be more effective in inhibiting SC-dependent DNA synthesis.

Correction: Antibacterial activity of a dual peptide targeting the Escherichia coli sliding clamp and the ribosome.

[This corrects the article DOI: 10.1039/D0CB00060D.].

Set1-dependent H3K4 methylation becomes critical for limiting DNA damage in response to changes in S-phase dynamics in Saccharomyces cerevisiae.

DNA replication is a highly regulated process that occurs in the context of chromatin structure and is sensitive to several histone post-translational modifications. In Saccharomyces cerevisiae, the histone methylase Set1 is responsible for the transcription-dependent deposition of H3K4 methylation (H3K4me) throughout the genome. Here we show that a combination of a hypomorphic replication mutation (orc5-1) with the absence of Set1 (set1Δ) compromises the progression through S-phase, and this is associated with a large increase in DNA damage. The ensuing DNA damage checkpoint activation, in addition to that of the spindle assembly checkpoint, restricts the growth of orc5-1 set1Δ. The opposite effects of the lack of RNase H activity and the reduction of histone levels on orc5-1 set1Δ viability are in agreement with their expected effects on replication fork progression. We propose that the role of H3K4 methylation during DNA replication becomes critical when the replication forks acceleration due to decreased origin firing in the orc5-1 background increases the risk for transcription replication conflicts. Furthermore, we show that an increase of reactive oxygen species levels, likely a consequence of the elevated DNA damage, is partly responsible for the lethality in orc5-1 set1Δ.

Optimal anchoring of a foldamer inhibitor of ASF1 histone chaperone through backbone plasticity.

Sequence-specific oligomers with predictable folding patterns, i.e., foldamers, provide new opportunities to mimic α-helical peptides and design inhibitors of protein-protein interactions. One major hurdle of this strategy is to retain the correct orientation of key side chains involved in protein surface recognition. Here, we show that the structural plasticity of a foldamer backbone may notably contribute to the required spatial adjustment for optimal interaction with the protein surface. By using oligoureas as α helix mimics, we designed a foldamer/peptide hybrid inhibitor of histone chaperone ASF1, a key regulator of chromatin dynamics. The crystal structure of its complex with ASF1 reveals a notable plasticity of the urea backbone, which adapts to the ASF1 surface to maintain the same binding interface. One additional benefit of generating ASF1 ligands with nonpeptide oligourea segments is the resistance to proteolysis in human plasma, which was highly improved compared to the cognate α-helical peptide.

Calcium Chelation by Phosphate Ions and Its Influence on Fouling Mechanisms of Whey Protein Solutions in a Plate Heat Exchanger.

Fouling of plate heat exchangers (PHEs) is a recurring problem when pasteurizing whey protein solutions. As Ca2+ is involved in denaturation/aggregation mechanisms of whey proteins, the use of calcium chelators seems to be a way to reduce the fouling of PHEs. Unfortunately, in depth studies investigating the changes of the whey protein fouling mechanism in the presence of calcium chelators are scarce. To improve our knowledge, reconstituted whey protein isolate (WPI) solutions were prepared with increasing amounts of phosphate, expressed in phosphorus (P). The fouling experiments were performed on a pilot-scale PHE, while monitoring the evolution of the pressure drop and heat transfer coefficient. The final deposit mass distribution and structure of the fouling layers were investigated, as well as the whey protein denaturation kinetics. Results suggest the existence of two different fouling mechanisms taking place, depending on the added P concentration in WPI solutions. For added P concentrations lower or equal to 20 mg/L, a spongy fouling layer consists of unfolded protein strands bound by available Ca2+. When the added P concentration is higher than 20 mg/L, a heterogeneously distributed fouling layer formed of calcium phosphate clusters covered by proteins in an arborescence structure is observed.

MRX Increases Chromatin Accessibility at Stalled Replication Forks to Promote Nascent DNA Resection and Cohesin Loading.

The recovery of stalled replication forks depends on the controlled resection of nascent DNA and on the loading of cohesin. These processes operate in the context of nascent chromatin, but the impact of nucleosome structure on a fork restart remains poorly understood. Here, we show that the Mre11-Rad50-Xrs2 (MRX) complex acts together with the chromatin modifiers Gcn5 and Set1 and the histone remodelers RSC, Chd1, and Isw1 to promote chromatin remodeling at stalled forks. Increased chromatin accessibility facilitates the resection of nascent DNA by the Exo1 nuclease and the Sgs1 and Chl1 DNA helicases. Importantly, increased ssDNA promotes the recruitment of cohesin to arrested forks in a Scc2-Scc4-dependent manner. Altogether, these results indicate that MRX cooperates with chromatin modifiers to orchestrate the action of remodelers, nucleases, and DNA helicases, promoting the resection of nascent DNA and the loading of cohesin, two key processes involved in the recovery of arrested forks.

Antibacterial activity of a dual peptide targeting the Escherichia coli sliding clamp and the ribosome.

The bacterial processivity factor, or sliding clamp (SC), is a target of choice for new antibacterial drugs development. We have previously developed peptides that target Escherichia coli SC and block its interaction with DNA polymerases in vitro. Here, one such SC binding peptide was fused to a Proline-rich AntiMicrobial Peptide (PrAMP) to allow its internalization into E. coli cells. Co-immunoprecipitation assays with a N-terminally modified bifunctional peptide that still enters the bacteria but fails to interact with the bacterial ribosome, the major target of PrAMPs, demonstrate that it actually interacts with the bacterial SC. Moreover, when compared to SC non-binding controls, this peptide induces a ten-fold higher antibacterial activity against E. coli, showing that the observed antimicrobial activity is linked to SC binding. Finally, an unmodified bifunctional compound significantly increases the survival of Drosophila melanogaster flies challenged by an E. coli infection. Our study demonstrates the potential of PrAMPs to transport antibiotics into the bacterial cytoplasm and validates the development of drugs targeting the bacterial processivity factor of Gram-negative bacteria as a promising new class of antibiotics.

Interaction of a Model Peptide on Gram Negative and Gram Positive Bacterial Sliding Clamps.

Bacterial sliding clamps control the access of DNA polymerases to the replication fork and are appealing targets for antibacterial drug development. It is therefore essential to decipher the polymerase-clamp binding mode across various bacterial species. Here, two residues of the E. coli clamp binding pocket, EcS346 and EcM362, and their cognate residues in M. tuberculosis and B. subtilis clamps, were mutated. The effects of these mutations on the interaction of a model peptide with these variant clamps were evaluated by thermodynamic, molecular dynamics, X-rays crystallography, and biochemical analyses. EcM362 and corresponding residues in Gram positive clamps occupy a strategic position where a mobile residue is essential for an efficient peptide interaction. EcS346 has a more subtle function that modulates the pocket folding dynamics, while the equivalent residue in B. subtilis is essential for polymerase activity and might therefore be a Gram positive-specific molecular marker. Finally, the peptide binds through an induced-fit process to Gram negative and positive pockets, but the complex stability varies according to a pocket-specific network of interactions.

Formation of ß-Lactoglobulin Aggregates from Quite, Unfolded Conformations upon Heat Activation.

In presence of calcium ions, ß-lactoglobulin (BLG) unfolds and subsequently aggregates after heating. This process has important pharmaceutical and agroalimentary applications. Nowadays, the molecular mechanism of unfolding and BLG aggregation, and the role of calcium in the mechanism, is poorly understood. Actually, in most studies, data have been acquired at room temperature, after heating and after aggregation, which makes it difficult to establish a clear causal-temporal relation between calcium binding, heat, and aggregation. Thus, the goal of the present study is to get accurate, nanoscale data about the molecular events leading to BLG unfolding and calcium-dependent aggregation. The molecular transformation of BLG during heating has been investigated, using the NMR pulse field gradient technique, operating in a high field (900 MHz). Thanks to this technique, the molecular conformation of newly formed unfolded BLG molecules can be distinguished in a large pool of native ones. The present work shows that BLG at neutral pH at 65 °C displays fast, cooperative-like unfolding, in which no long-lived intermediary state (as a molten globule one) is detected, before aggregation. These data also indicate that calcium ions bind unfolded BLG in specific sites which might be a necessary feature to form the aggregate. Finally, these data also provide an NMR-based methodology to monitor the rate of protein unfolding using NMR.

12/10-Helix in Mixed ß-Peptides Alternating Bicyclic and Acyclic ß-Amino Acids: Probing the Relationship between Bicyclic Side Chain and Helix Stability.

12/10-Helices constitute suitable templates that can be used to design original structures. Nevertheless, they often suffer from a weak stability in polar solvents because they exhibit a mixed hydrogen-bond network resulting in a small macrodipole. In this work, stable and functionalizable 12/10-helices were developed by alternating a highly constrained ß2, 3, 3 -trisubstituted bicyclic amino acid (S)-1-aminobicyclo[2.2.2]octane-2-carboxylic acid ((S)-ABOC) and an acyclic substituted ß-homologated proteinogenic amino acid (l-ß3 -hAA). Based on NMR spectroscopic analysis, it was shown that such mixed ß-peptides display well-defined right-handed 12/10-helices in polar, apolar, and chaotropic solvents; that are, CD3 OH, CDCl3 , and [D6 ]DMSO, respectively. The stability of the hydrogen bonds forming the C10 and C12 pseudocycles as well as the benefit provided by the use of the constrained bicyclic ABOC versus typical acyclic ß-amino acids sequences when designing 12/10-helix were investigated using NH/ND NMR exchange experiments and DFT calculations in various solvents. These studies showed that the ß3 -hAA/(S)-ABOC helix displayed a more stable hydrogen-bond network through specific stabilization of the C10 pseudocycles involving the bridgehead NH of the ABOC bicyclic scaffold.

Antifouling amphiphilic silicone coatings for dairy fouling mitigation on stainless steel.

Pasteurization of dairy products is plagued by fouling, which induces significant economic, environmental and microbiological safety concerns. Herein, an amphiphilic silicone coating was evaluated for its efficacy against fouling by a model dairy fluid in a pilot pasteurizer and against foodborne bacterial adhesion. The coating was formed by modifying an RTV silicone with a PEO-silane amphiphile comprised of a PEO segment and flexible siloxane tether ([(EtO)3Si-(CH2)2-oligodimethylsiloxanem-block-(OCH2CH2)n-OCH3]). Contact angle analysis of the coating revealed that the PEO segments were able to migrate to the aqueous interface. The PEO-modified silicone coating applied to pretreated stainless steel was exceptionally resistant to fouling. After five cycles of pasteurization, these coated substrata were subjected to a standard clean-in-place process and exhibited a minor reduction in fouling resistance in subsequent tests. However, the lack of fouling prior to cleaning indicates that harsh cleaning is not necessary. PEO-modified silicone coatings also showed exceptional resistance to adhesion by foodborne pathogenic bacteria.

Antifouling Biomimetic Liquid-Infused Stainless Steel: Application to Dairy Industrial Processing.

Fouling is a widespread and costly issue, faced by all food-processing industries. Particularly, in the dairy sector, where thermal treatments are mandatory to ensure product safety, heat-induced fouling represents up to 80% of the total production costs. Significant environmental impacts, due the massive consumption of water and energy, are also to deplore. Fouling control solutions are thus desperately needed, as they would lead to substantial financial gains as well as tremendous progress toward eco-responsible processes. This work aims at presenting a novel and very promising dairy fouling-mitigation strategy, inspired by nature, and to test its antifouling performances in real industrial conditions. Slippery liquid-infused surfaces were successfully designed directly on food grade stainless steel, via femtosecond laser ablation, followed by fluorosilanization and impregnation with an inert perfluorinated oil. Resulting hydrophobic surfaces (water contact angle of 112°) exhibited an extremely slippery nature (contact angle hysteresis of 0.6°). Outstanding fouling-release performances were obtained for these liquid-infused surfaces as absolutely no trace of dairy deposit was found after 90 min of pasteurization test in pilot-scale equipment followed by a short water rinse.

Targeting Binding Function-3 of the Androgen Receptor Blocks Its Co-Chaperone Interactions, Nuclear Translocation, and Activation.

The development of new antiandrogens, such as enzalutamide, or androgen synthesis inhibitors like abiraterone has improved patient outcomes in the treatment of advanced prostate cancer. However, due to the development of drug resistance and tumor cell survival, a majority of these patients progress to the refractory state of castration-resistant prostate cancer (CRPC). Thus, newer therapeutic agents and a better understanding of their mode of action are needed for treating these CRPC patients. We demonstrated previously that targeting the Binding Function 3 (BF3) pocket of the androgen receptor (AR) has great potential for treating patients with CRPC. Here, we explore the functional activity of this site by using an advanced BF3-specific small molecule (VPC-13566) that was previously reported to effectively inhibit AR transcriptional activity and to displace the BAG1L peptide from the BF3 pocket. We show that VPC-13566 inhibits the growth of various prostate cancer cell lines, including an enzalutamide-resistant cell line, and reduces the growth of AR-dependent prostate cancer xenograft tumors in mice. Importantly, we have used this AR-BF3 binder as a chemical probe and identified a co-chaperone, small glutamine-rich tetratricopeptide repeat (TPR)-containing protein alpha (SGTA), as an important AR-BF3 interacting partner. Furthermore, we used this AR-BF3-directed small molecule to demonstrate that inhibition of AR activity through the BF3 functionality can block translocation of the receptor into the nucleus. These findings suggest that targeting the BF3 site has potential clinical importance, especially in the treatment of CRPC and provide novel insights on the functional role of the BF3 pocket. Mol Cancer Ther; 15(12); 2936-45. ©2016 AACR.

12/14/14-Helix Formation in 2:1 α/ß-Hybrid Peptides Containing Bicyclo[2.2.2]octane Ring Constraints.

The highly constrained ß-amino acid ABOC induces different types of helices in ß urea and 1:1 α/ß amide oligomers. The latter can adopt 11/9- and 18/16-helical folds depending on the chain length in solution. Short peptides alternating proteinogenic α-amino acids and ABOC in a 2:1 α/ß repeat pattern adopted an unprecedented and stable 12/14/14-helix. The structure was established through extensive NMR, molecular dynamics, and IR studies. While the 1:1 α-AA/ABOC helices diverged from the canonical α-helix, the helix formed by the 9-mer 2:1 α/ß-peptide allowed the projection of the α-amino acid side chains in a spatial arrangement according to the α-helix. Such a finding constitutes an important step toward the conception of functional tools that use the ABOC residue as a potent helix inducer for biological applications.

Identification of a potent antiandrogen that targets the BF3 site of the androgen receptor and inhibits enzalutamide-resistant prostate cancer.

There has been a resurgence of interest in the development of androgen receptor (AR) inhibitors with alternative modes of action to overcome the development of resistance to current therapies. We demonstrated previously that one promising strategy for combatting mutation-driven drug resistance is to target the Binding Function 3 (BF3) pocket of the receptor. Here we report the development of a potent BF3 inhibitor, 3-(2,3-dihydro-1H-indol-2-yl)-1H-indole, which demonstrates excellent antiandrogen potency and anti-PSA activity and abrogates the androgen-induced proliferation of androgen-sensitive (LNCaP) and enzalutamide-resistant (MR49F) PCa cell lines. Moreover, this compound effectively reduces the expression of AR-dependent genes in PCa cells and effectively inhibits tumor growth in vivo in both LNCaP and MR49F xenograft models. These findings provide evidence that targeting the AR BF3 pocket represents a viable therapeutic approach to treat patients with advanced and/or resistant prostate cancer.

Unprecedented chain-length-dependent conformational conversion between 11/9 and 18/16†helix in α/ß-hybrid peptides.

α,ß-Hybrid oligomers of varying lengths with alternating proteogenic α-amino acid and the rigid ß(2,3,3) -trisubstituted bicyclic amino acid ABOC residues were studied using both X-ray crystal and NMR solution structures. While only an 11/9 helix was obtained in the solid state regardless of the length of the oligomers, conformational polymorphism as a chain-length-dependent phenomenon was observed in solution. Consistent with DFT calculations, we established that short oligomers adopted an 11/9 helix, whereas an 18/16 helix was favored for longer oligomers in solution. A rapid interconversion between the 11/9 helix and the 18/16 helix occurred for oligomers of intermediate length.

Spatially distinct functions of Clb2 in the DNA damage response.

In budding yeast four mitotic cyclins (Clb1-4) cooperate in a partially redundant manner to bring about M-phase specific events, including the apical isotropic switch that ends polarized bud growth initiated at bud emergence. How exactly this morphogenetic transition is regulated by mitotic CDKs remains poorly understood. We have taken advantage of the isotropic bud growth that prevails in cells responding to DNA damage to unravel the contribution of mitotic cyclins in this cellular context. We find that clb2∆, in contrast to the other mitotic cyclin mutants, inappropriately respond to the presence of DNA damage. This aberrant response is characterized by a Cdc42- and Bni1-dependent but Cln-independent resumption of polarized bud growth after a brief period of actin depolarization. Biochemical and genetic evidence is presented that formally excludes the possibility of indirect effects due for instance to unrestrained APC activity, untimely mitotic exit or Swe1-mediated CDK inhibition. Importantly, our data demonstrate that in order to maintain the characteristic dumbbell arrest phenotype upon checkpoint activation Clb2 needs to be efficiently exported into the cytoplasm. We propose that the inhibition of mitotic cyclin destruction by the DNA damage checkpoint pathway leads to a buildup of Clb2 in the cytoplasm where this cyclin can stabilize the apical isotropic switch throughout a G 2/M checkpoint arrest. Our study also unveils an essential role of nuclear Clb2 in both survival and adaptation to the DNA damage checkpoint, illustrating a spatially distinct dual function of this mitotic cyclin in the response to DNA damage.

Mixed oligoureas based on constrained bicyclic and acyclic ß-amino acids derivatives: on the significance of the subunit configuration for folding.

The combination of a non-functionalized constrained bicyclo[2.2.2]octane motif along with urea linkages allowed the formation of a highly rigid 2.5(12/14) helical system both in solution and the solid state. In this work, we aimed at developing stable and functionalized systems as promising materials for biological applications in investigating the impact of this constrained motif and its configuration on homo and heterochiral mixed-oligourea helix formation. Di-, tetra-, hexa-, and octa-oligoureas alternating the highly constrained bicyclic motif of (R) or (S) configuration with acyclic (S)-ß(3)-amino acid derivatives were constructed. Circular dichroism (CD), NMR experiments, and the X-ray crystal structure of the octamer unequivocally proved that the alternating heterochiral R/S sequences form a stable left-handed 2.5-helix in contrast to the mixed (S/S)-oligoureas, which did not adopt any defined secondary structure. We observed that the (-)-synclinal conformation around the C(α)-C(ß) bond of the acyclic residues, although sterically less favorable than the (+)-synclinal conformation, was imposed by the (R)-bicyclic amino carbamoyl (BAC) residue. This highlighted the strong ability of the BAC residue to drive helical folding in heterochiral compounds. The role of the stereochemistry of the BAC unit was assessed and a model was proposed to explain the misfolding of the S/S sequences.