DPP-4 Cleaves α/ß-Peptide Bonds: Substrate Specificity and Half-Lives.

The incorporation of ß-amino acids into a peptide sequence has gained particular attention as ß- and α/ß-peptides have shown remarkable proteolytic stability, even after a single homologation at the scissile bond. Several peptidases have been shown to cleave such bonds with high specificity but at a much slower rate compared to α-peptide bonds. In this study, a series of analogs of dipeptidyl peptidase-4 (DPP-4) substrate inhibitors were synthesized in order to investigate whether ß-amino acid homologation at the scissile bond could be a valid approach to improving peptide stability towards DPP-4 degradation. DPP-4 cleaved the α/ß-peptide bond after the N-terminal penultimate Pro with a broad specificity and retained full activity regardless of the ß3 -amino acid side chain and peptide length. Significantly improved half-lives were observed for ß3 Ile-containing peptides. Replacing the penultimate Pro with a conformationally constrained Pro mimetic led to proteolytic resistance. DPP-4 cleavage of α/ß-peptide bonds with a broad promiscuity represents a new insight into the stability of peptide analogs containing ß-amino acids as such analogs were thought to be stable towards enzymatic degradation.

Stapled Peptides as HIF-1α/p300 Inhibitors: Helicity Enhancement in the Bound State Increases Inhibitory Potency.

Protein-protein interactions (PPIs) control virtually all cellular processes and have thus emerged as potential targets for development of molecular therapeutics. Peptide-based inhibitors of PPIs are attractive given that they offer recognition potency and selectivity features that are ideal for function, yet, they do not predominantly populate the bioactive conformation, frequently suffer from poor cellular uptake and are easily degraded, for example, by proteases. The constraint of peptides in a bioactive conformation has emerged as a promising strategy to mitigate against these liabilities. In this work, using peptides derived from hypoxia-inducible factor 1 (HIF-1α) together with dibromomaleimide stapling, we identify constrained peptide inhibitors of the HIF-1α/p300 interaction that are more potent than their unconstrained sequences. Contrary to expectation, the increased potency does not correlate with an increased population of an α-helical conformation in the unbound state as demonstrated by experimental circular dichroism analysis. Rather, the ability of the peptide to adopt a bioactive α-helical conformation in the p300 bound state is better supported in the constrained variant as demonstrated by molecular dynamics simulations and circular dichroism difference spectra.

Recognition of ASF1 by Using Hydrocarbon-Constrained Peptides.

Inhibiting the histone H3-ASF1 (anti-silencing function 1) protein-protein interaction (PPI) represents a potential approach for treating numerous cancers. As an α-helix-mediated PPI, constraining the key histone H3 helix (residues 118-135) is a strategy through which chemical probes might be elaborated to test this hypothesis. In this work, variant H3118-135 peptides bearing pentenylglycine residues at the i and i+4 positions were constrained by olefin metathesis. Biophysical analyses revealed that promotion of a bioactive helical conformation depends on the position at which the constraint is introduced, but that the potency of binding towards ASF1 is unaffected by the constraint and instead that enthalpy-entropy compensation occurs.

Target-specific NMR detection of protein-ligand interactions with antibody-relayed 15N-group selective STD.

Fragment-based drug design has been successfully applied to challenging targets where the detection of the weak protein-ligand interactions is a key element. 1H saturation transfer difference (STD) NMR spectroscopy is a powerful technique for this work but it requires pure homogeneous proteins as targets. Monoclonal antibody (mAb)-relayed 15N-GS STD spectroscopy has been developed to resolve the problem of protein mixtures and impure proteins. A 15N-labelled target-specific mAb is selectively irradiated and the saturation is relayed through the target to the ligand. Tests on the anti-Gal-1 mAb/Gal-1/lactose system showed that the approach is experimentally feasible in a reasonable time frame. This method allows detection and identification of binding molecules directly from a protein mixture in a multicomponent system.

Predicting order and disorder for ß-peptide foldamers in water.

Following a quantitative validation approach, we tested the AMBER ff03 and GAFF force fields with the TIP3P explicit water model in molecular dynamic simulations of ß-peptide foldamers. The test sequences were selected to represent a wide range of folding behavior in water: compact helix, strand mimetic geometry, and the state of disorder. The combination AMBER ff03-TIP3P successfully predicted the experimentally observed conformational properties and reproduced the NOE distances and backbone (3)J coupling data at a good level. GAFF was unable to produce folded structures correctly due to its biased torsion potentials. We can recommend AMBER ff03-TIP3P for simulations involving ß-peptide sequences in aqueous media including ordered and disordered structures.

Site-directed allostery perturbation to probe the negative regulation of hypoxia inducible factor-1α.

The interaction between the intrinsically disordered transcription factor HIF-1α and the coactivator proteins p300/CBP is essential in the fast response to low oxygenation. The negative feedback regulator, CITED2, switches off the hypoxic response through a very efficient irreversible mechanism. The negative cooperativity with HIF-1α relies on the formation of a ternary intermediate that leads to allosteric structural changes in p300/CBP, in which the cooperative folding/binding of the CITED2 sequence motifs plays a key role. Understanding the contribution of a binding motif to the structural changes in relation to competition efficiency provides invaluable insights into the molecular mechanism. Our strategy is to site-directedly perturb the p300-CITED2 complex's structure without significantly affecting binding thermodynamics. In this way, the contribution of a sequence motif to the negative cooperativity with HIF-1α would mainly depend on the induced structural changes, and to a lesser extent on binding affinity. Using biophysical assays and NMR measurements, we show here that the interplay between the N-terminal tail and the rest of the binding motifs of CITED2 is crucial for the unidirectional displacement of HIF-1α. We introduce an advantageous approach for evaluating the roles of the different sequence parts with the help of motif-by-motif backbone perturbations.

Separation of ochratoxins by centrifugal partition chromatography.

The present research work was dedicated to developing an efficient method based on liquid-liquid chromatography (centrifugal partition chromatography, CPC) applicable to routine purifications of ochratoxins (OT) from the liquid culture of the strain A. albertensis SZMC 2107. The crude extract contained numerous components in addition to OTA (90.1 %,) and OTB (1.1 %,) according to HPLC examinations. For the separation of OTs by CPC, several tertiary systems based on acetonitrile, acetone, and short-chain alcohols were examined to find the most applicable biphasic system. The hexane/i-propanol/water 35:15:50 system supplemented with 0.1 % acetic acid was found to be the most efficient for use in CPC separation. Using liquid-liquid instrumental separation, the two OTs, namely OTA (2.23 mg) and OTB (0.031 mg), were successfully isolated with 96.3 % and-72.8 % purity, respectively, from 1 L ferment broth. The identities and purities of the purified components were confirmed and the performance parameters of each separation step and the whole procedure were determined. The developed method could be used effectively to purify OTs for analytical or toxicological applications.

Foldameric α/ß-peptide analogs of the ß-sheet-forming antiangiogenic anginex: structure and bioactivity.

The principles of ß-sheet folding and design for α-peptidic sequences are well established, while those for sheet mimetics containing homologated amino acid building blocks are still under investigation. To reveal the structure-function relations of ß-amino-acid-containing foldamers, we followed a top-down approach to study a series of α/ß-peptidic analogs of anginex, a ß-sheet-forming antiangiogenic peptide. Eight anginex analogs were developed by systematic α → ß(3) substitutions and analyzed by using NMR and CD spectroscopy. The foldamers retained the ß-sheet tendency, though with a decreased folding propensity. ß-Sheet formation could be induced by a micellar environment, similarly to that of the parent peptide. The destructuring effect was higher when the α → ß(3) exchange was located in the ß-sheet core. Analysis of the ß-sheet stability versus substitution pattern and the local conformational bias of the bulky ß(3)V and ß(3)I residues revealed that a mismatch between the H-bonding preferences of the α- and ß-residues played a minor role in the structure-breaking effect. Temperature-dependent CD and NMR measurements showed that the hydrophobic stabilization was scaled-down for the α/ß-peptides. Analysis of the biological activity of the foldamer peptides showed that four anginex derivatives dose-dependently inhibited the proliferation of a mouse endothelial cell line. The α → ß(3) substitution strategy applied in this work can be a useful approach to the construction of bioactive ß-sheet mimetics with a reduced aggregation tendency and improved pharmacokinetic properties.

Improved Metal-Free Approach for the Synthesis of Protected Thiol Containing Thymidine Nucleoside Phosphoramidite and Its Application for the Synthesis of Ligatable Oligonucleotide Conjugates.

Oligonucleotide conjugates are versatile scaffolds that can be applied in DNA-based screening platforms and ligand display or as therapeutics. Several different chemical approaches are available for functionalizing oligonucleotides, which are often carried out on the 5' or 3' end. Modifying oligonucleotides in the middle of the sequence opens the possibility to ligate the conjugates and create DNA strands bearing multiple different ligands. Our goal was to establish a complete workflow that can be applied for such purposes from monomer synthesis to templated ligation. To achieve this, a monomer is required with an orthogonal functional group that can be incorporated internally into the oligonucleotide sequence. This is followed by conjugation with different molecules and ligation with the help of a complementary template. Here, we show the synthesis and the application of a thiol-modified thymidine nucleoside phosphoramidite to prepare ligatable oligonucleotide conjugates. The conjugations were performed both in solution and on solid phase, resulting in conjugates that can be assembled into multivalent oligonucleotides decorated with tissue-targeting peptides using templated ligation.

Structural Adaptation of the Single-Stranded DNA-Binding Protein C-Terminal to DNA Metabolizing Partners Guides Inhibitor Design.

Single-stranded DNA-binding protein (SSB) is a bacterial interaction hub and an appealing target for antimicrobial therapy. Understanding the structural adaptation of the disordered SSB C-terminus (SSB-Ct) to DNA metabolizing enzymes (e.g., ExoI and RecO) is essential for designing high-affinity SSB mimetic inhibitors. Molecular dynamics simulations revealed the transient interactions of SSB-Ct with two hot spots on ExoI and RecO. The residual flexibility of the peptide-protein complexes allows adaptive molecular recognition. Scanning with non-canonical amino acids revealed that modifications at both termini of SSB-Ct could increase the affinity, supporting the two-hot-spot binding model. Combining unnatural amino acid substitutions on both segments of the peptide resulted in enthalpy-enhanced affinity, accompanied by enthalpy-entropy compensation, as determined by isothermal calorimetry. NMR data and molecular modeling confirmed the reduced flexibility of the improved affinity complexes. Our results highlight that the SSB-Ct mimetics bind to the DNA metabolizing targets through the hot spots, interacting with both of segments of the ligands.

Towards identification of protein-protein interaction stabilizers via inhibitory peptide-fragment hybrids using templated fragment ligation.

Using the hDMX/14-3-3 interaction, acylhydrazone-based ligand-directed fragment ligation was used to identify protein-protein interaction (PPI) inhibitory peptide-fragment hybrids. Separation of the peptide-fragment hybrids into the components yielded fragments that stabilized the hDMX/14-3-3 interaction.

The role of the Aspergillus nidulans high mobility group B protein HmbA, the orthologue of Saccharomyces cerevisiae Nhp6p.

The mammalian HMGB1 is a high-mobility-group B protein, which is both an architectural and functional element of chromatin. Nhp6p, the extensively studied fungal homologue of HMGB1 in Saccharomyces cerevisiae has pleiotropic physiological functions. Despite the existence of Nhp6p orthologues in filamentous ascomycetes, little is known about their physiological roles besides their contribution to sexual development. Here we study the function of HmbA, the Aspergillus nidulans orthologue of Nhp6p. We show that HmbA influences the utilization of various carbon- and nitrogen sources, stress tolerance, secondary metabolism, hyphae elongation and maintenance of polarized growth. Additionally, by conducting heterologous expression studies, we demonstrate that HmbA and Nhp6p are partially interchangeable. HmbA restores SNR6 transcription and fitness of nhp6AΔBΔ mutant and reverses its heat sensitivity. Nhp6Ap complements several phenotypes of hmbAΔ, including ascospore formation, utilization of various carbon- and nitrogen-sources, radial growth rate, hypha elongation by polarized growth. However, Nhp6Ap does not complement sterigmatocystin production in a hmbAΔ strain. Finally, we also show that HmbA is necessary for the normal expression of the endochitinase chiA, a cell wall re-modeller that is pivotal for the normal mode of maintenance of polar growth.

Understanding p300-transcription factor interactions using sequence variation and hybridization.

The hypoxic response is central to cell function and plays a significant role in the growth and survival of solid tumours. HIF-1 regulates the hypoxic response by activating over 100 genes responsible for adaptation to hypoxia, making it a potential target for anticancer drug discovery. Although there is significant structural and mechanistic understanding of the interaction between HIF-1α and p300 alongside negative regulators of HIF-1α such as CITED2, there remains a need to further understand the sequence determinants of binding. In this work we use a combination of protein expression, chemical synthesis, fluorescence anisotropy and isothermal titration calorimetry for HIF-1α sequence variants and a HIF-1α-CITED hybrid sequence which we term CITIF. We show the HIF-1α sequence is highly tolerant to sequence variation through reduced enthalpic and less unfavourable entropic contributions, These data imply backbone as opposed to side chain interactions and ligand folding control the binding interaction and that sequence variations are tolerated as a result of adopting a more disordered bound interaction or "fuzzy" complex.

A complete nicotinate degradation pathway in the microbial eukaryote Aspergillus nidulans.

Several strikingly different aerobic and anaerobic pathways of nicotinate breakdown are extant in bacteria. Here, through reverse genetics and analytical techniques we elucidated in Aspergillus nidulans, a complete eukaryotic nicotinate utilization pathway. The pathway extant in this fungus and other ascomycetes, is quite different from bacterial ones. All intermediate metabolites were identified. The cognate proteins, encoded by eleven genes (hxn) mapping in three clusters are co-regulated by a specific transcription factor. Several enzymatic steps have no prokaryotic equivalent and two metabolites, 3-hydroxypiperidine-2,6-dione and 5,6-dihydroxypiperidine-2-one, have not been identified previously in any organism, the latter being a novel chemical compound. Hydrolytic ring opening results in α-hydroxyglutaramate, a compound not detected in analogous prokaryotic pathways. Our earlier phylogenetic analysis of Hxn proteins together with this complete biochemical pathway illustrates convergent evolution of catabolic pathways between fungi and bacteria.

Identification of ß-strand mediated protein-protein interaction inhibitors using ligand-directed fragment ligation.

ß-Strand mediated protein-protein interactions (PPIs) represent underexploited targets for chemical probe development despite representing a significant proportion of known and therapeutically relevant PPI targets. ß-Strand mimicry is challenging given that both amino acid side-chains and backbone hydrogen-bonds are typically required for molecular recognition, yet these are oriented along perpendicular vectors. This paper describes an alternative approach, using GKAP/SHANK1 PDZ as a model and dynamic ligation screening to identify small-molecule replacements for tranches of peptide sequence. A peptide truncation of GKAP functionalized at the N- and C-termini with acylhydrazone groups was used as an anchor. Reversible acylhydrazone bond exchange with a library of aldehyde fragments in the presence of the protein as template and in situ screening using a fluorescence anisotropy (FA) assay identified peptide hybrid hits with comparable affinity to the GKAP peptide binding sequence. Identified hits were validated using FA, ITC, NMR and X-ray crystallography to confirm selective inhibition of the target PDZ-mediated PPI and mode of binding. These analyses together with molecular dynamics simulations demonstrated the ligands make transient interactions with an unoccupied basic patch through electrostatic interactions, establishing proof-of-concept that this unbiased approach to ligand discovery represents a powerful addition to the armory of tools that can be used to identify PPI modulators.

Query-guided protein-protein interaction inhibitor discovery.

Protein-protein interactions (PPIs) are central to biological mechanisms, and can serve as compelling targets for drug discovery. Yet, the discovery of small molecule inhibitors of PPIs remains challenging given the large and typically shallow topography of the interacting protein surfaces. Here, we describe a general approach to the discovery of orthosteric PPI inhibitors that mimic specific secondary protein structures. Initially, hot residues at protein-protein interfaces are identified in silico or from experimental data, and incorporated into secondary structure-based queries. Virtual libraries of small molecules are then shape-matched against the queries, and promising ligands docked to target proteins. The approach is exemplified experimentally using two unrelated PPIs that are mediated by an α-helix (p53/hDM2) and a ß-strand (GKAP/SHANK1-PDZ). In each case, selective PPI inhibitors are discovered with low µM activity as determined by a combination of fluorescence anisotropy and 1H-15N HSQC experiments. In addition, hit expansion yields a series of PPI inhibitors with defined structure-activity relationships. It is envisaged that the generality of the approach will enable discovery of inhibitors of a wide range of unrelated secondary structure-mediated PPIs.

Tunable Magnetic Hyperthermia Properties of Pristine and Mildly Reduced Graphene Oxide/Magnetite Nanocomposite Dispersions.

We present a study on the magnetic hyperthermia properties of graphene oxide/magnetite (GO/MNP) nanocomposites to investigate their heat production behavior upon the modification of the oxidation degree of the carbonaceous host. Avoiding the harsh chemical conditions of the regular in situ co-precipitation-based routes, the oppositely charged MNPs and GO nanosheets were combined by the heterocoagulation process at pH ~ 5.5, which is a mild way to synthesize composite nanostructures at room temperature. Nanocomposites prepared at 1/5 and 1/10 GO/MNP mass ratios were reduced by NaBH4 and L-ascorbic acid (LAA) under acidic (pH ~ 3.5) and alkaline conditions (pH ~ 9.3). We demonstrate that the pH has a crucial effect on the LAA-assisted conversion of graphene oxide to reduced GO (rGO): alkaline reduction at higher GO loadings leads to doubled heat production of the composite. Spectrophotometry proved that neither the moderately acidic nor alkaline conditions promote the iron dissolution of the magnetic core. Although the treatment with NaBH4 also increased the hyperthermic efficiency of aqueous GO/MNP nanocomposite suspensions, it caused a drastic decline in their colloidal stability. However, considering the enhanced heat production and the slightly improved stability of the rGO/MNP samples, the reduction with LAA under alkaline condition is a more feasible way to improve the hyperthermic efficiency of magnetically modified graphene oxides.

Separation and Purification of Aflatoxins by Centrifugal Partition Chromatography.

Aflatoxins are mycotoxins that are produced by several species of filamentous fungi. In the European Union, the concentration limits for this group of mycotoxins in food and feed products are very low (on the order of parts per billion). Thus, relatively high amounts of these substances in their pure forms are required as reference standards. Chromatographic techniques based on solid stationary phases are generally used to purify these molecules; however, liquid-liquid chromatographic separations may be a promising alternative. Therefore, this study proposes a liquid-liquid chromatographic method for the separation of four aflatoxins and impurities. To optimise the method, numerous biphasic solvent systems (chloroform-, acetone- and acetic acid-based systems) were tested and evaluated in terms of their effectiveness at partitioning aflatoxins; the toluene/acetic acid/water (30:24:50, v/v/v/%) system was found to be the most efficient for application in centrifugal partition chromatographic instrument. Using liquid-liquid instrumental separation, the four aflatoxins, namely B1 (400 mg), B2 (34 mg), G1 (817 mg) and G2 (100 mg), were successfully isolated with 96.3%-98.2% purity from 4.5 L of Aspergillus parasiticus fermented material in a 250 mL centrifugal partition chromatography column. The identities and purities of the purified components were confirmed, and the performance parameters of each separation step and the whole procedure was determined. The developed method could be effectively used to purify aflatoxins for analytical applications.

A catalytic protein-proteomimetic complex: using aromatic oligoamide foldamers as activators of RNase S.

Foldamers are abiotic molecules that mimic the ability of bio-macromolecules to adopt well-defined and organised secondary, tertiary or quaternary structure. Such templates have enabled the generation of defined architectures which present structurally defined surfaces that can achieve molecular recognition of diverse and complex targets. Far less explored is whether this mimicry of nature can extend to more advanced functions of biological macromolecules such as the generation and activation of catalytic function. In this work, we adopt a novel replacement strategy whereby a segment of protein structure (the S-peptide from RNase S) is replaced by a foldamer that mimics an α-helix. The resultant prosthetic replacement forms a non-covalent complex with the S-protein leading to restoration of catalytic function, despite the absence of a key catalytic residue. Thus this functional protein-proteomimetic complex provides proof that significant segments of protein can be replaced with non-natural building blocks that may, in turn, confer advantageous properties.

Predicting and Experimentally Validating Hot-Spot Residues at Protein-Protein Interfaces.

Protein-protein interactions (PPIs) are vital to all biological processes. These interactions are often dynamic, sometimes transient, typically occur over large topographically shallow protein surfaces, and can exhibit a broad range of affinities. Considerable progress has been made in determining PPI structures. However, given the above properties, understanding the key determinants of their thermodynamic stability remains a challenge in chemical biology. An improved ability to identify and engineer PPIs would advance understanding of biological mechanisms and mutant phenotypes and also provide a firmer foundation for inhibitor design. In silico prediction of PPI hot-spot amino acids using computational alanine scanning (CAS) offers a rapid approach for predicting key residues that drive protein-protein association. This can be applied to all known PPI structures; however there is a trade-off between throughput and accuracy. Here we describe a comparative analysis of multiple CAS methods, which highlights effective approaches to improve the accuracy of predicting hot-spot residues. Alongside this, we introduce a new method, BUDE Alanine Scanning, which can be applied to single structures from crystallography and to structural ensembles from NMR or molecular dynamics data. The comparative analyses facilitate accurate prediction of hot-spots that we validate experimentally with three diverse targets: NOXA-B/MCL-1 (an α-helix-mediated PPI), SIMS/SUMO, and GKAP/SHANK-PDZ (both ß-strand-mediated interactions). Finally, the approach is applied to the accurate prediction of hot-spot residues at a topographically novel Affimer/BCL-xL protein-protein interface.