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Mycobacterium tuberculosis dethiobiotin synthase (MtDTBS) is a crucial enzyme involved in the biosynthesis of biotin in the causative agent of tuberculosis, M. tuberculosis. Here, we report a binder of MtDTBS, cyclopentylacetic acid 2 (KD = 3.4 ± 0.4 mM), identified via in silico screening. X-ray crystallography showed that 2 binds in the 7,8-diaminopelargonic acid (DAPA) pocket of MtDTBS. Appending an acidic group to the para-position of the aromatic ring of the scaffold revealed compounds 4c and 4d as more potent binders, with KD = 19 ± 5 and 17 ± 1 μM, respectively. Further optimization identified tetrazole 7a as a particularly potent binder (KD = 57 ± 5 nM) and inhibitor (Ki = 5 ± 1 μM) of MtDTBS. Our findings highlight the first reported inhibitors of MtDTBS and serve as a platform for the further development of potent inhibitors and novel therapeutics for the treatment of tuberculosis.

Amyloid beta peptide (Aβ42) aggregation in the brain is thought to be responsible for the onset of Alzheimer's disease, an insidious condition without an effective treatment or cure. Hence, a strategy to prevent aggregation and subsequent toxicity is crucial. Bio-inspired peptide-based molecules are ideal candidates for the inhibition of Aβ42 aggregation, and are currently deemed to be a promising option for drug design. In this study, a hexapeptide containing a self-recognition component unique to Aβ42 was designed to mimic the β-strand hydrophobic core region of the Aβ peptide. The peptide is comprised exclusively of D-amino acids to enhance specificity towards Aβ42, in conjunction with a C-terminal disruption element to block the recruitment of Aβ42 monomers on to fibrils. The peptide was rationally designed to exploit the synergy between the recognition and disruption components, and incorporates features such as hydrophobicity, β-sheet propensity, and charge, that all play a critical role in the aggregation process. Fluorescence assays, native ion-mobility mass spectrometry (IM-MS) and cell viability assays were used to demonstrate that the peptide interacts with Aβ42 monomers and oligomers with high specificity, leading to almost complete inhibition of fibril formation, with essentially no cytotoxic effects. These data define the peptide-based inhibitor as a potentially potent anti-amyloid drug candidate for this hitherto incurable disease.

Proliferating cell nuclear antigen (PCNA) is an excellent inhibition target to shut down highly proliferative cells and thereby develop a broad-spectrum cancer therapeutic. It interacts with a wide variety of proteins through a conserved motif referred to as the PCNA-interacting protein (PIP) box. There is large sequence diversity between high-affinity PCNA binding partners, but with conservation of the binding structure-a well-defined 310 -helix. Herein, all current PIP-box peptides crystallised with human PCNA are collated to reveal common trends between binding structure and affinity. Key intra- and intermolecular hydrogen-bonding networks that stabilise the 310 -helix of PIP-box partners are highlighted and related back to the canonical PIP-box motif. High correlation with the canonical PIP-box sequence does not directly afford high affinity. Instead, we summarise key interactions that stabilise the binding structure that leads to enhanced PCNA binding affinity. These interactions also implicate the "non-conserved" residues within the PIP-box that have previously been overlooked. Such insights will allow a more directed approach to develop therapeutic PCNA inhibitors.

A series of dipeptide aldehydes containing different N-terminal heterocycles was prepared and assayed in vitro against α-chymotrypsin to ascertain the importance of the heterocycle in maintaining a β-strand geometry while also providing a hydrogen bond donor equivalent to the backbone amide nitrogen of the surrogate amino acid. The dipeptide containing a pyrrole constraint (10) was the most potent inhibitor, with >30-fold improved activity over dipeptides which lacked a nitrogen hydrogen bond donor (namely thiophene 11, furan 12 and pyridine 13). Molecular docking studies of 10 bound to α-chymotrypsin demonstrates a hydrogen bond between the pyrrole nitrogen donor and the backbone carbonyl of Gly216 located in the S3 pocket which is proposed to be critical for overall binding.

Oocyte activation, the dynamic transformation of an oocyte into an embryo, is largely driven by Ca2+ oscillations that vary in duration and amplitude across species. Previous studies have analysed intraoocyte Ca2+ oscillations in the absence of the oocyte’s supporting cumulus cells. Therefore, it is unknown whether cumulus cells also produce an ionic signal that reflects fertilisation success. Time-lapse confocal microscopy and image analysis on abattoir-derived cattle cumulus–oocyte complexes coincubated with spermatozoa revealed a distinct discharge of fluorescence from the cumulus vestment. This study demonstrated that this Ca2+ fluorescence discharge was an artefact induced by the imaging procedure independently of oocyte activation success. The fluorescence discharge was a direct result of cumulus cell membrane integrity loss, and future studies should consider the long-term effect of fluorescent labels on cells in time-lapse imaging. However, this study also demonstrated that the distinctive pattern of a coordinated fluorescence discharge was associated with both the presence of spermatozoa and subsequent embryo development to the morula stage, which was affected by Ca2+ chelation and a reduction in the active efflux of the fluorophore. This indicates that the cumulus vestment may have a relationship with oocyte activation at and beyond fertilisation that requires further investigation.

Inhibitors of biotin protein ligase (BPL) are novel antimicrobial compounds with the potential to treat infections caused by bacteria resistant to current antibiotics. A novel BPL inhibitor (12, Ki1.4 μM) was synthesized from biotin acetylene and an azide-functionalized analogue of fluorescent nitrobenzofurazan by Cu(I) catalysed cycloaddition and also by template guided synthesis using wild-type BPL from Staphylococcus aureus. LC/HRMS-based detection provides improved sensitivity over previous reports using a mutant BPL, with demonstrated applicability to other BPLs. Super-imaging fluorescence microscopy demonstrated the accumulation of 12 in the cytoplasm of S. aureus, but not Escherichia coli. This novel fluorescent probe can be used to gain new insights into the mechanism of uptake, efflux and metabolism of BPL inhibitors in S. aureus. Refereed/Peer-reviewed

The solvatochromic amino-acids 4-DMNA or 4-DAPA, were separately introduced at position 147, 150 or 151 of a short p21 peptide (141–155) known to bind sliding clamp protein PCNA. The ability of these peptides, 1a-3a and 1b-3b, to act as a turn-on fluorescent sensor for PCNA was then investigated. The 4-DMNA-containing peptides (1a-3a) displayed up to a 40-fold difference in fluorescence between a polar (Tris buffer) and a hydrophobic solvent (dioxane with 5 mM 18-crown-6), while the 4-DAPA-containing peptides (1b-3b) displayed a significantly enhanced (300-fold) increase in fluorescence from Tris buffer to dioxane with 18-crown-6. SPR analysis of the peptides against PCNA revealed that the 151-substituted peptides 3a and 3b interacted specifically with PCNA, with KD values of 921 nM and 1.28 μM, respectively. Analysis of the fluorescence of these peptides in the presence of increasing concentrations of PCNA revealed a 10-fold change in fluorescence for 3a at 2.5 equivalents of PCNA, compared to only a 3.5-fold change in fluorescence for 3b. Peptide 3a is an important lead for development of a PCNA-selective turn-on fluorescent sensor for application as a cell proliferation sensor to investigate diseases such as cancer.

Can we separate embryos cultured under either 7% or 20% oxygen atmospheres by measuring their metabolic heterogeneity?Metabolic heterogeneity and changes in metabolic profiles in morula exposed to two different oxygen concentrations were not detectable using traditional fluorophore and two-channel autofluorescence but were detectable using hyperspectral microscopy.Increased genetic and morphological blastomere heterogeneity is associated with compromised developmental competence of embryos and currently forms the basis for embryo scoring within the clinic. However, there remains uncertainty over the accuracy of current techniques, such as PGS and time-lapse microscopy, to predict subsequent pregnancy establishment.The impact of two oxygen concentrations (7% = optimal and 20% = stressed) during post-fertilisation embryo culture was assessed. Cattle embryos were exposed to the different oxygen concentrations for 8 days (D8; embryo developmental competence) or 5 days (D5; metabolism measurements). Between 3 and 4 experimental replicates were performed, with 40-50 embryos per replicate used for the developmental competency experiment, 10-20 embryos per replicate for the fluorophore and two-channel autofluorescence experiments and a total of 21-22 embryos used for the hyperspectral microscopy study.In-vitro produced (IVP) cattle embryos were utilised for this study. Post-fertilisation, embryos were exposed to 7% or 20% oxygen. To determine impact of oxygen concentrations on embryo viability, blastocyst development was assessed on D8. On D5, metabolic heterogeneity was assessed in morula (on-time) embryos using fluorophores probes (active mitochondria, hydrogen peroxide and reduced glutathione), two-channel autofluorescence (FAD and NAD(P)H) and 18-channel hyperspectral microscopy.Exposure to 20% oxygen following fertilisation significantly reduced total blastocyst, expanded and hatched blastocyst rates by 1.4-, 1.9- and 2.8-fold, respectively, compared to 7% oxygen (P0.05), demonstrating that atmospheric oxygen was a viable model for studying mild metabolic stress. The metabolic profiles of D5 embryos was determined and although metabolic heterogeneity was evident within the cleavage stage (i.e. arrested) embryos exposed to fluorophores, there were no detectable difference in fluorescence intensity and pattern localisation in morula exposed to the two different oxygen concentrations (P0.05). While there were no significant differences in two-channel autofluorescent profiles of morula exposed to 7% and 20% oxygen (main effect, P0.05), morula that subsequently progressed to the blastocyst stage had significantly higher levels of FAD and NAD(P)H fluorescence compared to arrested morula (P0.05), with no change in the redox ratio. Hyperspectral autofluorescence imaging (in 18-spectral channels) of the D5 morula revealed highly significant differences in four features of the metabolic profiles of morula exposed to the two different oxygen concentrations (P0.001). These four features were weighted and their linear combination revealed clear discrimination between the two treatment groups.Metabolic profiles were assessed at a single time point (morula), and as such further investigation is required to determine if differences in hyperspectral signatures can be detected in pre-compaction embryos and oocytes, using both cattle and subsequently human models. Furthermore, embryo transfers should be performed to determine the relationship between metabolic profiles and pregnancy success.Advanced autofluorescence imaging techniques, such as hyperspectral microscopy, may provide clinics with additional tools to improve the assessment of embryos prior to transfer.This study was funded by the Australian Research Council Centre of Excellence for Nanoscale BioPhotonics (CE140100003). The Fluoview FV10i confocal microscope was purchased as part of the Sensing Technologies for Advanced Reproductive Research (STARR) facility, funded by the South Australian Premier's Science and Research Fund. The authors declare there are no conflict of interest.

The human sliding clamp, Proliferating Cell Nuclear Antigen (hPCNA), interacts with over 200 proteins through a conserved binding motif, the PIP-box, to orchestrate DNA replication and repair. It is not clear how changes to the features of a PIP-box modulate protein binding and thus how they fine-tune downstream processes. Here, we present a systematic study of each position within the PIP-box to reveal how hPCNA-interacting peptides bind with drastically varied affinities. We synthesized a series of 27 peptides derived from the native protein p21 with small PIP-box modifications and another series of 19 peptides containing PIP-box binding motifs from other proteins. The hPCNA-binding affinity of all peptides, characterized as KD values determined by surface plasmon resonance, spanned a 4000-fold range, from 1.83 nM to 7.59 μM. The hPCNA-bound peptide structures determined by X-ray crystallography and modeled computationally revealed intermolecular and intramolecular interaction networks that correlate with high hPCNA affinity. These data informed rational design of three new PIP-box sequences, testing of which revealed the highest affinity hPCNA-binding partner to date, with a KD value of 1.12 nM, from a peptide with PIP-box QTRITEYF. This work showcases the sequence-specific nuances within the PIP-box that are responsible for high-affinity hPCNA binding, which underpins our understanding of how nature tunes hPCNA affinity to regulate DNA replication and repair processes. In addition, these insights will be useful to future design of hPCNA inhibitors.

Replacing the labile adenosinyl-substituted phosphoanhydride of biotinyl-5′-AMP with a N1-benzyl substituted 1,2,3-triazole gave a new truncated series of inhibitors of Staphylococcus aureus biotin protein ligase (SaBPL). The benzyl group presents to the ribose-binding pocket of SaBPL based on in silico docking. Halogenated benzyl derivatives (12t, 12u, 12w, and 12x) proved to be the most potent inhibitors of SaBPL. These derivatives inhibited the growth of S. aureus ATCC49775 and displayed low cytotoxicity against HepG2 cells. Refereed/Peer-reviewed

Here we describe an NMR and X-ray crystallography-based characterisation of the mechanism by which a new class of macrocyclic peptidomimetic aldehyde inhibits α-chymotrypsin. In particular, a (13)C-labelled analogue of the inhibitor was prepared and used in NMR experiments to confirm formation of a hemiacetal intermediate on binding with α-chymotrypsin. Analysis of an X-ray crystallographic structure in complex with α-chymotrypsin reveals that the backbone adopts a stable β-strand conformation as per its design. Binding is further stabilised by interaction with the oxyanion hole near the S1 subsite and multiple hydrogen bonds.

Constrained α-helical peptides are showing potential as biological probes and therapeutic agents that target protein-protein interactions. However, the factors that determine the optimal constraint locations are still largely unknown. Using the β-integrin/talin protein interaction as a model system, we examine the effect of constraint location on helical conformation, as well as binding affinity, using circular dichroism and NMR spectroscopy. Stapling increased the overall helical content of each integrin-based peptide tested. However, NMR analysis revealed that different regions within the peptide are stabilised, depending on constraint location, and that these differences correlate with the changes observed in talin binding mode and affinity. In addition, we show that examination of the atomic structure of the parent peptide provides insight into the appropriate placement of helical constraints.

The 26S proteasome and calpain are linked to a number of important human diseases. Here, we report a series of analogues of the prototypical tripeptide aldehyde inhibitor MG132 that show a unique combination of high activity and selectivity for calpains over proteasome. Tripeptide aldehydes (1–3) with an aromatic P3 substituent show enhanced activity and selectivity against ovine calpain 2 relative to chymotrypsin-like activity of proteasome. Docking studies reveal the key contacts between inhibitors and calpain to confirm the importance of the S3 pocket with respect to selectivity between calpains 1 and 2 and the proteasome Refereed/Peer-reviewed

This study reports on the real-time binding assessment between heavy metal ions and blood proteins immobilized onto nanoporous anodic alumina photonic crystals (NAA-PCs) by reflectometric interference spectroscopy (RIfS). The surface of NAA-PCs is chemically functionalized with γ-globulin (GG), transferrin (TFN), and serum albumin (HSA), the major proteins present in human blood plasma. Protein-modified NAA-PC platforms are exposed to analytical solutions of mercury ions of different concentrations. Dynamic changes in the effective optical thickness of protein-modified NAA-PCs in response to heavy metal ions are assessed in real time to evaluate the binding kinetics, affinity, and mechanism. Protein molecules undergo conformational changes upon exposure to mercury ions, with HSA exhibiting the strongest affinity. The combination of protein-modified NAA-PCs with RIfS allows real-time monitoring of protein-heavy metal ions interactions under dynamic flow conditions. This system is capable of detecting dynamic conformational changes in these proteins upon exposure to heavy metal ions. Our results provide new insights into these binding events, which could enable new methodologies to study the toxicity of heavy metal ions and other biomolecular interactions.

Fil: Sutton Mcdowall, Melanie L.. University Of Adelaide; Australia. Australian Research Council; Australia

We report the synthesis and evaluation of 5-halogenated-1,2,3-Triazoles as inhibitors of biotin protein ligase from Staphylococcus aureus. The halogenated compounds exhibit significantly improved antibacterial activity over their nonhalogenated counterparts. Importantly, the 5-fluoro-1,2,3-Triazole compound 4c displays antibacterial activity against S. aureus ATCC49775 with a minimum inhibitory concentration (MIC) of 8 μg/mL. Refereed/Peer-reviewed

The onset of new multidrug-resistant strains of bacteria demands continuous development of antibacterial agents with new chemical scaffolds and mechanisms of action. We present the first structure–activity relationship (SAR) study of 16 derivatives of a structurally novel antibiotic merochlorin A that were designed using a biosynthetic blueprint. Our lead compounds are active against several Gram-positive bacteria such as Staphylococcus aureus (SA), methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant Enterococcus faecium (VRE) and Bacillus subtilis, inhibit intracellular growth of Mycobacterium bovis, and are relatively nontoxic to human cell lines. Furthermore, derivative 12 c {(±)-(3aR,4S,5R,10bS)-5-bromo-7,9-dimethoxy-4-methyl-4-(4-methylpent-3-en-1-yl)-2-(propan-2-ylidene)-1,2,3,3a,4,5-hexahydro-6H-5,10b-methanobenzo[e]azulene-6,11-dione} was found to inhibit the growth of Bacillus Calmette–Guérin (BCG)-infected cells at concentrations similar to rifampicin. These results outperform the natural product, underscoring the potential of merochlorin analogues as a new class of antibiotics. Refereed/Peer-reviewed

There is a real need for simple structures that define a β-strand conformation, a secondary structure that is central to peptide-protein interactions. For example, protease substrates and inhibitors almost universally adopt this geometry on active site binding. A planar pyrrole is used to replace two amino acids of a peptide backbone to generate a simple macrocycle that retains the required geometry for active site binding. The resulting β-strand templates have reduced peptide character and provide potent protease inhibitors with the attachment of an appropriate amino aldehyde to the C-terminus. Picomolar inhibitors of cathepsin L and S are reported and the mode of binding of one example to the model protease chymotrypsin is defined by X-ray crystallography.

The essential metabolic enzyme biotin protein ligase (BPL) is a potential target for the development of new antibiotics required to combat drug-resistant pathogens. Staphylococcus aureus BPL (SaBPL) is a bifunctional protein, possessing both biotin ligase and transcription repressor activities. This positions BPL as a key regulator of several important metabolic pathways. Here, we report the structural analysis of both holo- and apo-forms of SaBPL using X-ray crystallography. We also present small-angle X-ray scattering data of SaBPL in complex with its biotin-carboxyl carrier protein substrate as well as the SaBPL:DNA complex that underlies repression. This has revealed the molecular basis of ligand (biotinyl-5′-AMP) binding and conformational changes associated with catalysis and repressor function. These data provide new information to better understand the bifunctional activities of SaBPL and to inform future strategies for antibiotic discovery.

Ketamine is primarily metabolized to norketamine by hepatic CYP2B6 and CYP3A4-mediated N-demethylation. However, the relative contribution from each enzyme remains controversial. The CYP2B6*6 allele is associated with reduced enzyme expression and activity that may lead to interindividual variability in ketamine metabolism. We examined the N-demethylation of individual ketamine enantiomers using human liver microsomes (HLMs) genotyped for the CYP2B6*6 allele, insect cell-expressed recombinant CYP2B6 and CYP3A4 enzymes, and COS-1 cell-expressed recombinant CYP2B6.1 and CYP2B6.6 protein variant. Effects of CYP-selective inhibitors on norketamine formation were also determined in HLMs. The two-enzyme Michaelis-Menten model best fitted the HLM kinetic data. The Michaelis-Menten constants (K(m)) for the high-affinity enzyme and the low-affinity enzyme were similar to those for the expressed CYP2B6 and CYP3A4, respectively. The intrinsic clearance for both ketamine enantiomers by the high-affinity enzyme in HLMs with CYP2B6*1/*1 genotype were at least 2-fold and 6-fold higher, respectively, than those for CYP2B6*1/*6 genotype and CYP2B6*6/*6 genotype. The V(max) and K(m) values for CYP2B6.1 were approximately 160 and 70% of those for CYP2B6.6, respectively. N,N'N'-triethylenethiophosphoramide (thioTEPA) (CYP2B6 inhibitor, 25 μM) and the monoclonal antibody against CYP2B6 but not troleandomycin (CYP3A4 inhibitor, 25 μM) or the monoclonal antibody against CYP3A4 inhibited ketamine N-demethylation at clinically relevant concentrations. The degree of inhibition was significantly reduced in HLMs with the CYP2B6*6 allele (gene-dose P < 0.05). These results indicate a major role of CYP2B6 in ketamine N-demethylation in vitro and a significant impact of the CYP2B6*6 allele on enzyme-ketamine binding and catalytic activity.

Insulin-like growth factor II (IGF-II) is a protein with high structural and sequence similarity to insulin. Unlike insulin, it binds both the type 1 IGF receptor and the exon 11- isoform of the insulin receptor with high affinity. The overexpression and up regulation of IGF-II has been associated with the progression of various forms of cancer. The exact binding mechanism of IGF-II to its high affinity receptors is still not completely understood. Herein we describe the successful synthesis of a novel fluorescent IGF-II protein (F19Cou IGF-II), where residue 19 (phenylalanine) has been replaced by a fluorescent chromophore (coumaryl glycine). This novel coumaryl IGF-II analogue will be a useful tool for analysing the receptor interaction mechanisms in future studies.

There is a desperate need for novel antibiotic classes to combat the rise of drug resistant pathogenic bacteria, such as Staphylococcus aureus. Inhibitors of the essential metabolic enzyme biotin protein ligase (BPL) represent a promising drug target for new antibacterials. Structural and biochemical studies on the BPL from S. aureus have paved the way for the design and development of new antibacterial chemotherapeutics. BPL employs an ordered ligand binding mechanism for the synthesis of the reaction intermediate biotinyl-5′-AMP from substrates biotin and ATP. Here we review the structure and catalytic mechanism of the target enzyme, along with an overview of chemical analogues of biotin and biotinyl-5′-AMP as BPL inhibitors reported to date. Of particular promise are studies to replace the labile phosphoroanhydride linker present in biotinyl-5′-AMP with alternative bioisosteres. A novel in situ click approach using a mutant of S. aureus BPL as a template for the synthesis of triazole-based inhibitors is also presented. These approaches can be widely applied to BPLs from other bacteria, as well as other closely related metabolic enzymes and antibacterial drug targets. Refereed/Peer-reviewed

The interaction of IGF-II with the insulin receptor (IR) and type 1 insulin-like growth factor receptor (IGF-1R) has recently been identified as potential therapeutic target for the treatment of cancer. Understanding the interactions of IGF-II with these receptors is required for the development of potential anticancer therapeutics. This work describes an efficient convergent synthesis of native IGF-II and two non-native IGF-II analogues with coumarin fluorescent probes incorporated at residues 19 and 28. These fluorescent analogues bind with nanomolar affinities to the IGF-1R and are suitable for use in fluorescence resonance energy transfer (FRET) studies. From these studies the F19Cou IGF-II and F28Cou IGF-II proteins were identified as good probes for investigating the binding interactions of IGF-II with the IGF-1R and its other high affinity binding partners.

Reduced oocyte quality has been associated with poor fertility of high-performance dairy cows during peak lactation, due to negative energy balance. We examined the role of nonesterified fatty acids (NEFAs), known to accumulate within follicular fluid during under- and overnutrition scenarios, in causing endoplasmic reticulum (ER) stress of in vitro maturated cattle cumulus-oocyte complexes (COCs). NEFA concentrations were: palmitic acid (150 μM), oleic acid (200 μM), and steric acid (75 μM). Abattoir-derived COCs were randomly matured for 24 h in the presence of NEFAs and/or an ER stress inhibitor, salubrinal. Total and hatched blastocyst yields were negatively impacted by NEFA treatment compared with controls, but this was reversed by salubrinal. ER stress markers, activating transcription factor 4 (Atf4) and heat shock protein 5 (Hspa5), but not Atf6, were significantly up-regulated by NEFA treatment within whole COCs but reversed by coincubation with salubrinal. Likewise, glucose uptake and lactate production, measured in spent medium samples, showed a similar pattern, suggesting that cumulus cell metabolism is sensitive to NEFAs via an ER stress-mediated process. In contrast, while mitochondrial DNA copy number was recovered in NEFA-treated oocytes, oocyte autofluorescence of the respiratory chain cofactor, FAD, was lower following NEFA treatment of COCs, and this was not reversed by salubrinal, suggesting the negative impact was via reduced mitochondrial function. These results reveal the significance of NEFA-induced ER stress on bovine COC developmental competence, revealing a potential therapeutic target for improving oocyte quality during peak lactation.

Calpain inhibitors are possible therapeutic agents in the treatment of cataracts. These covalent inhibitors contain an electrophilic anchor (“warhead”), an aldehyde that reacts with the active site cysteine. Whilst high throughput docking of such ligands into high resolution protein structures (e.g. calpain) is a standard computational approach in drug discovery, there is no docking program that consistently achieves low rates of both false positives (FPs) and negatives (FNs) for ligands that react covalently (via irreversible interactions) with the target protein. Schroedinger’s GLIDE score, widely used to screen ligand libraries, is known to give high false classification, however a two-level Self Organizing Map (SOM) artificial neural network (ANN) algorithm, with KM clustering proved that the addition of two structural components of the calpain molecule, number hydrogen bonds and warhead distance, combined with GLIDE score (or its partial energy subcomponents) provide a superior predictor set for classification of true molecular binding strength (IC50). SOM ANN/KM significantly reduced the number of FNs by 64 % and FPs by 26 %, compared to the glide score alone. FPs were shown to be mostly esters and amides plus alcohols and non-classical, and FNs mainly aldehydes and ketones, masked aldehydes and ketones and Michael.

Reactive Oxygen Species (ROS) cause DNA damage and defective function in sperm and also affects the developmental competence of embryos. It is therefore critical to monitor ROS in sperm, oocytes and developing embryos. In particular, hydrogen peroxide (H2O2) is a ROS important to normal cell function and signalling as well as its role in oxidative stress. Here we report the development of a fluorescent sensor for H2O2 using carboxyperoxyfluor-1 (CPF1) in solution and attached to a glass slide or multi-mode optical fibre. CPF1 increases in fluorescence upon reaction with H2O2 to non-invasively detect H2O2 near developing embryos. These probes are constructed by immobilising CPF1 to the optical fibre tip a polyacrylamide layer. Also reported is a new dual optical fibre sensor for detecting both H2O2 and pH that is functional at biologically concentrations of H2O2 and can sense pH to 0.1 units. This research shows promise for the use of optical fibre sensors for monitoring the health of developing embryos. Furthermore, these sensors are applicable for use beyond embryos such as detecting stress in endothelial cells involved in cardiovascular dysfunction. Refereed/Peer-reviewed

The 26S proteasome has emerged over the past decade as an attractive therapeutic target in the treatment of cancers. Here, we report new tripeptide aldehydes that are highly specific for the chymotrypsin-like catalytic activity of the proteasome. These new specific proteasome inhibitors demonstrated high potency and specificity for sarcoma cells, with therapeutic windows superior to those observed for benchmark proteasome inhibitors, MG132 and Bortezomib. Constraining the peptide backbone into the β-strand geometry, known to favor binding to a protease, resulted in decreased activity in vitro and reduced anticancer activity. Using these new proteasome inhibitors, we show that the presence of an intact p53 pathway significantly enhances cytotoxic activity, thus suggesting that this tumor suppressor is a critical downstream mediator of cell death following proteasomal inhibition.

Acetyl-CoA carboxylase (ACC) catalyses the first committed step in fatty acid biosynthesis: a metabolic pathway required for several important biological processes including the synthesis and maintenance of cellular membranes. ACC employs a covalently attached biotin moiety to bind a carboxyl anion and then transfer it to acetyl-CoA, yielding malonyl-CoA. These activities occur at two different subsites: the biotin carboxylase (BC) and carboxyltransferase (CT). Structural biology, together with small molecule inhibitor studies, has provided new insights into the molecular mechanisms that govern ACC catalysis, specifically the BC and CT subunits. Here, we review these recent findings and highlight key differences between the bacterial and eukaryotic isozymes with a view to establish those features that provide an opportunity for selective inhibition. Especially important are examples of highly selective small molecule inhibitors capable of differentiating between ACCs from different phyla. The implications for early stage antibiotic discovery projects, stemming from these studies, are discussed. Refereed/Peer-reviewed

DNA methylation, which often occurs at the cytosine residue of cytosine-guanine dinucleotides, is critical for the control of gene expression and mitotic inheritance in eukaryotes. DNA methylation silences gene expression either by directly hindering the access of transcription factors to the target DNA, or through recruitment of histone deacetylases to remodel the chromatin structure to an inactive state. Aberrant hypermethylation of tumor suppressor genes is commonly associated with the development of cancer. A number of anti-cancer agents have been developed that function through demethylation, reversing regional hypermethylation to restore the expression of tumor suppressor genes. Azacitidine and decitabine are used in the clinic, but their applications are limited to myelodysplastic syndrome and other blood-related diseases. Despite the potency of these drugs, their broader clinical application is restricted by cytotoxicity, nonspecific targeting, structural instability, catabolism, and poor bioavailability. Further improvements in the delivery systems for these drugs could overcome the issues associated with inefficient bioavailability, whilst facilitating the administration of combinations of demethylating agents and histone deacetylase inhibitors to enhance efficacy. This review focuses on the current limitations of existing demethylating agents and highlights possible approaches using recent developments in drug delivery systems to improve the clinical potential of these drugs.

The Kyn-containing peptide FP-Kyn-L(NH2) is an unusual minor component of the skin peptide profile of the Australian red tree frog Litoria rubella collected from an area within a 20 kilometre radius of Alice Springs in central Australia. The structure was determined by electrospray mass spectrometry and synthesis. The major component of the skin secretion is the analogous tryptophyllin peptide FPWL(NH2). Both peptides show opioid activity at 10–7 M, and are likely to act via the μ opioid receptor. Copyright © 2011 John Wiley & Sons, Ltd.

Victoria Peddie, Markus Pietsch, Karen M. Bromfield, Robert N. Pike, Peter J. Duggan, Andrew D. Abell

The physiological roles of calpains are discussed, as are the associated pathological disorders that result from their over-activation. We also present practical information for establishing functional inhibition assays and an overview of X-ray crystal structures of calpain-inhibitor complexes to aid inhibitor design. These structures reveal the expected extended beta-strand conformation for the inhibitor backbone, a geometry that has been engineered into inhibitors with the introduction of either an N-terminal heterocycle or a macrocycle that links the P(1) and P(3) residues. The structure and function of all the main classes of inhibitors are reviewed, with most examples being classified according to the nature of the C-terminal reactive warhead group that reacts with the active site cysteine of calpains. These inhibitor classes include epoxysuccinate derivatives, aldehydes, aldehyde prodrugs (hemiacetals) and alpha-keto carbonyl compounds. Inhibitors derived from the endogenous inhibitor calpastatin and examples lacking a warhead, are now known and these are also discussed.

The aim of this study is to evaluate the therapeutic potential of a newly synthesized calpain inhibitor, CAT0059, using a naturally occurring in vivo sheep cataract model.The selectivity of CAT0059 was investigated by an in vitro protease assay. The efficacy of CAT0059 in preventing proteolysis of lens cytoskeletal proteins by calpain 2 was investigated using a lens-based cell-free method. The cytotoxicity and stability of CAT0059 in physiological conditions were examined using cultured sheep lenses. Protein binding of CAT0059 by ocular proteins was assessed and quantified by a modified high-performance liquid chromatography assay. CAT0059 was formulated in an eye drop solution and as an eye ointment. These were applied in vivo daily to one eye of the cataract lambs, over a 67- and 97-day trial period, respectively. The progression of cataracts in the treated and untreated eyes was assessed by an independent veterinary ophthalmologist using a slit-lamp microscope.In vitro assays revealed that CAT0059 was selective for cysteine proteases and also protected lens cytoskeletal proteins from degradation. CAT0059 was stable in physiological conditions and non-toxic to the lens. Only 15% of CAT0059 is bound to proteins in the aqueous humour but90% bound to lens homogenate. The 67-day CAT0059 eye drop treatment was not effective in slowing the rate of cataract development. However, application of CAT0059 in an eye ointment initially slowed cataract development compared with the untreated eye. This effect was temporary.In vitro assays confirmed CAT0059 to be a potent calpain inhibitor. The two in vivo trials addressed the ability of CAT0059 to reach the lens and established its limitations as a therapeutic molecule for cataract treatment.

A series of peptidomimetic trifluoromethylketones containing the photoisomerisable azobenzene group have been synthesised, photoisomerised and assayed against the serine protease alpha-chymotrypsin. All are inhibitors of the enzyme and exhibit up to a fivefold increase in activity on UV irradiation. Visible irradiation returns activity to close to that of the original sample. These results suggest the trifluoromethylketone group to be the best electrophilic enzyme binding group for use in photoswitch inhibitors of alpha-chymotrypsin.

Three new compounds (3-5) based on a dipeptide non-covalent inhibitor (1) of α-chymotrypsin have been synthesised and assayed against the serine protease α-chymotrypsin. The stability of the compounds to α-chymotrypsin catalysed hydrolysis has been measured. All three compounds were inactive with a retained stability to enzyme catalysed hydrolysis.

Filling the pocket: Biaryl inhibitors of type II dehydroquinase are described, designed to bind to both the active site and a subsidiary pocket. Compounds exhibited nanomolar inhibition of the enzyme, which is rationalised by molecular docking and protein crystallographic studies.

4′-Substituted bicyclic pyridones were prepared and evaluated as non-steroidal inhibitors of type 1 and 2 steroid 5α-reductase (SR). A range of 4′-substituents were incorporated into the bicyclic scaffold to investigate SAR within and across different classes of non-steroidal inhibitors of SR. Bicyclic pyridones containing a 4′-benzoyl or long carbon chain tether showed more potent inhibition against type 1 SR than inhibitors with N-substituted acetamide groups in the 4′-position. SAR derived from 4′-substituted bicyclic pyridones reported here do not correlate with SAR derived from known potent 4′-substituted biaryl acid SR inhibitors. A 4′-benzoyl group is favoured by the active site in both isozymes.

An improved synthesis of biotinol-5′-AMP, an acyl-AMP mimic of the natural reaction intermediate of biotin protein ligase (BPL), is reported. This compound was shown to be a pan inhibitor of BPLs from a series of clinically important bacteria, particularly Staphylococcus aureus and Mycobacterium tuberculosis, and kinetic analysis revealed it to be competitive against the substrate biotin. Biotinol-5′-AMP also exhibits antibacterial activity against a panel of clinical isolates of S. aureus and M. tuberculosis with MIC values of 1-8 and 0.5-2.5 μg/mL, respectively, while being devoid of cytotoxicity to human HepG2 cells. Refereed/Peer-reviewed

A series of peptidomimetic boronate esters containing the photoisomerisable azobenzene group has been synthesised and assayed against the serine protease alpha-chymotrypsin. Compounds with borophenylalanine inhibition groups were found to be inhibitors with micromolar activity, while those with aryl boronate inhibition groups were inactive. Selected compounds were isomerised by UV or visible light to obtain enriched states of the (Z) or (E) isomers, respectively, and assayed. A change in activity on photoisomerisation was observed, however some decomposition of the boronate group on irradiation was also observed, limiting reversibility.

Two vitamin K analogues bearing a carboxylic acid side chain (9a and its deuterated analogue 9b) were each synthesised in six steps from commercially available menadione. Analogue 9b was conjugated to lysozyme and bovine serum albumin (BSA) using EDCI/HOBT and by prior formation of its activated succinimidyl ester 11. Quantification of the thus formed conjugates by ESMS and LC-MS revealed that the number of equivalents of the analogue used in the couplings systematically controls the number of analogues that conjugate to the protein.

Biotin protein ligase (BPL) represents a promising target for the discovery of new antibacterial chemotherapeutics. Here we review the central role of BPL for the survival and virulence of clinically important Staphylococcus aureus in support of this claim. X-ray crystallography structures of BPLs in complex with ligands and small molecule inhibitors provide new insights into the mechanism of protein biotinylation, and a template for structure guided approaches to the design of inhibitors for antibacterial discovery. Most BPLs employ an ordered ligand binding mechanism for the synthesis of the reaction intermediate biotinyl-5′-AMP from substrates biotin and ATP. Recent studies reporting chemical analogs of biotin and biotinyl-5′-AMP as BPL inhibitors that represent new classes of anti-S. aureus agents are reviewed. We highlight strategies to selectively inhibit bacterial BPL over the mammalian equivalent using a 1,2,3-triazole isostere to replace the labile phosphoanhydride naturally present in biotinyl-5′-AMP. A novel in situ approach to improve the detection of triazole-based inhibitors is also presented that could potentially be widely applied to other protein targets. Refereed/Peer-reviewed

The design, synthesis, photoisomerism and biological testing of two peptide-based photoswitchable inhibitors of α-chymotrypsin are presented. The use of a dipeptide recognition sequence gave a ‘slow-tight binding’ inhibitor, while the introduction of a carbamate linker to the azobenzene gave a modest enhancement in photoswitching of enzyme activity for the photostationary state enriched in the ( Z )-isomer over the ( E )-isomer.

N-Acyl and N-sulfonylhydroxymetyhylpyrroles have been synthesised and shown to inhibit alpha-chymotrypsin. A hydrophobic group in the N-substituent has been shown to be required for activity.

Three photoswitchable, peptidomimetic inhibitors of α-chymotrypsin have been synthesised. The compounds comprise an azobenzene, an α-ketoester and l -phenylalanine. The compounds were photoisomerised to give enriched states of the (E) and (Z) isomers and these states were assayed against α-chymotrypsin. The inhibitors were shown to be moderately active with switching ability of between two- and three-fold between the two isomer-enriched states. The behaviour of the inhibitors in solution was examined; specifically, their hydration and configurational stability.

The four stereoisomers of 3-(N-acetylleucylamino)-3-benzyl-1-[(methylsulfonyl)oxy]succinimid e have been prepared and shown to inhibit alpha-chymotrypsin and human neutrophil elastase. The structural and stereochemical features that affect the potency and selectivity of inhibition are discussed.

A series of ascorbic acid and isoascorbic acid derivatives has been evaluated as inhibitors of malt, bacterial, fungal, pancreatic and salivary alpha-amylases using a simple and quick assay procedure. The results demonstrate that the enediol moiety of ascorbic acid is essential for alpha-amylase inhibition. Acylation of the primary and secondary alcohols, and the absolute configuration of the secondary alcohol, do not affect the potency of inhibition.

The 4-nitrophenoxy-based epoxide 6a, conveniently synthesized using Sharpless epoxidation chemistry, has been shown to be an irreversible inhibitor of HIV-1 protease (Kinact 1.8 μM, pH 6.5, I = 0.1 M). Related analogues, differing in the mode of attachment to the epoxide core, have also been prepared and assayed against HIV-1 protease.

A synthesis of a new, tetrazole-based, cis-constrained hydroxyethylamine peptide bond isostere is reported. This has been used to produce a new class of HIV-1 protease inhibitor.

The key structural features associated with the potent cytotoxicity observed in the mycalamide, onnamide, pederin and theopederin series have been defined on the basis of structure–activity studies. A model pharmacophore structure has been proposed and selected examples, with modest bioactivity, synthesized.