Identification of Ribonuclease Inhibitors for the Control of Pathogenic Bacteria.

Bacteria are known to be constantly adapting to become resistant to antibiotics. Currently, efficient antibacterial compounds are still available; however, it is only a matter of time until these compounds also become inefficient. Ribonucleases are the enzymes responsible for the maturation and degradation of RNA molecules, and many of them are essential for microbial survival. Members of the PNPase and RNase II families of exoribonucleases have been implicated in virulence in many pathogens and, as such, are valid targets for the development of new antibacterials. In this paper, we describe the use of virtual high-throughput screening (vHTS) to identify chemical compounds predicted to bind to the active sites within the known structures of RNase II and PNPase from Escherichia coli. The subsequent in vitro screening identified compounds that inhibited the activity of these exoribonucleases, with some also affecting cell viability, thereby providing proof of principle for utilizing the known structures of these enzymes in the pursuit of new antibacterials.

Novel Paracrine Action of Endothelium Enhances Glucose Uptake in Muscle and Fat.

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Site-directed M2 proton channel inhibitors enable synergistic combination therapy for rimantadine-resistant pandemic influenza.

Pandemic influenza A virus (IAV) remains a significant threat to global health. Preparedness relies primarily upon a single class of neuraminidase (NA) targeted antivirals, against which resistance is steadily growing. The M2 proton channel is an alternative clinically proven antiviral target, yet a near-ubiquitous S31N polymorphism in M2 evokes resistance to licensed adamantane drugs. Hence, inhibitors capable of targeting N31 containing M2 (M2-N31) are highly desirable. Rational in silico design and in vitro screens delineated compounds favouring either lumenal or peripheral M2 binding, yielding effective M2-N31 inhibitors in both cases. Hits included adamantanes as well as novel compounds, with some showing low micromolar potency versus pandemic "swine" H1N1 influenza (Eng195) in culture. Interestingly, a published adamantane-based M2-N31 inhibitor rapidly selected a resistant V27A polymorphism (M2-A27/N31), whereas this was not the case for non-adamantane compounds. Nevertheless, combinations of adamantanes and novel compounds achieved synergistic antiviral effects, and the latter synergised with the neuraminidase inhibitor (NAi), Zanamivir. Thus, site-directed drug combinations show potential to rejuvenate M2 as an antiviral target whilst reducing the risk of drug resistance.

PTPRD and DCC Are Novel BACE1 Substrates Differentially Expressed in Alzheimer's Disease: A Data Mining and Bioinformatics Study.

The ß-site Amyloid precursor protein Cleaving Enzyme 1 (BACE1) is an extensively studied therapeutic target for Alzheimer's disease (AD), owing to its role in the production of neurotoxic amyloid beta (Aß) peptides. However, despite numerous BACE1 inhibitors entering clinical trials, none have successfully improved AD pathogenesis, despite effectively lowering Aß concentrations. This can, in part, be attributed to an incomplete understanding of BACE1, including its physiological functions and substrate specificity. We propose that BACE1 has additional important physiological functions, mediated through substrates still to be identified. Thus, to address this, we computationally analysed a list of 533 BACE1 dependent proteins, identified from the literature, for potential BACE1 substrates, and compared them against proteins differentially expressed in AD. We identified 15 novel BACE1 substrates that were specifically altered in AD. To confirm our analysis, we validated Protein tyrosine phosphatase receptor type D (PTPRD) and Netrin receptor DCC (DCC) using Western blotting. These findings shed light on the BACE1 inhibitor failings and could enable the design of substrate-specific inhibitors to target alternative BACE1 substrates. Furthermore, it gives us a greater understanding of the roles of BACE1 and its dysfunction in AD.

Affimer proteins as a tool to modulate fibrinolysis, stabilize the blood clot, and reduce bleeding complications.

Bleeding complications secondary to surgery, trauma, or coagulation disorders are important causes of morbidity and mortality. Although fibrin sealants are considered to minimize blood loss, this is not widely adopted because of its high cost and/or risk for infection. We present a novel methodology employing nonantibody fibrinogen-binding proteins, termed Affimers, to stabilize fibrin networks with the potential to control excessive bleeding. Two fibrinogen-specific Affimer proteins, F5 and G2, were identified and characterized for their effects on clot structure/fibrinolysis, using turbidimetric and permeation analyses and confocal and electron microscopy. Binding studies and molecular modeling identified interaction sites, whereas plasmin generation assays determined effects on plasminogen activation. In human plasma, F5 and G2 prolonged clot lysis time from 9.8 ± 1.1 minutes in the absence of Affimers to 172.6 ± 7.4 and more than 180 minutes (P < .0001), respectively, and from 7.6 ± 0.2 to 28.7 ± 5.8 (P < .05) and 149.3 ± 9.7 (P < .0001) minutes in clots made from purified fibrinogen. Prolongation in fibrinolysis was consistent across plasma samples from healthy control patients and individuals at high bleeding risk. F5 and G2 had a differential effect on clot structure and G2 profoundly altered fibrin fiber arrangement, whereas F5 maintained physiological clot structure. Affimer F5 reduced fibrin-dependent plasmin generation and was predicted to bind fibrinogen D fragment close to tissue plasminogen activator (tPA; residues γ312-324) and plasminogen (α148-160) binding sites, thus interfering with tPA-plasminogen interaction and representing 1 potential mechanism for modulation of fibrinolysis. Our Affimer proteins provide a novel methodology for stabilizing fibrin networks with potential future clinical implications to reduce bleeding risk.

Fibrinogen interaction with complement C3: a potential therapeutic target to reduce thrombosis risk.

Complement C3 binds fibrinogen and compromises fibrin clot lysis thereby enhancing thrombosis risk. We investigated the role of fibrinogen-C3 interaction as a novel therapeutic target to reduce thrombosis risk by analysing: i) consistency in the fibrinolytic properties of C3, ii) binding sites between fibrinogen and C3 and iii) modulation of fibrin clot lysis by manipulating fibrinogen-C3 interactions. Purified fibrinogen and C3 from the same individuals (n=24) were used to assess inter-individual variability in the anti-fibrinolytic effects of C3. Microarray screening and molecular modelling evaluated C3 and fibrinogen interaction sites. Novel synthetic conformational proteins, termed Affimers, were used to modulate C3-fibrinogen interaction and fibrinolysis. C3 purified from patients with type 1 diabetes showed enhanced prolongation of fibrinolysis compared with healthy control protein [195±105 and 522±166 seconds, respectively (p=0.04)], with consistent effects but a wider range (5-51% and 5-18% lysis prolongation, respectively). Peptide microarray screening identified 2 potential C3-fibrinogen interactions sites within fibrinogen ß chain (residues 424-433, 435-445). One fibrinogen-binding Affimer was isolated that displayed sequence identity with C3 in an exposed area of the protein. This Affimer abolished C3-induced prolongation of fibrinolysis (728±25.1 seconds to 632±23.7 seconds, p=0.005) and showed binding to fibrinogen in the same region that is involved in C3-fibrinogen interactions. Moreover, it shortened plasma clot lysis of patients with diabetes, cardiovascular disease or controls by 7-11%. C3 binds fibrinogen ß-chain and disruption of fibrinogen-C3 interaction using Affimer proteins enhances fibrinolysis, which represents a potential novel target tool to reduce thrombosis in high risk individuals.

Divergent effects of genetic and pharmacological inhibition of Nox2 NADPH oxidase on insulin resistance-related vascular damage.

Insulin resistance leads to excessive endothelial cell (EC) superoxide generation and accelerated atherosclerosis. The principal source of superoxide from the insulin-resistant endothelium is the Nox2 isoform of NADPH oxidase. Here we examine the therapeutic potential of Nox2 inhibition on superoxide generation in saphenous vein ECs (SVECs) from patients with advanced atherosclerosis and type 2 diabetes and on vascular function, vascular damage, and lipid deposition in apolipoprotein E-deficient (ApoE-/-) mice with EC-specific insulin resistance (ESMIRO). To examine the effect of genetic inhibition of Nox2, ESMIRO mice deficient in ApoE-/- and Nox2 (ESMIRO/ApoE-/-/Nox2-/y) were generated and compared with ESMIRO/ApoE-/-/Nox2+/y littermates. To examine the effect of pharmacological inhibition of Nox2, we administered gp91dstat or scrambled peptide to ESMIRO/ApoE-/- mice. SVECs from diabetic patients had increased expression of Nox2 protein with concomitant increase in superoxide generation, which could be reduced by the Nox2 inhibitor gp91dstat. After 12 wk Western diet, ESMIRO/ApoE-/-/Nox2-/y mice had reduced EC superoxide generation and greater aortic relaxation to acetylcholine. ESMIRO/ApoE-/-/Nox2-/y mice developed more lipid deposition in the thoraco-abdominal aorta with multiple foci of elastin fragmentation at the level of the aortic sinus and greater expression of intercellular adhesion molecule-1 (ICAM-1). Gp91dstat reduced EC superoxide and lipid deposition in the thoraco-abdominal aorta of ESMIRO/ApoE-/- mice without causing elastin fragmentation or increased ICAM-1 expression. These results demonstrate that insulin resistance is characterized by increased Nox2-derived vascular superoxide. Complete deletion of Nox2 in mice with EC insulin resistance exacerbates, whereas partial pharmacological Nox2 inhibition protects against, insulin resistance-induced vascular damage.

Extending enzyme molecular recognition with an expanded amino acid alphabet.

Natural enzymes are constructed from the 20 proteogenic amino acids, which may then require posttranslational modification or the recruitment of coenzymes or metal ions to achieve catalytic function. Here, we demonstrate that expansion of the alphabet of amino acids can also enable the properties of enzymes to be extended. A chemical mutagenesis strategy allowed a wide range of noncanonical amino acids to be systematically incorporated throughout an active site to alter enzymic substrate specificity. Specifically, 13 different noncanonical side chains were incorporated at 12 different positions within the active site of N-acetylneuraminic acid lyase (NAL), and the resulting chemically modified enzymes were screened for activity with a range of aldehyde substrates. A modified enzyme containing a 2,3-dihydroxypropyl cysteine at position 190 was identified that had significantly increased activity for the aldol reaction of erythrose with pyruvate compared with the wild-type enzyme. Kinetic investigation of a saturation library of the canonical amino acids at the same position showed that this increased activity was not achievable with any of the 20 proteogenic amino acids. Structural and modeling studies revealed that the unique shape and functionality of the noncanonical side chain enabled the active site to be remodeled to enable more efficient stabilization of the transition state of the reaction. The ability to exploit an expanded amino acid alphabet can thus heighten the ambitions of protein engineers wishing to develop enzymes with new catalytic properties.

Molecular mechanism of ligand recognition by membrane transport protein, Mhp1.

The hydantoin transporter Mhp1 is a sodium-coupled secondary active transport protein of the nucleobase-cation-symport family and a member of the widespread 5-helix inverted repeat superfamily of transporters. The structure of Mhp1 was previously solved in three different conformations providing insight into the molecular basis of the alternating access mechanism. Here, we elucidate detailed events of substrate binding, through a combination of crystallography, molecular dynamics, site-directed mutagenesis, biochemical/biophysical assays, and the design and synthesis of novel ligands. We show precisely where 5-substituted hydantoin substrates bind in an extended configuration at the interface of the bundle and hash domains. They are recognised through hydrogen bonds to the hydantoin moiety and the complementarity of the 5-substituent for a hydrophobic pocket in the protein. Furthermore, we describe a novel structure of an intermediate state of the protein with the external thin gate locked open by an inhibitor, 5-(2-naphthylmethyl)-L-hydantoin, which becomes a substrate when leucine 363 is changed to an alanine. We deduce the molecular events that underlie acquisition and transport of a ligand by Mhp1.

Inhibition of complement C3 and fibrinogen interaction: a potential novel therapeutic target to reduce cardiovascular disease in diabetes.

BACKGROUND: Enhanced complement C3 incorporation into the fibrin network in diabetes is one mechanism for impaired fibrinolysis and increased thrombosis risk in this condition. Our aim was to develop new strategies to modulate fibrinolysis in diabetes by interfering with fibrin-C3 interaction. METHODS: To modulate interaction between fibrinogen and C3 we used a novel technique by screening fibrinogen with a phage display library of 3 billion random, conformational 9AA peptides (termed adhirons). The effect of high affinity fibrinogen binding adhirons, released by the addition of excess C3, on fibrin clot lysis and structure was assessed in turbidimetric assays. Fibrinogen-C3 interactions were further studied by peptide microarray techniques and modelled with the website PepSite2. FINDINGS: Ten high affinity fibrinogen binding adhirons, released by C3, were available for turbidimetric analysis. One adhiron (A6) was found to have a sequence homology with C3 and studied further. In the absence of C3, adhiron A6 failed to modulate fibrin clot lysis time (mean 644 s [SE 13] and 620 [14] without and with adhiron A6, respectively). However, adhiron A6 abolished C3-induced prolongation of clot lysis, reducing mean lysis time from 728 s (SE 25) to 632 (24) (p=0·01). The peptide microarray screening of C3 identified two peptide motifs within the ß chain of fibrinogen (residues 424-433, 435-445) that bound to C3. PepSite2 predicted that adhiron A6 binds to similar areas on the ß chain of fibrinogen. INTERPRETATION: Using a novel phage display system, we discovered an adhiron that shared sequence homology with C3 and abolished C3-induced prolongation of fibrin clot lysis by interfering with C3-fibrinogen interaction within the ß chain. This technique offers a unique method to identify new therapeutic targets for the reduction of diabetes-specific thrombosis risk. FUNDING: Sir Jules Thorn Charitable Trust.

Cyclic dinucleotides bind the C-linker of HCN4 to control channel cAMP responsiveness.

cAMP mediates autonomic regulation of heart rate by means of hyperpolarization-activated cyclic nucleotide-gated (HCN) channels, which underlie the pacemaker current If. cAMP binding to the C-terminal cyclic nucleotide binding domain enhances HCN open probability through a conformational change that reaches the pore via the C-linker. Using structural and functional analysis, we identified a binding pocket in the C-linker of HCN4. Cyclic dinucleotides, an emerging class of second messengers in mammals, bind the C-linker pocket (CLP) and antagonize cAMP regulation of the channel. Accordingly, cyclic dinucleotides prevent cAMP regulation of If in sinoatrial node myocytes, reducing heart rate by 30%. Occupancy of the CLP hence constitutes an efficient mechanism to hinder ß-adrenergic stimulation on If. Our results highlight the regulative role of the C-linker and identify a potential drug target in HCN4. Furthermore, these data extend the signaling scope of cyclic dinucleotides in mammals beyond their first reported role in innate immune system.

Docking of competitive inhibitors to the P2X7 receptor family reveals key differences responsible for changes in response between rat and human.

The P2X7 receptor is a calcium permeable cationic channel activated by extracellular ATP, playing a role in chronic pain, osteoporosis and arthritis. A number of potential lead compounds are inactive against the rat isoform, despite good activity against the human homologue, making animal model studies problematic. Here we have produced P2X7 models and docked three structurally distinct inhibitors using in silico approaches and show they have a similar mode of binding in which Phe95 plays a key role by forming pi-stacking interactions. Importantly this residue is replaced by Leu in the rat P2X7 receptor resulting in a significantly reduced binding affinity. This work provides new insights into binding of P2X7 inhibitors and shows the structural difference in human and rat P2X7 receptors which results in a difference in affinity. Such information is useful both for the rational design of inhibitors based on these scaffolds and also the way in which these compounds are tested in animal models.

Applications of structure-based design to antibacterial drug discovery.

In recent years bacterial resistance has been observed against many of our current antibiotics, for instance most worryingly against the cephalosporins which are typically the last line of defence against many bacterial infections. Additionally the failure of high throughput screening in the discovery of new antibacterial drug leads has led to a decline in the number of antibacterial agents reaching the market. Alternative methods of drug discovery including structure based drug design are needed to meet the threats caused by the emergence of resistance. In this review we explore the latest advancements in the identification of new antibacterial agents through the use of a number of structure based drug design programs.

A virtual high-throughput screening approach to the discovery of novel inhibitors of the bacterial leucine transporter, LeuT.

Membrane proteins are intrinsically involved in both human and pathogen physiology, and are the target of 60% of all marketed drugs. During the past decade, advances in the studies of membrane proteins using X-ray crystallography, electron microscopy and NMR-based techniques led to the elucidation of over 250 unique membrane protein crystal structures. The aim of the European Drug Initiative for Channels and Transporter (EDICT) project is to use the structures of clinically significant membrane proteins for the development of lead molecules. One of the approaches used to achieve this is a virtual high-throughput screening (vHTS) technique initially developed for soluble proteins. This paper describes application of this technique to the discovery of inhibitors of the leucine transporter (LeuT), a member of the neurotransmitter:sodium symporter (NSS) family.

Using site-directed mutagenesis to further the understanding of insulin receptor-insulin like growth factor-1 receptor heterodimer structure.

Type 2 diabetes is characterised by the disruption of insulin and insulin-like growth factor (IGF) signalling. The key hubs of these signalling cascades - the Insulin receptor (IR) and Insulin-like growth factor 1 receptor (IGF1R) - are known to form functional IR-IGF1R hybrid receptors which are insulin resistant. However, the mechanisms underpinning IR-IGF1R hybrid formation are not fully understood, hindering the ability to modulate this for future therapies targeting this receptor. To pinpoint suitable sites for intervention, computational hotspot prediction was utilised to identify promising epitopes for targeting with point mutagenesis. Specific IGF1R point mutations F450A, R391A and D555A show reduced affinity of the hybrid receptor in a BRET based donor-saturation assay, confirming hybrid formation could be modulated at this interface. These data provide the basis for rational design of more effective hybrid receptor modulators, supporting the prospect of identifying a small molecule that specifically interacts with this target.

Small molecule modulation of insulin receptor-insulin like growth factor-1 receptor heterodimers in human endothelial cells.

OBJECTIVES: The insulin receptor (IR) and insulin like growth factor-1 receptor (IGF-1R) are heterodimers consisting of two extracellular α-subunits and two transmembrane ß -subunits. Insulin αß and insulin like growth factor-1 αß hemi-receptors can heterodimerize to form hybrids composed of one IR αß and one IGF-1R αß. The function of hybrids in the endothelium is unclear. We sought insight by developing a small molecule capable of reducing hybrid formation in endothelial cells. METHODS: We performed a high-throughput small molecule screening, based on a homology model of the apo hybrid structure. Endothelial cells were studied using western blotting and qPCR to determine the effects of small molecules that reduced hybrid formation. RESULTS: Our studies unveil a first-in-class quinoline-containing heterocyclic small molecule that reduces hybrids by >50% in human umbilical vein endothelial cells (HUVECs) with no effects on IR or IGF-1R. This small molecule reduced expression of the negative regulatory p85α subunit of phosphatidylinositol 3-kinase, increased basal phosphorylation of the downstream target Akt and enhanced insulin/insulin-like growth factor-1 and shear stress-induced serine phosphorylation of Akt. In primary saphenous vein endothelial cells (SVEC) from patients with type 2 diabetes mellitus undergoing coronary artery bypass (CABG) surgery, hybrid receptor expression was greater than in patients without type 2 diabetes mellitus. The small molecule significantly reduced hybrid expression in SVEC from patients with type 2 diabetes mellitus. CONCLUSIONS: We identified a small molecule that decreases the formation of IR: IGF-1R hybrid receptors in human endothelial cells, without significant impact on the overall expression of IR or IGF-1R. In HUVECs, reduction of IR: IGF-1R hybrid receptors leads to an increase in insulin-induced serine phosphorylation of the critical downstream signalling kinase, Akt. The underpinning mechanism appears, at least in part to involve the attenuation of the inhibitory effect of IR: IGF-1R hybrid receptors on PI3-kinase signalling.

The ß-Secretase BACE1 Drives Fibroblast Activation in Systemic Sclerosis through the APP/ß-Catenin/Notch Signaling Axis.

BACE1 is well-known for its role in the development of Alzheimer's disease. Recent publications, including our own, have demonstrated a role for this enzyme in other chronic diseases. The aim of this study was to investigate the role of BACE1 in the autoimmune disease systemic sclerosis (SSc). BACE1 protein levels were elevated in the skin of patients with SSc. Inhibition of BACE1 with small-molecule inhibitors or small interfering RNA blocked SSc and fibrotic stimuli-mediated fibroblast activation. Furthermore, we show that BACE1 regulation of dermal fibroblast activation is dependent on ß-catenin and Notch signaling. The neurotropic factor brain-derived neurotrophic factor negatively regulates BACE1 expression and activity in dermal fibroblasts. Finally, sera from patients with SSc show higher ß-amyloid and lower brain-derived neurotrophic factor levels than healthy controls. The ability of BACE1 to regulate SSc fibroblast activation reveals a therapeutic target in SSc. Several BACE1 inhibitors have been shown to be safe in clinical trials for Alzheimer's disease and could be repurposed to ameliorate fibrosis progression.

Effectiveness of 2 methods of chlorhexidine bathing.

The purposes of this study were to examine the impact of chlorhexidine on the transmission of methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococcus (VRE) on an inpatient oncology unit, compare the cost of 2 chlorhexidine bath delivery methods, and evaluate nursing time and satisfaction to administer the baths. MRSA and VRE transmission rates decreased from those during the previous years. Costs associated with bathing increased, but time to administer the bath decreased with the chlorhexidine cloths, and nursing staff reported satisfaction with their use.

Plasmin Inhibitor in Health and Diabetes: Role of the Protein as a Therapeutic Target.

The vascular obstructive thrombus is composed of a mesh of fibrin fibers with blood cells trapped in these networks. Enhanced fibrin clot formation and/or suppression of fibrinolysis are associated with an increased risk of vascular occlusive events. Inhibitors of coagulation factors and activators of plasminogen have been clinically used to limit fibrin network formation and enhance lysis. While these agents are effective at reducing vascular occlusion, they carry a significant risk of bleeding complications. Fibrin clot lysis, essential for normal hemostasis, is controlled by several factors including the incorporation of antifibrinolytic proteins into the clot. Plasmin inhibitor (PI), a key antifibrinolytic protein, is cross-linked into fibrin networks with higher concentrations of PI documented in fibrin clots and plasma from high vascular risk individuals. This review is focused on exploring PI as a target for the prevention and treatment of vascular occlusive disease. We first discuss the relationship between the PI structure and antifibrinolytic activity, followed by describing the function of the protein in normal physiology and its role in pathological vascular thrombosis. Subsequently, we describe in detail the potential use of PI as a therapeutic target, including the array of methods employed for the modulation of protein activity. Effective and safe inhibition of PI may prove to be an alternative and specific way to reduce vascular thrombotic events while keeping bleeding risk to a minimum. Key Points Plasmin inhibitor (PI) is a key protein that inhibits fibrinolysis and stabilizes the fibrin network.This review is focused on discussing mechanistic pathways for PI action, role of the molecule in disease states, and potential use as a therapeutic target.

Recent developments in the structural characterisation of the IR and IGF1R: implications for the design of IR-IGF1R hybrid receptor modulators.

The insulin receptor (IR) and insulin-like growth factor 1 receptor (IGF1R) are dimeric disulfide-linked receptor tyrosine kinases, whose actions regulate metabolic and mitogenic signalling pathways inside the cell. It is well documented that in tissues co-expressing the IR and IGF1R, their respective monomers can heterodimerise to form IR-IGF1R hybrid receptors. Increased populations of the IR-IGF1R hybrid receptors are associated with several disease states, including type 2 diabetes and cancer. Recently, progress in the structural biology of IR and IGF1R has given insights into their structure-function relationships and mechanism of action. However, challenges in isolating IR-IGF1R hybrid receptors mean that their structural properties remain relatively unexplored. This review discusses the advances in the structural understanding of the IR and IGF1R, and how these discoveries can inform the design of small-molecule modulators of the IR-IGF1R hybrid receptors to understand their role in cell biology.