Cathepsin X deficiency alters the processing and localisation of cathepsin L and impairs cleavage of a nuclear cathepsin L substrate.

Proteases function within sophisticated networks. Altering the activity of one protease can have sweeping effects on other proteases, leading to changes in their activity, structure, specificity, localisation, stability, and expression. Using a suite of chemical tools, we investigated the impact of cathepsin X, a lysosomal cysteine protease, on the activity and expression of other cysteine proteases and their inhibitors in dendritic cells. Among all proteases examined, cathepsin X gene deletion specifically altered cathepsin L levels; pro-cathepsin L and its single chain accumulated while the two-chain form was unchanged. This effect was recapitulated by chemical inhibition of cathepsin X, suggesting a dependence on its catalytic activity. We demonstrated that accumulation of pro- and single chain cathepsin L was not due to a lack of direct cleavage by cathepsin X or altered glycosylation, secretion, or mRNA expression but may result from changes in lysosomal oxidative stress or pH. In the absence of active cathepsin X, nuclear cathepsin L and cleavage of the known nuclear cathepsin L substrate, Lamin B1, were diminished. Thus, cathepsin X activity selectively regulates cathepsin L, which has the potential to impact the degree of cathepsin L proteolysis, the nature of substrates that it cleaves, and the location of cleavage.

Disrupting the platelet internal membrane via PI3KC2α inhibition impairs thrombosis independently of canonical platelet activation.

Arterial thrombosis causes heart attacks and most strokes and is the most common cause of death in the world. Platelets are the cells that form arterial thrombi, and antiplatelet drugs are the mainstay of heart attack and stroke prevention. Yet, current drugs have limited efficacy, preventing fewer than 25% of lethal cardiovascular events without clinically relevant effects on bleeding. The key limitation on the ability of all current drugs to impair thrombosis without causing bleeding is that they block global platelet activation, thereby indiscriminately preventing platelet function in hemostasis and thrombosis. Here, we identify an approach with the potential to overcome this limitation by preventing platelet function independently of canonical platelet activation and in a manner that appears specifically relevant in the setting of thrombosis. Genetic or pharmacological targeting of the class II phosphoinositide 3-kinase (PI3KC2α) dilates the internal membrane reserve of platelets but does not affect activation-dependent platelet function in standard tests. Despite this, inhibition of PI3KC2α is potently antithrombotic in human blood ex vivo and mice in vivo and does not affect hemostasis. Mechanistic studies reveal this antithrombotic effect to be the result of impaired platelet adhesion driven by pronounced hemodynamic shear stress gradients. These findings demonstrate an important role for PI3KC2α in regulating platelet structure and function via a membrane-dependent mechanism and suggest that drugs targeting the platelet internal membrane may be a suitable approach for antithrombotic therapies with an improved therapeutic window.

Heterodimeric Analogues of the Potent Y1R Antagonist 1229U91, Lacking One of the Pharmacophoric C-Terminal Structures, Retain Potent Y1R Affinity and Show Improved Selectivity over Y4R.

The cyclic dimeric peptide 1229U91 (GR231118) has an unusual structure and displays potent, insurmountable antagonism of the Y1 receptor. To probe the structural basis for this activity, we have prepared ring size variants and heterodimeric compounds, identifying the specific residues underpinning the mechanism of 1229U91 binding. The homodimeric structure was shown to be dispensible, with analogues lacking key pharmacophoric residues in one dimer arm retaining high antagonist affinity. Compounds 11d-h also showed enhanced Y1R selectivity over Y4R compared to 1229U91.

Application of a Sulfoxonium Ylide Electrophile to Generate Cathepsin X-Selective Activity-Based Probes.

Cathepsin X/Z/P is cysteine cathepsin with unique carboxypeptidase activity. Its expression is associated with cancer and neurodegenerative diseases, although its roles during normal physiology are still poorly understood. Advances in our understanding of its function have been hindered by a lack of available tools that can specifically measure the proteolytic activity of cathepsin X. We present a series of activity-based probes that incorporate a sulfoxonium ylide warhead, which exhibit improved specificity for cathepsin X compared to previously reported probes. We apply these probes to detect cathepsin X activity in cell and tissue lysates, in live cells and in vivo, and to localize active cathepsin X in mouse tissues by microscopy. Finally, we utilize an improved method to generate chloromethylketones, necessary intermediates for synthesis of acyloxymethylketones probes, by way of sulfoxonium ylide intermediates. In conclusion, the probes presented in this study will be valuable for investigating cathepsin X pathophysiology.

The first total synthesis and solution structure of a polypeptin, PE2, a cyclic lipopeptide with broad spectrum antibiotic activity.

The first total synthesis of a polypeptin, PE2, as well as its solution structure is reported. Synthesis in optically pure form confirms the proposed stereochemistry of the polypeptins at the 3-position on the 3-hydroxy depsipeptide moiety. We have also determined the NMR structure of PE2 in aqueous solution, showing it to form a stable ring conformation. The synthetic peptide shows anti-bacterial activity consistent with reports for naturally derived counterparts.

Development of single and mixed isoform selectivity PI3Kδ inhibitors by targeting Asn836 of PI3Kδ.

A series of PI3Kδ inhibitors derived from the pan-PI3K inhibitor ZSTK474 was prepared that target a non-conserved region of the catalytic site. Dependent upon the substituents present, these analogues show different levels of isoform selectivity and sensitivity to the mutation N836D in PI3Kδ. As a marker of 'on-target' activity and permeability, a selection of the most potent PI3Kδ inhibitors were shown to inhibit pAkt production in the Nawalma Burkitt lymphoma cell line.

Identification of a Cyanine-Dye Labeled Peptidic Ligand for Y1R and Y4R, Based upon the Neuropeptide Y C-Terminal Analogue, BVD-15.

Traceable truncated Neuropeptide Y (NPY) analogues with Y1 receptor (Y1R) affinity and selectivity are highly desirable tools in studying receptor location, regulation, and biological functions. A range of fluorescently labeled analogues of a reported Y1R/Y4R preferring ligand BVD-15 have been prepared and evaluated using high content imaging techniques. One peptide, [Lys(2)(sCy5), Arg(4)]BVD-15, was characterized as an Y1R antagonist with a pKD of 7.2 measured by saturation analysis using fluorescent imaging. The peptide showed 8-fold lower affinity for Y4R (pKD = 6.2) and was a partial agonist at this receptor. The suitability of [Lys(2)(sCy5), Arg(4)]BVD-15 for Y1R and Y4R competition binding experiments was also demonstrated in intact cells. The nature of the label was shown to be critical with replacement of sCy5 by the more hydrophobic Cy5.5 resulting in a switch from Y1R antagonist to Y1R partial agonist.

Discovery and antiplatelet activity of a selective PI3Kß inhibitor (MIPS-9922).

A series of amino-substituted triazines were developed and examined for PI3Kß inhibition and anti-platelet function. Structural adaptations of a morpholine ring of the prototype pan-PI3K inhibitor ZSTK474 yielded PI3Kß selective compounds, where the selectivity largely derives from an interaction with the non-conserved Asp862 residue, as shown by site directed mutagenesis. The most PI3Kß selective inhibitor from the series was studied in detail through a series of in vitro and in vivo functional studies. MIPS-9922, 10 potently inhibited ADP-induced washed platelet aggregation. It also inhibited integrin αIIbß3 activation and αIIbß3 dependent platelet adhesion to immobilized vWF under high shear. It prevented arterial thrombus formation in the in vivo electrolytic mouse model of thrombosis without inducing prolonged bleeding or excess blood loss.

Optically Pure, Structural, and Fluorescent Analogues of a Dimeric Y4 Receptor Agonist Derived by an Olefin Metathesis Approach.

The dimeric peptide 1 (BVD-74D, as a diastereomeric mixture) is a potent and selective neuropeptide Y Y4 receptor agonist. It represents a valuable candidate in developing traceable ligands for pharmacological studies of Y4 receptors and as a lead compound for antiobesity drugs. Its optically pure stereoisomers along with analogues and fluorescently labeled variants were prepared by exploiting alkene metathesis reactions. The (2R,7R)-diaminosuberoyl containing peptide, (R,R)-1, had markedly higher affinity and agonist efficacy than its (S,S)-counterpart. Furthermore, the sulfo-Cy5 labeled (R,R)-14 retained high agonist potency as a novel fluorescent ligand for imaging Y4 receptors.

Class II but Not Second Class-Prospects for the Development of Class II PI3K Inhibitors.

The Class II PI3 kinases are emerging from the shadows of their Class I cousins. The data emerging from PIK3C2 genetic modification studies and from siRNA knockdown suggest important roles in physiology and pathology. With some well-studied Class I isoform inhibitors showing strong Class II activity and a wealth of crystallographic information available, the structural similarity of these isoforms to Class I provides both the opportunity and the challenge in design of selective pharmacological inhibitors.

Design, synthesis and evaluation of pyridine-based chromatographic adsorbents for antibody purification.

The structure-based design and synthesis of four series of adsorbents for antibody purification by affinity chromatography has been investigated. The structures of 10 ligands were based on pyridine compounds that possessed thioalkyl substituents containing a primary amine, which was required for immobilisation of the ligands onto an epoxy-activated matrix (epoxy-Sepharose Fast Flow(®)). These new adsorbents were screened in monoclonal antibody binding assays in order to determine optimal buffer conditions for capture and elution under static and dynamic adsorption conditions. From batch binding measurements, the binding affinities, KD's, were found to be in the range of 3-5µM and the maximum capacities, qm's were between 12 and 30mgmAb/mL resin, depending on the substitution pattern of the thioalkylamine in the N-heterocyclic ring structure of the ligands. The amount of monoclonal antibody bound and eluted under overload conditions was influenced by the concentration of the sample loaded, the flow rate at which the sample was applied and the loading/volume. Further, the ability of these new adsorbents to selectively capture monoclonal antibodies of the class IgG1 from supernatants derived from genetically engineered CHO cells cultured in chemically defined media was investigated, documenting efficient capture and recovery of the mAb.

N-Heterocyclic-based adsorbents for antibody purification-effect of ligand structure.

A new set of ligands based on substituted pyridine and other N-heterocyclic structures, possessing an aliphatic primary amino group tether and an exocyclic sulphur atom, has been prepared and immobilized onto epoxy-activated matrices such as Sepharose 6 Fast Flow®. The derived adsorbents have been evaluated for their utility to capture and purify humanized monoclonal antibodies. Favourable binding properties were assessed from screening assays to determine optimal conditions for the capture and elution of the monoclonal antibodies. Static and dynamic binding experiments were employed to derive the equilibrium dissociation constants KD 's and binding capacities Qmax 's. Typically, the KD values were in the range of 2-5 µM and the Qmax values between 20 and 75 mg mAb/ml resin, depending on the stereo-electronic properties of the substituent in the N-heterocyclic ring structure. The effect of ligand structure on the selectivity of these adsorbents was also investigated, and criteria for their use in the purification of monoclonal antibodies from cell culture supernatants established.

Synthetic routes to the Neuropeptide Y Y1 receptor antagonist 1229U91 and related analogues for SAR studies and cell-based imaging.

The potent Y1 receptor antagonist, 1229U91 has an unusual cyclic dimer structure that makes syntheses of analogue series quite challenging. We have examined three new routes to the synthesis of such peptides that has given access to novel structural variants including heterodimeric compounds, ring size variants and labelled conjugates. These compounds, including a fluorescently labelled analogue VIII show potent antagonism that can be utilised in studying Y1 receptor pharmacology.

Synthesis, structure-activity relationships and brain uptake of a novel series of benzopyran inhibitors of insulin-regulated aminopeptidase.

Peptide inhibitors of insulin-regulated aminopeptidase (IRAP) enhance fear avoidance and spatial memory and accelerate spatial learning in a number of memory paradigms. Using a virtual screening approach, a series of benzopyran compounds was identified that inhibited the catalytic activity of IRAP, ultimately resulting in the identification of potent and specific inhibitors. The present study describes the medicinal chemistry campaign that led to the development of the lead candidate, 3, highlighting the key structural features considered as critical for binding. Furthermore, the in vivo pharmacokinetics and brain uptake of compounds (1 and 3) were assessed in rats and were complemented with in vitro human and rat microsomal stability studies. Following intravenous administration to rodents, 3 exhibits brain exposure, albeit it is rapidly converted to 1, a compound which also exhibits potent inhibition of IRAP.

Identification and development of specific inhibitors for insulin-regulated aminopeptidase as a new class of cognitive enhancers.

Two structurally distinct peptides, angiotensin IV and LVV-haemorphin 7, both competitive high-affinity inhibitors of insulin-regulated aminopeptidase (IRAP), were found to enhance aversion-associated and spatial memory in normal rats and to improve performance in a number of memory tasks in rat deficits models. These findings provide compelling support for the development of specific, high-affinity inhibitors of the enzyme as new cognitive enhancing agents. Different classes of IRAP inhibitors have been developed including peptidomimetics and small molecular weight compounds identified through in silico screening with a homology model of the catalytic domain of IRAP. The proof of principal that inhibition of IRAP activity results in facilitation of memory has been obtained by the demonstration that the small-molecule IRAP inhibitors also exhibit memory-enhancing properties.

Morpholinone mediated oxazolone-free C-terminus amide coupling permitting a convergent strategy for peptide synthesis.

3-Substituted-5-phenylmorpholinones have been demonstrated to act as N-protected C-terminus activated alpha-amino acids capable of undergoing solution phase N-terminus peptide extension following standard coupling procedures. The N-acylated morpholinones do not undergo epimerisation of the stereocentre of the C-terminus amino acid residue as oxazolone formation is sterically prevented, although C-terminus peptide coupling is still possible. This convergent approach to peptide synthesis is exemplified by the preparation of L-ala-L-ala-L-ala and L-ala-D-ala-L-ala.