Guy Y. Krippner

Structure activity relationship of dendrimer microbicides with dual action antiviral activity.

Background Topical microbicides, used by women to prevent the transmission of HIV and other sexually transmitted infections are urgently required. Dendrimers are highly branched nanoparticles being developed as microbicides. However, the anti-HIV and HSV structure-activity relationship of dendrimers comprising benzyhydryl amide cores and lysine branches, and a comprehensive analysis of their broad-spectrum anti-HIV activity and mechanism of action have not been published. Methods and Findings Dendrimers with optimized activity against HIV-1 and HSV-2 were identified with respect to the number of lysine branches (generations) and surface groups. Antiviral activity was determined in cell culture assays. Time-of-addition assays were performed to determine dendrimer mechanism of action. In vivo toxicity and HSV-2 inhibitory activity were evaluated in the mouse HSV-2 susceptibility model. Surface groups imparting the most potent inhibitory activity against HIV-1 and HSV-2 were naphthalene disulfonic acid (DNAA) and 3,5-disulfobenzoic acid exhibiting the greatest anionic charge and hydrophobicity of the seven surface groups tested. Their anti-HIV-1 activity did not appreciably increase beyond a second-generation dendrimer while dendrimers larger than two generations were required for potent anti-HSV-2 activity. Second (SPL7115) and fourth generation (SPL7013) DNAA dendrimers demonstrated broad-spectrum anti-HIV activity. However, SPL7013 was more active against HSV and blocking HIV-1 envelope mediated cell-to-cell fusion. SPL7013 and SPL7115 inhibited viral entry with similar potency against CXCR4-(X4) and CCR5-using (R5) HIV-1 strains. SPL7013 was not toxic and provided at least 12 h protection against HSV-2 in the mouse vagina. Conclusions Dendrimers can be engineered with optimized potency against HIV and HSV representing a unique platform for the controlled synthesis of chemically defined multivalent agents as viral entry inhibitors. SPL7013 is formulated as VivaGel® and is currently in clinical development to provide protection against HIV and HSV. SPL7013 could also be combined with other microbicides.

Circulating microparticles generate and transport monomeric C-reactive protein in patients with myocardial infarction

AIMS Elevated serum C-reactive protein (CRP) following myocardial infarction (MI) is associated with poor outcomes. Although animal studies have indicated a direct pathogenic role of CRP, the mechanism underlying this remains elusive. Dissociation of pentameric CRP (pCRP) into pro-inflammatory monomers (mCRP) may directly link CRP to inflammation. We investigated whether cellular microparticles (MPs) can convert pCRP to mCRP and transport mCRP following MI. METHODS AND RESULTS MPs enriched in lysophosphatidylcholine were obtained from cell cultures and patient whole-blood samples collected following acute MI and control groups. Samples were analysed by native western blotting and flow cytometry. MPs were loaded with mCRP in vitro and incubated with endothelial cells prior to staining with monoclonal antibodies. In vitro experiments demonstrated that MPs were capable of converting pCRP to mCRP which could be inhibited by the anti-CRP compound 1,6 bis-phosphocholine. Significantly more mCRP was detected on MPs from patients following MI compared with control groups by western blotting and flow cytometry (P = 0.0005 for association). MPs containing mCRP were able to bind to the surface of endothelial cells and generate pro-inflammatory signals in vitro, suggesting a possible role of MPs in transport and delivery of pro-inflammatory mCRP in vascular disease. CONCLUSION Circulating MPs can convert pCRP to pro-inflammatory mCRP in patients following MI, demonstrating for the first time mCRP generation in vivo and its detection in circulating blood. MPs can bind to cell membranes and transfer mCRP to the cell surface, suggesting a possible mCRP transport/delivery role of MPs in the circulation.

GPVI and GPIbα Mediate Staphylococcal Superantigen-Like Protein 5 (SSL5) Induced Platelet Activation and Direct toward Glycans as Potential Inhibitors

Background Staphylococcus aureus (S. aureus) is a common pathogen capable of causing life-threatening infections. Staphylococcal superantigen-like protein 5 (SSL5) has recently been shown to bind to platelet glycoproteins and induce platelet activation. This study investigates further the interaction between SSL5 and platelet glycoproteins. Moreover, using a glycan discovery approach, we aim to identify potential glycans to therapeutically target this interaction and prevent SSL5-induced effects. Methodology/Principal Findings In addition to platelet activation experiments, flow cytometry, immunoprecipitation, surface plasmon resonance and a glycan binding array, were used to identify specific SSL5 binding regions and mediators. We independently confirm SSL5 to interact with platelets via GPIbα and identify the sulphated-tyrosine residues as an important region for SSL5 binding. We also identify the novel direct interaction between SSL5 and the platelet collagen receptor GPVI. Together, these receptors offer one mechanistic explanation for the unique functional influences SSL5 exerts on platelets. A role for specific families of platelet glycans in mediating SSL5-platelet interactions was also discovered and used to identify and demonstrate effectiveness of potential glycan based inhibitors in vitro. Conclusions/Significance These findings further elucidate the functional interactions between SSL5 and platelets, including the novel finding of a role for the GPVI receptor. We demonstrate efficacy of possible glycan-based approaches to inhibit the SSL5-induced platelet activation. Our data warrant further work to prove SSL5-platelet effects in vivo.

An Orally Available 3-Ethoxybenzisoxazole Capsid Binder with Clinical Activity against Human Rhinovirus.

Respiratory infections caused by human rhinovirus are responsible for severe exacerbations of underlying clinical conditions such as asthma in addition to their economic cost in terms of lost working days due to illness. While several antiviral compounds for treating rhinoviral infections have been discovered, none have succeeded, to date, in reaching approval for clinical use. We have developed a potent, orally available rhinovirus inhibitor 6 that has progressed through early clinical trials. The compound shows favorable pharmacokinetic and activity profiles and has a confirmed mechanism of action through crystallographic studies of a rhinovirus-compound complex. The compound has now progressed to phase IIb clinical studies of its effect on natural rhinovirus infection in humans.

Glycosylation of pramlintide: synthetic glycopeptides that display in vitro and in vivo activities as amylin receptor agonists.

Amylin is a 37-amino acid peptide that is co-secreted with insulin by pancreatic b-cells; it was first isolated from amyloid deposits in the pancreases of patients suffering from type II diabetes. Amylin acts as a partner hormone to insulin and is absent or significantly reduced in patients with type I diabetes, and furthermore, as with insulin, mealstimulated amylin secretion is markedly reduced in patients with type II diabetes. Administration of amylin either alone or in conjunction with insulin would therefore be expected to have beneficial effects for sufferers of either type I or II diabetes. However, amylin has particularly unfavourable physiochemical properties, such as poor solubility and a tendency to self-aggregate, which precludes its clinical use. A synthetic version of amylin with superior properties called pramlintide was, therefore, developed by Amylin Pharmaceuticals, in which comparison with the structure of rat amylin led to replacement of three amino acids present in human amylin (A25 and S28 and S29) with proline, and resulted in a peptide with improved physiochemical properties. Pramlintide (Symlin) was approved in 2005 by the FDA for use in US in patients with type I and II diabetes in conjunction with administration of prandial insulin to improve postprandial glycemic control. Pramlintide still has significant limitations, typical of other peptide drugs, including low circulatory half-life and poor solubility/bioavailability issues, which means that it currently has to be administered as a separate injection to insulin. For several years it has been proposed that glycosylation may have beneficial effects on the pharmacokinetic properties of peptide and protein drugs, for example, by improving solubility/increasing oral bioavailability and/or by increasing circulatory lifetime. Glycosylation of several synthetic peptides relevant to the treatment of diabetes has been attempted to improve their pharmacokinetic properties, including insulin, GLP-1, and exendin-4. Glycosylation of amylin or amylin analogues, such as pramlintide may, therefore, be expected to produce glycopeptides with superior properties. Native amylin itself circulates in nonglycosylated (~50 %) and also glycosylated forms in which O-glycans are attached to threonine residues near the N terminus. The latter are known to be biologically inactive, and it could, therefore, be assumed that glycosylation at either T6 or T9 completely ablates agonist activity. It is perhaps for this reason that there has been no reported synthesis or biological study of glycosylated amylin analogues to date. Nevertheless amylin comprises of multiple amino acids to which glycans might be attached. The potential benefit from site-specific glycosylation warranted an exploration of a structure–activity relationship. A program was initiated to study the effect of Nlinked glycosylation on the properties of pramlintide. Standard synthetic approaches to glycopeptides utilize either elaborate glycoamino acid building blocks during peptide synthesis or sequential modification of a glycopeptide chain with glycosyl transferases (GTases) to provide short nonnatural glycans. However, recent developments into the use of endo-b-N-acetylglucosaminidases (ENGases) have revealed their application to be a powerful method for the convergent assembly of complex glycoconjugates. Key recent developments in the field include the production of glycosynthase mutants of these enzymes, and access to N-glycan oxazolines as efficient donors, compounds that may now be accessed directly from the corresponding reducing sugars in water. Although it has been demonstrated that ENGases will in certain cases catalyse the transfer of N-glycan oligosaccharides to other substrates, in general a pre-existing GlcNAc residue is required at the site of the attachment of N-glycans. We elected to prepare two pramlintide glycopeptides in which a GlcNAc residue was attached to either the Asn residue at position 3 or 21 by solidphase peptide synthesis (SPPS, Scheme 1). Removal of the protecting groups from the sugar and amino acid side chains, cleavage from the resin, and cyclic disulfide formation produced two glycopeptides 3 and 4, with a GlcNAc at N3 and N21, respectively. These were [a] Dr. Y. Tomabechi, Dr. M. A. Squire, Prof. A. J. Fairbanks Department of Chemistry, University of Canterbury Private Bag 4800, Christchurch 8140 (New Zealand) E-mail : antony.fairbanks@canterbury.ac.nz [b] Dr. G. Krippner Baker IDI Heart and Diabetes Institute, 75 Commercial Road Melbourne, Victoria 3004 (Australia) [c] Dr. P. M. Rendle Callaghan Innovation, PO Box 31310, 69 Gracefield Road Lower Hutt 5040 (New Zealand) Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/chem.201303303.

A Versatile Approach for the Site‐Specific Modification of Recombinant Antibodies Using a Combination of Enzyme‐Mediated Bioconjugation and Click Chemistry

A unique two-step modular system for site-specific antibody modification and conjugation is reported. The first step of this approach uses enzymatic bioconjugation with the transpeptidase Sortase A for incorporation of strained cyclooctyne functional groups. The second step of this modular approach involves the azide-alkyne cycloaddition click reaction. The versatility of the two-step approach has been exemplified by the selective incorporation of fluorescent dyes and a positron-emitting copper-64 radiotracer for fluorescence and positron-emission tomography imaging of activated platelets, platelet aggregates, and thrombi, respectively. This flexible and versatile approach could be readily adapted to incorporate a large array of tailor-made functional groups using reliable click chemistry whilst preserving the activity of the antibody or other sensitive biological macromolecules.

The Modified Selenenyl Amide, M-hydroxy Ebselen, Attenuates Diabetic Nephropathy and Diabetes-Associated Atherosclerosis in ApoE/GPx1 Double Knockout Mice

Seleno-organic glutathione peroxidase (GPx) mimetics, including ebselen (Eb), have been tested in in vitro studies for their ability to scavenge reactive oxygen and nitrogen species, including hydrogen peroxide and peroxynitrite. In this study, we investigated the efficacies of two Eb analogues, m-hydroxy ebselen (ME) and ethanol-ebselen (EtE) and compared these with Eb in cell based assays. We found that ME is superior in attenuating the activation of hydrogen peroxide-induced pro-inflammatory mediators, ERK and P38 in human aortic endothelial cells. Consequently, we investigated the effects of ME in an in vivo model of diabetes, the ApoE/GPx1 double knockout (dKO) mouse. We found that ME attenuates plaque formation in the aorta and lesion deposition within the aortic sinus of diabetic dKO mice. Oxidative stress as assessed by 8-OHdG in urine and nitrotyrosine immunostaining in the aortic sinus and kidney tubules, was reduced by ME in diabetic dKO mice. ME also attenuated diabetes-associated renal injury which included tubulointerstitial fibrosis and glomerulosclerosis. Furthermore, the bioactivity of the pro-fibrotic cytokine transforming growth factor-β (TGF-β) as assessed by phospho-Smad2/3 immunostaining was attenuated after treatment with ME. TGF-β-stimulated increases in collagen I and IV gene expression and protein levels were attenuated by ME in rat kidney tubular cells. However, in contrast to the superior activity of ME in in vitro and cell based assays, ME did not further augment the attenuation of diabetes-associated atherosclerosis and renal injury in our in vivo model when compared with Eb. In conclusion, this study strengthens the notion that bolstering GPx-like activity using synthetic mimetics may be a useful therapeutic strategy in lessening the burden of diabetic complications. However, these studies highlight the importance of in vivo analyses to test the efficacies of novel Eb analogues, as in vitro and cell based assays are only partly predictive of the in vivo situation.

An Activation-Specific Platelet Inhibitor That Can Be Turned On/Off by Medically Used Hypothermia

Objective— Therapeutic hypothermia is successfully used, for example, in cardiac surgery to protect organs from ischemia. Cardiosurgical procedures, especially in combination with extracorporeal circulation, and hypothermia itself are potentially prothrombotic. Despite the obvious need, the long half-life of antiplatelet drugs and thus the risk of postoperative bleedings have restricted their use in cardiac surgery. We describe here the design and testing of a unique recombinant hypothermia-controlled antiplatelet fusion protein with the aim of providing increased safety of hypothermia, as well as cardiac surgery. Methods and Results— An elastin-mimetic polypeptide was fused to an activation-specific glycoprotein (GP) IIb/IIIa-blocking single-chain antibody. In silico modeling illustrated the sterical hindrance of a &bgr;-spiral conformation of elastin-mimetic polypeptide preventing the single-chain antibody from inhibiting GPIIb/IIIa at 37°C. Circular dichroism spectra demonstrated reverse temperature transition, and flow cytometry showed binding to and blocking of GPIIb/IIIa at hypothermic body temperature (⩽32°C) but not at normal body temperature. In vivo thrombosis in mice was selectively inhibited at hypothermia but not at 37°C. Conclusion— This is the first description of a broadly applicable pharmacological strategy by which the activity of a potential drug can be controlled by temperature. In particular, this drug steerability may provide substantial benefits for antiplatelet therapy.

Concise Synthesis of Enantiomerically Pure (1'S,2'R)- and (1'R,2'S)-2S-Amino-3-(2'-aminomethyl-cyclopropyl)propionic Acid: Two E-Diastereoisomers of 4,5-Methano-L-lysine

A concise synthesis of both E-isomers of 2S-amino-3-(2′-aminomethyl-cyclopropyl)propionic acid, new methano-l-lysines, is described. The synthetic route includes nine steps from l-methionine, with a key step involving the cyclopropanation of an intermediate E-allylic alcohol. The resultant hydroxymethylcyclopropanes were readily separated and converted into the title α-amino acids. The stereochemistry around the cyclopropane rings was deduced by conducting the cyclopropanation in the presence of N,N,N′,N′-tetramethyl-d-tartaric acid diamide butylboronate, a chiral controller which is known to favour the production of S-hydroxymethyl cyclopropanes from allylic alcohols.