Long-lasting enfuvirtide carrier pentasaccharide conjugates with potent anti-human immunodeficiency virus type 1 activity.

Enfuvirtide (also known as Fuzeon, T-20, or DP-178) is an antiretroviral fusion inhibitor which prevents human immunodeficiency virus type 1 (HIV-1) from entering host cells. This linear 36-mer synthetic peptide is indicated, in combination with other antiretroviral agents, for the treatment of HIV-1-infected individuals and AIDS patients with multidrug-resistant HIV infections. Although enfuvirtide is an efficient anti-HIV-1 drug, its clinical use is limited by a short plasma half-life, i.e., approximately 2 h, which requires twice-daily subcutaneous injections, often resulting in skin sensitivity reaction side effects at the injection sites. Ultimately, 80% of patients stop enfuvirtide treatment within 6 months because of these side effects. We report on the development of long-lasting enfuvirtide conjugates by the use of the site-specific conjugation of enfuvirtide to an antithrombin-binding carrier pentasaccharide (CP) through polyethylene glycol (PEG) linkers of various lengths. These conjugates showed consistent and broad anti-HIV-1 activity in the nanomolar range. The coupling of the CP to enfuvirtide only moderately affected the in vitro anti-HIV-1 activity in the presence of antithrombin. Most importantly, one of these conjugates, enfuvirtide-PEG(12)-CP (EP40111), exhibited a prolonged elimination half-life of more than 10 h in rat plasma compared to the half-life of native enfuvirtide, which was 2.8 h. On the basis of the pharmacokinetic properties of antithrombin-binding pentasaccharides, the anticipated half-life of EP40111 in humans would putatively be about 120 h, which would allow subcutaneous injection once a week instead of twice daily. In conclusion, EP40111 is a promising compound with strong potency as a novel long-lasting anti-HIV-1 drug.

Regio- and chemoselective covalent immobilization of proteins through unnatural amino acids.

A general approach was developed for the regio- and chemoselective covalent immobilization of soluble proteins on glass surfaces through an unnatural amino acid created by post-translationally modifying the cysteine residue in a CaaX recognition motif with functional groups suitable for "click" chemistry or a Staudinger ligation. Farnesyl diphosphate analogues bearing omega-azide or omega-alkyne moieties were attached to the cysteine residue in Cys-Val-Ile-Ala motifs at the C-termini of engineered versions of green fluorescent protein (GFP) and glutathione S-transferase (GST) by protein farnesyltransferase. The derivatized proteins were attached to glass slides bearing linkers containing azide ("click" chemistry) or phosphine (Staudinger ligation) groups. "Click"-immobilized proteins were detected by fluorescently labeled antibodies and remained attached to the slide through two cycles of stripping under stringent conditions at 80 degrees C. GFP immobilized by a Staudinger ligation was detected by directly imagining the GFP fluorophore over a period of 6 days. These methods for covalent immobilization of proteins should be generally applicable. CaaX recognition motifs can easily be appended to the C-terminus of a cloned protein by a simple modification of the corresponding gene, and virtually any soluble protein or peptide bearing a CaaX motif is a substrate for protein farnesyltransferase.

Design and synthesis of diversely substituted azacyclic inhibitors of endothelin converting enzyme.

A series of azacyclic phosphonic acids were synthesized from L-pyroglutamic acid, 6-oxo-L-pipecolic acid, and their enantiomers. The objective was to study the effect of constraining acyclic inhibitors of endothelin converting enzyme on inhibitory activity. Potential pharmacophoric tethers were introduced by stereocontrolled reactions to give highly substituted pyrrolidine- and piperidine-alpha-phosphonic acids. Weak inhibitory activity was observed for one diastereomer in each series having the same relative orientation of substituents.

The use of enzyme immunoassays for the detection of abzymatic activities. Application to an enantioselective thioacetal hydrolysis activity.

Relying on the particularly high specificity displayed by antibodies, enzyme immunoassays have proved to be one of the most efficient tools for early detection of the catalytic activities displayed by antibodies. We took advantage of such an assay, namely the Cat-enzyme-linked immunoassay (EIA) approach developed in our laboratories, both to exhibit and characterise an antibody-catalysed thioacetal hydrolysis. Monoclonal antibody (mAb) H3-32 was thus identified to accelerate the hydrolysis reaction of thioacetal substrate (NC9) to vanillylmandelic acid (VMA), with a k(cat) of 0.148 h(-1) (k(uncat) = 6.85 x 10(-5) h(-1)), and a K(M) of 720 microM. Taking advantage of the enantiomeric discrimination between (R)- and (S)-VMA displayed by some of the anti-H3 monoclonal antibodies, we were also able to determine that (S)-VMA was preferentially formed during this abzymatic hydrolysis with a 47% enantiomeric excess. All these EIA measurements were confirmed through HPLC analyses.