The emergence and spread of multidrug-resistant gram-positive bacteria represent a serious clinical problem. Telavancin is a novel lipoglycopeptide antibiotic that possesses rapid in vitro bactericidal activity against a broad spectrum of clinically relevant gram-positive pathogens. Here we demonstrate that telavancin's antibacterial activity derives from at least two mechanisms. As observed with vancomycin, telavancin inhibited latestage peptidoglycan biosynthesis in a substrate-dependent fashion and bound the cell wall, as it did the lipid II surrogate tripeptide N,N-diacetyl-L-lysinyl-D-alanyl-D-alanine, with high affinity. Telavancin also perturbed bacterial cell membrane potential and permeability. In methicillin-resistant Staphylococcus aureus, telavancin caused rapid, concentration-dependent depolarization of the plasma membrane, increases in permeability, and leakage of cellular ATP and K ؉ . The timing of these changes correlated with rapid, concentration-dependent loss of bacterial viability, suggesting that the early bactericidal activity of telavancin results from dissipation of cell membrane potential and an increase in membrane permeability. Binding and cell fractionation studies provided direct evidence for an interaction of telavancin with the bacterial cell membrane; stronger binding interactions were observed with the bacterial cell wall and cell membrane relative to vancomycin. We suggest that this multifunctional mechanism of action confers advantageous antibacterial properties.The emergence and spread of bacterial resistance to vancomycin, an important antibiotic used to treat serious infections caused by gram-positive bacteria, has prompted active research to discover new glycopeptides and semisynthetic analogs with improved antimicrobial properties. Vancomycin and related glycopeptide antibiotics inhibit cell wall synthesis in susceptible bacteria by binding with high specificity to peptidoglycan precursors containing the C-terminal D-alanyl-D-alanine (D-Ala-DAla) motif (8). The peptide portion of glycopeptide antibiotics forms a carboxylate binding pocket that imparts, through a combination of five hydrogen bonds plus favorable hydrophobic interactions, strong affinity for the D-Ala-D-Ala-containing terminus of lipid II (8,46,54). Rational approaches toward the design of glycopeptides with improved antimicrobial activities have been described previously (for reviews, see references 35 and 36). One promising approach has been the discovery of lipoglycopeptides, analogs containing hydrophobic groups substituted at the amine position of the disaccharide moiety (20,39,40,45).Telavancin, a semisynthetic derivative of vancomycin possessing a hydrophobic (decylaminoethyl) side chain appended to the vancosamine sugar and a hydrophilic [(phosphonomethyl)aminomethyl] group on the resorcinol-like 4Ј position of amino acid 7 (33), is in late-stage clinical development for the treatment of serious gram-positive infections. Telavancin and other lipoglycopeptides exhibit superior in vitro activity compa...

The pharmacokinetics, tolerability, and serum inhibitory and bactericidal titers of telavancin, a new rapidly bactericidal lipoglycopeptide with multiple mechanisms of action against gram-positive pathogens, were assessed in a two-part, randomized, double-blind, placebo-controlled, ascending-dose study with 54 healthy men. In part 1, single ascending intravenous doses of 0.25 to 15 mg/kg of body weight were studied. In part 2, multiple ascending doses (30-min infusions of 7.5 to 15 mg/kg/day) were studied over 7 days. Following the administration of multiple doses, steady state was achieved by days 3 to 4. At day 7 after the administration of telavancin at 7.5, 12.5, and 15 mg/kg/day, peak concentrations in plasma were 96.7, 151.3, and 202.5 g/ml, respectively, and steady-state area-under-the-curve values were 700, 1,033, and 1,165 g ⅐ h/ml, respectively. The elimination half-life ranged from 6.9 to 9.1 h following the administration of doses >5 mg/kg. Most adverse events were mild in severity. At 24 h postinfusion, serum from subjects given telavancin demonstrated potent bactericidal activity against methicillin-resistant Staphylococcus aureus and penicillin-resistant Streptococcus pneumoniae strains. The results suggest that telavancin may be an effective once-daily therapy for serious bacterial infections caused by these pathogens. , abstr. F-2108, 2003). Data from in vitro and animal studies indicate that telavancin has superior activity to ␤-lactams, older glycopeptides (e.g., vancomycin), and the oxazolidinone linezolid (6). This antimicrobial agent is rapidly bactericidal against staphylococci, with Ͼ99.9% killing achieved within 4 h of exposure at concentrations achievable in vivo (8).Unlike most glycopeptides, telavancin has multiple mechanisms of action. In addition to inhibiting peptidoglycan synthesis by blocking the transglycosylation step, telavancin also interacts with the bacterial membrane, dissipating the membrane potential and effecting changes in cell permeability. The multiple mechanisms of action may lead to a lower frequency of resistance (Debavov et al., 43rd ICAAC; K. Krause, D. Debabov, J. Pace, and K. Kaniga, Abstr. 43rd Intersci. Conf. Antimicrob. Agents Chemother., abstr. C1-1810, 2003). The rapid bactericidal activity of this agent may result in a reduction in the duration of therapy and improved clinical outcomes. This paper reports the results of a two-part, randomized, double-blind, placebo-controlled study of sequentially ascending doses of telavancin with healthy adult male subjects. The study objectives were to assess the safety, pharmacokinetics, and pharmacodynamics (serum inhibitory and bactericidal titers) of intravenous (i.v.) telavancin. Study subjects. Eligible male subjects (age, 18 to 50 years; weight, 50 to 100 kg) with no clinically relevant abnormal physical or laboratory findings and normal electrocardiograms (ECGs), blood pressure, and heart rate were included in the study. Subjects had to be able to comply with the study requirements and to provide written inf...

An affinity fingerprint database, which provides a rich source of data defining operational similarities among proteins, can be used to test theories of cryptic homology unexpected from current understanding of protein structure. Practical applications to drug design include efficient pre-screening of large numbers of compounds against target proteins using fingerprint similarities, supplemented by a small number of empirical measurements, to select promising compounds for further study.

We evaluated an elderly patient with a lifelong history of severe bleeding after surgery or trauma and with evidence of persistent hyperfibrinolysis. Routine coagulation studies were normal. Serum plasminogen (40%, normal 72-128%) and a2-antiplasmin (55%, normal 70-145%) activities were decreased. Euglobulin clot lysis was abnormally shortened (50 min) and normalized in vitro with e-aminocaproic acid (EACA). The patient was treated with EACA with prompt cessation of bleeding. Patient tissue-plasminogen activator (t-PA) levels in serum were normal (4.7 ng/ml, control 3.5-7.2) as detected by a two-site immunoradiometric assay (IRMA). Patient fibrinolytic inhibitor activities were assessed by incubating 11I-labeled t-PA with either whole blood or serum followed by SDS-PAGE and autoradiography to identify the resultant protease/ protease inhibitor complexes. In comparison to blood samples obtained from normal donors, patient plasma and serum demonstrated reduced binding of a fast-acting plasminogen activator inhibitor to '251-labeled t-PA. Immunoprecipitation experiments indicated diminished complex formation between type 1 plasminogen activator inhibitor (PAI-1) in patient serum and 1251-labeled t-PA. Low patient PAI-1 activity was confirmed in serum (0.36 U/ml, control 0.87-1.81; n = 3) and in platelet lysates using a functional IRMA to quantitate PAI-1 binding to immobilized t-PA. However, patient serum PAI-1 antigen was within the normal range when analyzed by IRMA (31.8 ng/ml, control 19.642.2); this result was confirmed in both serum and platelets by Western blot (n = 3). Mixing experiments using purified PAI-1 as well as patient and control sera did not show evidence for an inhibitor against PAI-1. We conclude that this patient's bleeding diathesis was due to hyperfibrinolysis and defective PAI-1. This patient provides the first demonstration of a link between decreased in vivo PAI-1 activity and disordered hemostasis, and supports a role for PAI-1 in control of in vivo fibrinolysis.

Activation of the zymogen form of a serine protease is associated with a conformational change that follows proteolysis at a specific site. Tissue-type plasminogen activator (t-PA) is homologous to mammalian serine proteases and contains an apparent activation cleavage site at arginine-275. To clarify the functional consequences of cleavage at arginine-275 of t-PA, site-specific mutagenesis was performed to convert arginine-275 to a glutamic acid. The mutant enzyme (designated Arg-275----Glu t-PA) could be converted to the two-chain form by Staphylococcus aureus V8 protease but not by plasmin. The one-chain form was 8 times less active against the tripeptide substrate H-D-isoleucyl-L-prolyl-L-arginine-p-nitroanilide (S-2288), and the ability of the enzyme to activate plasminogen in the absence of fibrinogen was reduced 20-50 times compared to the two-chain form. In contrast, one-chain Arg-275----Glu t-PA has equal activity to the two-chain form when assayed in the presence of physiological levels of fibrinogen and plasminogen. Fibrin bound significantly more of the one-chain form of t-PA than the two-chain form for both the wild-type and mutated enzymes. One- and two-chain forms of the wild-type and mutated plasminogen activators slowly formed complexes with plasma protease inhibitors, although the one-chain forms showed decreased complex formation with alpha 2-macroglobulin. The one-chain form of t-PA therefore is fully functional under physiologic conditions and has an increased fibrin binding compared to the two-chain form.