Drugs with prolonged, on-target residence time often show superior efficacy, yet general strategies for optimizing drug-target residence time are lacking. Here, we demonstrate progress toward this elusive goal by targeting a noncatalytic cysteine in Bruton's tyrosine kinase (BTK) with reversible covalent inhibitors. Utilizing an inverted orientation of the cysteine-reactive cyanoacrylamide electrophile, we identified potent and selective BTK inhibitors that demonstrate biochemical residence times spanning from minutes to 7 days. An inverted cyanoacrylamide with prolonged residence time in vivo remained bound to BTK more than 18 hours after clearance from the circulation. The inverted cyanoacrylamide strategy was further utilized to discover fibroblast growth factor receptor (FGFR) kinase inhibitors with residence times of several days, demonstrating generalizability of the approach. Targeting noncatalytic cysteines with inverted cyanoacrylamides may serve as a broadly applicable platform that facilitates “residence time by design”, the ability to modulate and improve the duration of target engagement in vivo.

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...

One of the most important factors limiting the universal application of matrix-assisted laser desorption/ionization mass spectrometry (MALDI) to analytical problems is the preparation of the analyte in a proper matrix. This problem is particularly exacerbated in working with organic-soluble polymers since nearly all matrix/solvent systems have been developed for the analysis of materials of biological interest that have significant solubility in aqueous environments.'s2 Previous work in this area has shown that water-soluble polymers can be analyzed using conditions similar to those of the biopolymer~.~ Additionally, MALDI on polygly~ols.'-~ poly(styrene),6 and poly(methylmethacrylate)6*8 have been shown using solvent mixtures and salt addition in the preparation. During the solvent removal from the matrix/analyte mixture the solubility of the polymer often is such that it will precipitate out of solution before crystallization of the matrix, preventing good conditions for MALDI. In order to ensure more interaction between the polymer and the matrix we have moved to simpler solvent systems than the typical water, acetonitrile, ethanol, etc. mixtures and will present results here showing that good polymer spectra can be obtained using the common matrices and organic solvents.All measurements were performed on the Bruker Reflex time-of-flight mass spectrometer with 30 or 35 kV acceleration and detection in either the linear or reflected mode. The samples were ionized by irradiation with the output of an N, laser (337 nm, 5 ns) with power levels slightly above threshold (-106-107 W cn-'). Low mass ions were removed with pulsed deflection and 100-200 transients were summed for the spectra shown. All matrices are from Aldrich except the 7amino-4-methylcarbostyril which is from Kodak. The poly(methy1 methacrylate) (pMMA) molecular weight stanr.i. 0.40 0.30 0.20dards are from Polymer Laboratories (28k, 59k, 127k and 260k) and American Polymer Standards Corp. (10k). The pMMA was dissolved in acetone at a concentration of 1 x lo-' M for the 260k, 2 x lo-' M for the 127k, and 1 x M for the other samples. This solution was mixed with a five-fold excess (v/v) of matrix solution which was generally 2 x lo-' M in acetone and 1 p1 of this was placed on the probe and dried under ambient conditions.The mass spectrum of the pMMA 10k standard in t-3indoleacrylic acid acquired in the reflected mode is shown in Fig. 1. The molecular ions result from the cationization of the pMMA oligomers by fortuitous sodium and potassium with the periodicity of m/z 100 corresponding to the mass of the methyl methacrylate. The peak of the molecular ion distribution, M,, occurs at 5990 as opposed to the value of 9600 (determined by vapor-phase osmometry and intrinsic viscosity) claimed by the supplier. We suspected that selective cationization of the smaller oligomers could play a role here since the binding efficiency of polymers for alkali metal ions varies with the size of the polymer and nature of the ion for polyacrylates' and other polyelectrol...

The analysis of large molecules by matrix-assisted laser desorption time-of-flight mass spectrometry (MALD/TOF) is established as a powerful technique for the determination of molecular mass.',' The analysis of biopolymers such as prot e i n~,~-~ oligonucleotide^,^-^ and polysaccharideslO*'l by MALD/TOF provides molecular mass information faster and with more accuracy than previously available techniques such as polyacrylamide gel electrophoresis." Another class of large molecules for which this technique should provide important information is synthetic organic polymers. MALD/TOF has several advantages over conventional polymer molecular mass techniques such as gel permeation chromatography and vis-~0 m e t r y . I~ It provides absolute molecular masses as opposed to relative values, the entire distribution is determined instead of an average value for the molecular mass and molecular composition information is obtained since the molecular masses of single oligomers are measured. Previous results on polygly~ols~*'~ have been encouraging. Here we show results demonstrating the power of MALD/TOF for the determination of molecular mass distribution, polymer composition, and end group for selected water-soluble polymers.Measurements were performed using a Bruker REFLEXTM MALD/TOF mass spectrometer. This time-of-flight instrument is fitted with a reflectron and dual microchannel plate detector for high resolution analysis and with a postacceleration detector in the linear mode for lower resolution molecular mass distribution measurements. Samples were prepared in a sinapinic acid matrix at a mole ratio of to with a total loading of polymer around 20 to 100 pico-moles (this is based on the manufacturer's value for the average molecular mass). Ions were formed by laser desorption at 337 nm (N2 laser, 3 ns pulse width, lo7 to lo* 0.2 mm2 spot) and accelerated with 10-33 kV. The negative ions were detected in all cases, and ions less than m/z 1000 were removed with a pulsed deflector. The poly(acry1ic acid) sample (weight-average molecular mass," M, = 3000 Da) is supplied from Polysciences, Inc. and poly(styrene sulfonate) (peak-average molecular mass,I5 M, = 200000 Da) is from Polymer Laboratories. The polymers were supplied as the neutralized sodium salts of the acids. Since salts have been shown to have a deleterious effect on MALD ionization,6 the polymers were prepared in the acid form by ion-exchange with a column of Amberlite IRN-77 (Rohm and Haas Co.) ion-exchange resin followed by freeze-drying. The MALD/TOF mass spectrum of poly(styrene sulfonic acid) is shown in Fig.

PRN1008 was safe and well-tolerated following oral administration, and achieved high, sustained levels of BTK occupancy in peripheral blood mononuclear cells.