Tomas Fex

Design and evaluation of pilicides: potential novel antibacterial agents directed against uropathogenic Escherichia coli.

Design and evaluation of Pilicides : Potential novel antibacterial agents directed against Uropathogenic Escherichia coli

Weak affinity chromatography as a new approach for fragment screening in drug discovery.

Fragment-based drug design (FBDD) is currently being implemented in drug discovery, creating a demand for developing efficient techniques for fragment screening. Due to the intrinsic weak or transient binding of fragments (mM-μM in dissociation constant (K(D))) to targets, methods must be sensitive enough to accurately detect and quantify an interaction. This study presents weak affinity chromatography (WAC) as an alternative tool for screening of small fragments. The technology was demonstrated by screening of a selected 23-compound fragment collection of documented binders, mostly amidines, using trypsin and thrombin as model target protease proteins. WAC was proven to be a sensitive, robust, and reproducible technique that also provides information about affinity of a fragment in the range of 1 mM-10 μM. Furthermore, it has potential for high throughput as was evidenced by analyzing mixtures in the range of 10 substances by WAC-MS. The accessibility and flexibility of the technology were shown as fragment screening can be performed on standard HPLC equipment. The technology can further be miniaturized and adapted to the requirements of affinity ranges of the fragment library. All these features of WAC make it a potential method in drug discovery for fragment screening.

Label-free primary screening and affinity ranking of fragment libraries using parallel analysis of protein panels.

The authors present fragment screening data obtained using a label-free parallel analysis approach where the binding of fragment library compounds to 4 different target proteins can be screened simultaneously using surface plasmon resonance detection. They suggest this method as a first step in fragment screening to identify and select binders, reducing the demanding requirements on subsequent X-ray or nuclear magnetic resonance studies, and as a valuable “clean-up” tool to eliminate unwanted promiscuous binders from libraries. A small directed fragment library of known thrombin binders and a general 500-compound fragment library were used in this study. Thrombin, blocked thrombin, carbonic anhydrase, and glutathione-S-transferase were immobilized on the sensor chip surface, and the direct binding of the fragments was studied in real time. Only 12 µg of each protein is needed for screening of a 3000-compound fragment library. For screening, a binding site-blocked target as reference facilitates the identification of binding site-selective hits and the signals from other reference proteins for the elimination of false positives. The scope and limitations of this screening approach are discussed for both target-directed and general fragment libraries. (Journal of Biomolecular Screening 2008:202-209)

Use of 19F NMR spectroscopy to evaluate reactions in solid phase organic synthesis

Abstract Gel-phase 19F NMR spectroscopy has been used to characterize products from a variety of reactions of fluorinated aromatics linked to a TentaGel resin. High quality spectra were obtained in a few minutes using an ordinary NMR spectrometer, and the 19F chemical shifts of the support-bound compounds closely matched those of soluble references. In addition, substantial chemical shift differences were obtained for almost all of the synthetic transformations, illustrating the potential of 19F NMR for rapid monitoring of reactions in solid-phase organic synthesis.

High-Throughput Fragment Screening by Affinity LC-MS

Fragment screening, an emerging approach for hit finding in drug discovery, has recently been proven effective by its first approved drug, vemurafenib, for cancer treatment. Techniques such as nuclear magnetic resonance, surface plasmon resonance, and isothemal titration calorimetry, with their own pros and cons, have been employed for screening fragment libraries. As an alternative approach, screening based on high-performance liquid chromatography separation has been developed. In this work, we present weak affinity LC/MS as a method to screen fragments under high-throughput conditions. Affinity-based capillary columns with immobilized thrombin were used to screen a collection of 590 compounds from a fragment library. The collection was divided into 11 mixtures (each containing 35 to 65 fragments) and screened by MS detection. The primary screening was performed in <4 h (corresponding to >3500 fragments per day). Thirty hits were defined, which subsequently entered a secondary screening using an active site-blocked thrombin column for confirmation of specificity. One hit showed selective binding to thrombin with an estimated dissociation constant (KD) in the 0.1 mM range. This study shows that affinity LC/MS is characterized by high throughput, ease of operation, and low consumption of target and fragments, and therefore it promises to be a valuable method for fragment screening.

Compounds Binding to the S2−S3 Pockets of Thrombin

A set of compounds designed to bind to the S2-S3 pockets of thrombin was prepared. These compounds included examples with no interactions in the S1 pocket. Proline, a common P2 in many thrombin inhibitors, was combined with known P3 residues and P1 substituents of varying size and lipophilicity. Binding constants were determined using surface plasmon resonance (SPR) biosensor technology and were found to be in good agreement with results from an enzyme assay. A dramatic increase in affinity (100-1000 times) was seen for compounds incorporating an amino group capable of forming a hydrogen bond with gly216 in the protein backbone. The ligand efficiency was increased by including substituents that form stronger hydrophobic interactions with the P1 pocket. The binding mode was confirmed by X-ray analysis, which revealed the anticipated binding motif that included hydrogen bonds as well as a tightly bound water molecule. A QSAR model indicated that hydrogen bonding and lipophilicity were important for the prediction of binding constants. The results described here may have implications for how directed compound libraries for shallow protein pockets, like S2 and S3 in serine proteases, can be designed.

Fluorinated linkers for monitoring solid-phase synthesis using gel-phase 19F NMR spectroscopy

Abstract Three fluorinated linkers which are analogues of linkers commonly used in solid-phase peptide synthesis have been prepared. Using 19F NMR spectroscopy, the fluorine atom of the linker allowed monitoring of several transformations in the solid-phase synthesis of a peptoid having a coumarin moiety. Especially, attachment of the linker to the solid phase, coupling of the first building block to the linker and cleavage of the product were efficiently monitored and optimised.

Design and synthesis of macrocyclic indoles targeting blood coagulation cascade Factor XIa

The synthesis of a series of novel macrocyclic compounds designed to target blood coagulation Factor XIa is described. The compounds were evaluated for their inhibition of a small set of serine proteases. Several compounds displayed modest activity and good selectivity for Factor XIa. Within the series, a promising lead structure for developing novel macrocyclic inhibitors of thrombin was identified.

Discovery of the Fibrinolysis Inhibitor AZD6564, Acting via Interference of a Protein–Protein Interaction

A class of novel oral fibrinolysis inhibitors has been discovered, which are lysine mimetics containing an isoxazolone as a carboxylic acid isostere. As evidenced by X-ray crystallography the inhibitors bind to the lysine binding site in plasmin thus preventing plasmin from binding to fibrin, hence blocking the protein-protein interaction. Optimization of the series, focusing on potency in human buffer and plasma clotlysis assays, permeability, and GABAa selectivity, led to the discovery of AZD6564 (19) displaying an in vitro human plasma clot lysis IC50 of 0.44 μM, no detectable activity against GABAa, and with DMPK properties leading to a predicted dose of 340 mg twice a day oral dosing in humans.

3-aminopiperidine-based peptide analogues as the first selective noncovalent inhibitors of the bacterial cysteine protease IdeS

A series of eight peptides corresponding to the amino acid sequence of the hinge region of IgG and 17 newly synthesized peptide analogues containing a piperidine moiety as a replacement of a glycine residue were tested as potential inhibitors of the bacterial IgG degrading enzyme of Streptococcus pyogenes, IdeS. None of the peptides showed any inhibitory activity of IdeS, but several piperidine-based analogues were identified as inhibitors. Two different analysis methods were used: an SDS-PAGE based assay to detect IgG cleavage products and a surface plasmon resonance spectroscopy based assay to quantify the degree of inhibition. To investigate the selectivity of the inhibitors for IdeS, all compounds were screened against two other related cysteine proteases (SpeB and papain). The selectivity results show that larger analogues that are active inhibitors of IdeS are even more potent as inhibitors of papain, whereas smaller analogues that are active inhibitors of IdeS inhibit neither SpeB nor papain. Two compounds were identified that exhibit high selectivity against IdeS and will be used for further studies.