Non-covalent inhibitors of rhinovirus 3C protease.

The first known non-covalent inhibitors of rhinovirus 3C protease (3CP) have been identified through fragment based screening and hit identification activities.

Identification of cyclic peptides able to mimic the functional epitope of IgG1-Fc for human Fc gammaRI.

Identification of short, structured peptides able to mimic potently protein-protein interfaces remains a challenge in drug discovery. We report here the use of a naive cyclic peptide phage display library to identify peptide ligands able to recognize and mimic IgG1-Fc functions with Fc gammaRI. Selection by competing off binders to Fc gammaRI with IgG1 allowed the isolation of a family of peptides sharing the common consensus sequence TX(2)CXXthetaPXLLGCPhiXE (theta represents a hydrophobic residue, Phi is usually an acidic residue, and X is any residue) and able to inhibit IgG1 binding to Fc gammaRI. In soluble form, these peptides antagonize superoxide generation mediated by IgG1. In complexed form, they trigger phagocytosis and a superoxide burst. Unlike IgG, these peptides are strictly Fc gammaRI-specific among the Fc gammaRs. Molecular modeling studies suggest that these peptides can adopt 2 distinct and complementary conformers, each able to mimic the discontinuous interface contacts constituted by the Cgamma2-A and -B chains of Fc for Fc gammaRI. In addition, by covalent homodimerization, we engineered a synthetic bivalent 37-mer peptide that retains the ability to trigger effector functions. We demonstrate here that it is feasible to maintain IgG-Fc function within a small structured peptide. These peptides represent a new format for modulation of effector functions.

Novel prostaglandin D synthase inhibitors generated by fragment-based drug design.

We describe the discovery of novel inhibitors of prostaglandin D2 synthase (PGDS) through fragment-based lead generation and structure-based drug design. A library of 2500 low-molecular-weight compounds was screened using 2D nuclear magnetic resonance (NMR), leading to the identification of 24 primary hits. Structure determination of protein-ligand complexes with the hits enabled a hit optimization process, whereby we harvested increasingly more potent inhibitors out of our corporate compound collection. Two iterative cycles were carried out, comprising NMR screening, molecular modeling, X-ray crystallography, and in vitro biochemical testing. Six novel high-resolution PGDS complex structures were determined, and 300 hit analogues were tested. This rational drug design procedure culminated in the discovery of 24 compounds with an IC 50 below 1 microM in the in vitro assay. The best inhibitor (IC 50 = 21 nM) is one of the most potent inhibitors of PGDS to date. As such, it may enable new functional in vivo studies of PGDS and the prostaglandin metabolism pathway.

An improved model for fragment-based lead generation at AstraZeneca.

Modest success rates in fragment-based lead generation (FBLG) projects at AstraZeneca (AZ) prompted operational changes to improve performance. In this review, we summarize these changes, emphasizing the construction and composition of the AZ fragment library, screening practices and working model. We describe the profiles of the screening method for specific fragment subsets and statistically assess our ability to follow up on fragment hits through near-neighbor selection. Performance analysis of our second-generation fragment library (FL2) in screening campaigns illustrates the complementary nature of flat and 3D fragments in exploring protein-binding pockets and highlights our ability to deliver fragment hits using multiple screening techniques for various target classes. The new model has had profound impact on the successful delivery of lead series to drug discovery projects.

Small molecule binding sites on the Ras:SOS complex can be exploited for inhibition of Ras activation.

Constitutively active mutant KRas displays a reduced rate of GTP hydrolysis via both intrinsic and GTPase-activating protein-catalyzed mechanisms, resulting in the perpetual activation of Ras pathways. We describe a fragment screening campaign using X-ray crystallography that led to the discovery of three fragment binding sites on the Ras:SOS complex. The identification of tool compounds binding at each of these sites allowed exploration of two new approaches to Ras pathway inhibition by stabilizing or covalently modifying the Ras:SOS complex to prevent the reloading of Ras with GTP. Initially, we identified ligands that bound reversibly to the Ras:SOS complex in two distinct sites, but these compounds were not sufficiently potent inhibitors to validate our stabilization hypothesis. We conclude by demonstrating that covalent modification of Cys118 on Ras leads to a novel mechanism of inhibition of the SOS-mediated interaction between Ras and Raf and is effective at inhibiting the exchange of labeled GDP in both mutant (G12C and G12V) and wild type Ras.

Homology modelling and docking studies on Varicella Zoster Virus Thymidine kinase.

Thymidine kinase (TK) is the key enzyme in antiviral and suicide gene therapies. While herpes simplex virus type 1 thymidine kinase has been widely studied and crystallised less is known on Varicella Zoster Virus thymidine kinase (VZV TK) and its three-dimensional structure. In this paper we report the model of the three-dimensional structure of VZV TK resulting from a homology modelling study. Subsequent docking studies of the natural substrate deoxythymidine (dT) and known antiviral drugs were performed and shaded new light on the binding characteristics of the enzyme.

Synthesis and in vitro characterization of organometallic rhenium and technetium glucose complexes against Glut 1 and hexokinase.

A series of nine organometallic technetium-99m and rhenium complexes of glucose are presented and characterized in vitro regarding their potential as surrogates of [18F]-2-fluoro-desoxy glucose ([18F]-FDG). The glucose derivatives are functionalized at positions C-1, C-2, C-3, and C-6. Different spacer lengths and chelating systems have been introduced at these sites. For the (radio)labeling, the organometallic precursors [99mTc(H2O)3(CO)3]+ and [ReBr3(CO)3](2-) respectively have been used. The resulting complexes have been characterized chemically and radiochemically. The formation of uniform products has been observed on the macroscopic (Re) and no-carrier-added level (99mTc). The Tc-99m complexes revealed good inertness against ligand exchange (Cys and His) and excellent stability in physiological buffered saline as well as in human plasma over a period of 24 h at 37 degrees C. The rhenium complexes have been tested for competitive inhibition of the (yeast) hexokinase. Only for C-2 derivatized glucose complexes with extended spacer functionalities Ki values in the millimolar and sub-millimolar range have been observed. In silico molecular docking experiments supported these experimental findings. However, the competitive inhibitors are not recognized as a pseudosubstrate of hexokinase. The cellular uptake of all 99mTc-complexes into HT-29 colon carcinoma cells via Glut1 was generally low and unspecific independent of the position at the hexose ring, the chelating systems, or the overall charge of the corresponding metal complexes. The current results seem to preclude the use of these compounds as [18F]-FDG surrogates primarily due to the low cellular uptake via Glut1.

Design of novel nicotinic ligands through 3D database searching.

A series of quinoline derivatives have been designed on the basis of results from a 3D search of the Cambridge Structural Database using the nicotinic pharmacophore as a query and further modified using molecular modeling. Some of the synthesized compounds show nanomolar affinity for the central nicotinic receptor on rat cerebral cortex.