Current Drug Discovery Technologies - Volume 2, Issue 1, 2005

Efficiently scouting the chemical space is one of the major challenges for lead discovery for drug development. In recent times some shifts have been made away from HTS and combinatorial chemistry to more focused approaches. Combinatorial chemistry was the starting point for the development of synthesis concepts that were intended to cover and explore the chemical space without having to prepare every individual compound. In this review, these lead finding approaches will be discussed comparing virtual and synthesized libraries. In addition we discuss the concepts and relationships of evolutionary libraries using genetic algorithms and dynamic combinatorial chemistries, as well as templated fragment ligation concepts. Taking a more abstract view of all approaches, the concepts may loop back into Combinatorial Chemistry allowing a more educated choice of building blocks and chemistries.

A database of 106 human A3 adenosine receptor antagonists was used to derive two alternative PLS models: one starting from CoMFA descriptors and the other starting from the autocorrelation descriptors. The peculiarity of this work is the introduction of autocorrelation vectors as molecular descriptors for the PLS analysis. The autocorrelation allows comparing molecules (and their properties) with different structures and with different spatial orientation without any previous alignment. In particular, Molecular Electrostatic Potential (MEP) was the property computed and its information encoded in autocorrelation vectors. The 3D spatial distribution and the values of the electrostatic potential is in fact largely responsible for the binding of a substrate to its receptor binding site. Validation was done with an external test set and the results of the two models were compared. Interestingly, our preliminary results seem to indicate that this new alternative approach could robustly compete with the already well consolidated CoMFA approach. In particular, we have suggested that it could be a very interesting tool to filter large structural database in several virtual screening applications.

String synthesis [1-3] is an efficient and cheap manual method for preparation of combinatorial libraries by using macroscopic solid support units. Sorting the units between two synthetic steps is an important operation of the procedure. The software developed to guide sorting can be used only when complete combinatorial libraries are prepared. Since very often only selected components of the full libraries are needed, new software was constructed that guides sorting in preparation of non-complete combinatorial libraries. Application of the software is described in details.

Uremic patients have a higher risk of infection and malignancy than normal subjects. Previous studies have deomonstrated that monocytes isolated from uremic patients display an increased apoptosis rate compared to normal subjects; furthermore uremic plasma can increase apoptosis rates on U937, a human monocytic cell line. In several pathological conditions, precipitation of uric acid crystals can lead to renal insufficiency or acute renal failure by different mechanisms. In recent studies uric acid has been shown to induce inflammatory response from monocytes and it has been suggested to be involved in cell dysfunction. Rasburicase is a new recombinant urate oxidase developed to prevent and treat hyperuricaemia in patients with cancer or renal failure; it degrades uric acid to allantoin, a less toxic and more soluble product. In the present study, we aimed at determining whether uric acid may be a factor affecting U937 apoptosis, and whether urate oxidase may reduces or even prevent uric acid induced cell apoptosis. Hoechst staining and internucleosome ledder fragmentation of DNA showed that uric acid increased the percentage of apoptotic cells comparing to the control and that when the U937 cells were incubated with uric acid and urate oxidase the percentage of apoptosis significantly decreased (from 43±7% to 19± 3%, p<0.05). Also, the activity of caspase-8 and caspase-3 showed the same trend (caspase 3: from 2.7±0.53 to 1.6±0.42; caspase-8: from 2.2±0.43 to 1.3±0.57). A reduction of intracellular reduced glutathione (GSH) concentration was found in uric acid treated cells while the addition of urate oxidase in the uric acid incubated cells decreased the GSH extrusion. The concentration of TNF-α was increased in the sample incubated with uric acid comparing to the control. Uric acid is an inducer of apoptosis on U937 cell line, and therefore it may be a component of the mosaic of uremic toxins both in acute and chronic renal disease. We can hypothesize that uric acid might be directly involved in the apoptotic process trough the activation of both death receptor and mitochondrial-mediated pathways. We have, also, demonstrated that urate oxidase is able to prevent at least in part, the effect of uric acid on U937 apoptosis. This effect might be a result of different mechanisms of action.

Type I collagen is the major fibrous protein of mammals being needed to strengthen and organise the extracellular matrix (ECM). Connective tissue components are modulated by matrix metalloproteinases, which are critical for disintegration and remodelling of ECM under physiological and pathological conditions. Cardosin A is an abundant aspartic proteinase (AP) from Cynara cardunculus L. that has been shown to be able to hydrolyse fibrillar collagen within the α-chains. The aim of this work is the characterisation of collagen degradation by cardosin A, since in the native state fibrillar collagen is resistant to most proteolytic enzymes. The pattern of type I collagen hydrolysis by cardosin A is defined and maintained for at least 24 hours of digestion, suggesting that cardosin A can hydrolyse collagen at a small number of specific peptide bonds. N-terminal sequencing of hydrolysis products identified one cleavage site as being Phe464-Gln465 in the a2 chain of collagen I. Two peptides were synthesised correspondent to collagen I specific sequences, in order to produce two polyclonal antibodies, that allowed the identification of three collagen fragments following cardosin A cleavage. Defining the mechanism of collagen cleavage by collagenases and other enzymes, like cardosin A, is important for the comprehension of physiological and pathological processes affecting the ECM. To our knowledge, this is the first study of in vitro collagenolytic activity of a plant AP. Therefore, in view of the cardosin A restricted specificity towards collagen this enzyme may be proposed for an eventual medical or technical procedures assisting ECM remodelling.