SAR and X-ray. A New Approach Combining Fragment-Based Screening and Rational Drug Design:  Application to the Discovery of Nanomolar Inhibitors of Src SH2

Abstract

(pp60)Src is a protein involved in signal transduction and is mainly expressed in neurones, platelets, and osteoclasts. Its precise biological role was recently discovered with the KO experiments by Soriano that gave rise to no other apparent phenotype than osteopetrosis, a disease resulting in excedent bone formation. The SH2 domain of the Src family specifically recognizes a sequence of tetrapeptide featuring a phosphotyrosine and a lipophilic aminoacid at the +1 and +3 positions. Recently we engaged in the search for SH2 ligands via modular peptidomimicry of this tetrapetide. This gave rise to several families of nanomolar inhibitors; the best one incorporated a caprolactam scaffold, a biphenyl moiety, and a phosphotyrosine. However, these inhibitors still incorporated the phosphate group that confers good binding affinity to the protein. Phosphates have undesirable features for drug candidates, namely, high rate of hydrolysis of the phosphate group by phosphatases and high charge content precluding cell penetration. Therefore, while searching for optimal non-peptide ligands for Src SH2, we looked for phosphate replacements. For this, we have designed an SAR by fragment crystallography approach. The start of this work resulted from two experimental observations. First, the fact that phenyl phosphate itself displayed detectable binding affinity for Src SH2 permitted us to perform a screening of small aromatic compounds as phenyl phosphate surrogates. Second, the obtention of large Src SH2 crystals displaying a channel large enough for soaking purposes allowed structure determination of over 40 of these small aromatic compounds bound in the phosphotyrosine binding pocket. This search and the way it gave rise to low nanomolar range Src SH2 inhibitors devoid of phosphate groups will be the subject of the present paper.

Hit clustering can improve virtual fragment screening: CDK2 and PARP1 case studies

“…At the same time less common PARP inhibition assay needed a validation on some known fragment inhibitors. We have chosen two known PARP inhibitors: 4-aminobenzamide (IC 50 NAD + [66]). Experimentally obtained IC 50 values were 17 and 49 μM (600 μM NAD + ) respectively, which were in good agreement with the literature data.…”

The challenges of developing novel antiparasitic drugs

“…Therefore, if the compounds entering the lead discovery process are already too drug-like, the lead optimisation process will further increase MW and lipophilicity, potentially reducing the drug-like properties. Fragmentbased screening (Lesuisse et al 2002;Carr and Hann 2002;Rees et al 2004) employs significantly smaller and functionally simpler compounds, the advantage being that with fewer compounds a more efficient evaluation of the chemical space is achieved leading to a more rapid hit-to-lead optimisation phase. The downside, however, is that these fragments tend to have very low potencies (millimolar) and may be difficult to detect in standard assay systems.…”

Probing the Tyrosine Phosphorylation State in Breast Cancer by Src Homology 2 Domain Binding

“…Until now, osteoporosis has generally been treated with two categories of drugs: bone resorption inhibitors such as biphosphonate, and stimulants of bone formation, such as parathyroid hormone and prostaglandin E2 [216]. In recent years several pharmaceutical companies including Glaxo, ARIAD and Aventis have been working to develop Src SH2 inhibitors, with the rationale that Src is a validated therapeutic target for bone resorption [178,217,218].…”