Peptidylarginine deiminases 2 and 4 modulate innate and adaptive immune responses in TLR-7-dependent lupus.

The peptidylarginine deiminases PAD2 and PAD4 are implicated in the pathogenesis of several autoimmune diseases. PAD4 may be pathogenic in systemic lupus erythematosus (SLE) through its role in neutrophil extracellular trap (NET) formation that promotes autoantigen externalization, immune dysregulation, and organ damage. The role of this enzyme in mouse models of autoimmunity remains unclear, as pan-PAD chemical inhibitors improve clinical phenotype, whereas PAD4-KO models have given conflicting results.

Benzamide capped peptidomimetics as non-ATP competitive inhibitors of CDK2 using the REPLACE strategy.

Inhibition of cyclin dependent kinase 2 (CDK2) in complex with cyclin A in G1/S phase of the cell cycle has been shown to promote selective apoptosis of cancer cells through the E2F1 pathway. An alternative approach to catalytic inhibition is to target the substrate recruitment site also known as the cyclin binding groove (CBG) to generate selective non-ATP competitive inhibitors. The REPLACE strategy has been applied to identify fragment alternatives and substituted benzoic acid derivatives were evaluated as a promising scaffold to present appropriate functionality to mimic key peptide determinants.

Efficient soluble expression of active recombinant human cyclin A2 mediated by E. coli molecular chaperones.

Bacterial expression of human proteins continues to present a critical challenge in protein crystallography and drug design. While human cyclin A constructs have been extensively characterized in complex with cyclin dependent kinase 2 (CDK2), efforts to express the monomeric human cyclin A2 in Escherichia coli in a stable form, without the kinase subunit, have been laden with technical difficulties, including solubility, yield and purity. Here, optimized conditions are described with the aim of generating for first time, sufficient quantities of human recombinant cyclin A2 in a soluble and active form for crystallization and ligand characterization purposes.

Iterative conversion of cyclin binding groove peptides into druglike CDK inhibitors with antitumor activity.

The cyclin groove is an important recognition site for substrates of the cell cycle cyclin dependent kinases and provides an opportunity for highly selective inhibition of kinase activity through a non-ATP competitive mechanism. The key peptide residues of the cyclin binding motif have been studied in order to precisely define the structure-activity relationship for CDK kinase inhibition. Through this information, new insights into the interactions of peptide CDK inhibitors with key subsites of the cyclin binding groove provide for the replacement of binding determinants with more druglike functionality through REPLACE, a strategy for the iterative conversion of peptidic blockers of protein-protein interactions into pharmaceutically relevant compounds.

Fragment based discovery of arginine isosteres through REPLACE: towards non-ATP competitive CDK inhibitors.

In order to develop non-ATP competitive CDK2/cyclin A inhibitors, the REPLACE strategy has been applied to generate fragment alternatives for the N-terminal tetrapeptide of the cyclin binding motif (HAKRRLIF) involved in substrate recruitment prior to phosphotransfer. The docking approach used for the prediction of small molecule mimics for peptide determinants was validated through reproduction of experimental binding modes of known inhibitors and provides useful information for evaluating binding to protein-protein interaction sites. Further to this, potential arginine isosteres predicted using the validated LigandFit docking method were ligated to the truncated C-terminal peptide, RLIF using solid phase synthesis and evaluated in a competitive binding assay.

Optimization of non-ATP competitive CDK/cyclin groove inhibitors through REPLACE-mediated fragment assembly.

A major challenge in drug discovery is to develop and improve methods for targeting protein-protein interactions. Further exemplification of the REPLACE (REplacement with Partial Ligand Alternatives through Computational Enrichment) strategy for generating inhibitors of protein-protein interactions demonstrated that it can be used to optimize fragment alternatives of key determinants, to combine these in an effective way, and this was achieved for compounds targeting the cyclin-dependent kinase 2 (CDK2) substrate recruitment site on the cyclin regulatory subunit. Phenylheterocyclic isosteres replacing a critical charge-charge interaction provided new structural insights for binding to the cyclin groove.

Structural and functional analysis of cyclin D1 reveals p27 and substrate inhibitor binding requirements.

An alternative strategy for inhibition of the cyclin dependent kinases (CDKs) in antitumor drug discovery is afforded through the substrate recruitment site on the cyclin positive regulatory subunit. Critical CDK substrates such as the Rb and E2F families must undergo cyclin groove binding before phosphorylation, and hence inhibitors of this interaction also block substrate specific kinase activity. This approach offers the potential to generate highly selective and cell cycle specific CDK inhibitors and to reduce the inhibition of transcription mediated through CDK7 and 9, commonly observed with ATP competitive compounds.