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. Fragment Ligated Inhibitory Peptides (FLIPs) are described which potently inhibit both CDK2/cyclin A and CDK4/cyclin D1 and have preliminary anti-tumor activity. A structural rationale for binding was obtained through molecular modeling further demonstrating their potential for further development as next generation non ATP competitive CDK inhibitors.

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. The studies involve implementation of a His-tagged heterologous expression system under conditions of auto-induction and mediated by molecular chaperone-expressing plasmids. A high yield of human cyclin A2 was obtained in natively folded and soluble form, through co-expression with groups of molecular chaperones from E. coli in various combinations. A one-step affinity chromatography method was utilized to purify the fusion protein products to homogeneity, and the biological activity confirmed through ligand-binding affinity to inhibitory peptides, representing alternatives for the key determinants of the CDK2 substrate recruitment site on the cyclin regulatory subunit. As a whole, obtaining the active cyclin A without the CDK partner (referred to as monomeric in this work) in a straightforward and facile manner will obviate protein--production issues with the CDK2/cyclin A complex and enable drug discovery efforts for non-ATP competitive CDK inhibition through the cyclin groove.

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. After testing, identified fragments were shown to represent not only appropriate mimics for a critical arginine residue but also to interact effectively with a minor hydrophobic pocket present in the binding groove. Further evaluation of binding modes was undertaken to optimize the potency of these compounds. Through further application of the REPLACE strategy in this study, peptide-small molecule hybrid CDK2 inhibitors were identified that are more drug-like and suitable for further optimization as anti-tumor therapeutics.

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. The role of PAD2 in SLE has not been investigated. The differential roles of PAD2 and PAD4 in TLR-7-dependent lupus autoimmunity were examined. Padi4-/- displayed decreased autoantibodies, type I IFN responses, immune cell activation, vascular dysfunction, and NET immunogenicity. Padi2-/- mice showed abrogation of Th subset polarization, with some disease manifestations reduced compared with WT but to a lesser extent than Padi4-/- mice. RNA sequencing analysis revealed distinct modulation of immune-related pathways in PAD-KO lymphoid organs. Human T cells express both PADs and, when exposed to either PAD2 or PAD4 inhibitors, displayed abrogation of Th1 polarization. These results suggest that targeting PAD2 and/or PAD4 activity modulates dysregulated TLR-7-dependent immune responses in lupus through differential effects of innate and adaptive immunity. Compounds that target PADs may have potential therapeutic roles in T cell-mediated diseases.

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. As a result, REPLACE is further exemplified in combining optimized peptidic sequences with effective N-terminal capping groups to generate more stable compounds possessing antitumor activity consistent with on-target inhibition of cell cycle CDKs. The compounds described here represent prototypes for a next generation of kinase therapeutics with high efficacy and kinome selectivity, thus avoiding problems observed with first generation CDK inhibitors.

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. In particular, these results shed light onto the key contributions of a H-bond observed in crystal structures of N-terminally capped peptides. Furthermore, the structure-activity relationship of a bis(aryl) ether C-terminal capping group mimicking dipeptide interactions was probed through ring substitutions, allowing increased complementarity with the primary hydrophobic pocket. This study further validates REPLACE as an effective strategy for converting peptidic compounds to more pharmaceutically relevant compounds.