Development Of PDE? Inhibitors With Fragment-Bsased Drug Discovery

Ras proteins couple extracellular,growth-promoting signals to intracellular effector pathways and have a key role in the control of vital cellular processes such as cell differentiation,proliferation and survival.Mutations in Ras proto-oncogenes are found in approximately 30%of all human tumors,thereby placing the Ras variants among the most prevalent drivers of cancer.In particular,K-Ras,the most frequently mutated Ras isoform,is considered one of the most important but?undruggable?targets in cancer research.Picomolar nucleotide affinities of Ras combined with millimolar intracellular nucleotide pools impede the use of GTP-competitive inhibi-tors.Recently,new insight into the process that transports K-Ras4B to the plasma membrane has opened up a similar unprecedented opportunity.To interfere with K-Ras4B localization,inhibitors of the K-Ras4B-PDE?interaction were developed.While those inhibitors reveal good molecular level activity,it reveals low cellular activity.Fragment-based ligand discovery was applied to identify new ligand scaffolds.NMR assay was used to investigate the PDE?binding effects of a library of fragment compounds,resulting in fragment F1 as a confirmed hit.Consistent with LC-MS analysis,high resolution co-crystal structures with PDE?revealed that the fragment F1 binds at the internal pocket.Under the guidance of co-crystal,a series of A compounds whose core contain triazole were designed and synthesized.Compound24 binds to PDE?with high nanomolar affinity in the fluorescence polarization assay(IC₅₀=26 nM),but shows SW480 cellular activity only at micromolar concentrations(IC₅₀=8?M).The crystal structure of PDE?in complex with the compound 11,revealing a new binding mode was that just the reverse of what we expected.Through analysis of this crystal structure,B series of compounds were designed and synthesized.Compound 80 bind to PDE?with high nanomolar affinity in vitro(IC₅₀=23.6 nM).Subsequent optimization was focus on improving cellular activity and metabolic stability.