Bifunctional Ligands for Inhibition of Tight-Binding ProteinProtein Interactions
11 The acknowledged potential of small-molecule therapeutics targeting disease-related 12 protein-protein interactions (PPIs) has promoted active research in this field. The strategy of 13 using small molecule inhibitors (SMIs) to fight strong (tight-binding) PPIs tends to fall short 14 due to the flat and wide interfaces of PPIs. Here we propose a biligand approach for disruption 15 of strong PPIs. The potential of this approach was realized for disruption of the tight-binding 16 (KD = 100 pM)
... ng 16 (KD = 100 pM) tetrameric holoenzyme of cAMP-dependent protein kinase (PKA). Supported 17 by X-ray analysis of co-crystals, bifunctional inhibitors (ARC-inhibitors) were constructed that 18 simultaneously associated with both the ATP-pocket and PPI interface area of the catalytic 19 subunit of PKA (PKAc). Bifunctional inhibitor ARC-1411, possessing KD value of 3 pM 20 towards PKAc induced the dissociation of the PKA holoenzyme with a low-nanomolar IC50, 21 whereas the ATP-competitive inhibitor H89 bound to the PKA holoenzyme without disruption 22 of the protein tetramer. the past two decades. 10 All small-molecule PK inhibitors currently approved by FDA as cancer 48 drugs bind to the deep and largely hydrophobic ATP-pocket of the PK. However, high 49 conservation of the nucleotide-binding site across more than 500 human PKs presents a 50 challenge for the construction of PK inhibitors that are suitable for specific therapeutic and 51 research applications. Nevertheless, as an indication of the success of cancer drug development 52 efforts, more than 30 small-molecule PK inhibitors have been approved for clinical use. 11 53 By definition, all PKs inevitably participate in PPIs: the bisubstrate mechanism of the 54 PK-catalyzed protein phosphorylation reaction involves formation of the molecular triple 55 complex incorporating the PK, a substrate protein, and ATP. Usually the interactions between 56 PKs and protein substrates are relatively weak, possessing KD-s in micromolar range. 12 In present work, supported by structure-affinity studies and X-ray analysis of inhibitor:PK co-91 crystals, we developed bifunctional ARC-inhibitors whose affinity towards PKAc extended to 92 one-digit picomolar range (KD = 3 pM). Area of binding of PKAc with ARC-compounds partly 93 overlaps with the contact area of interaction of PKAc with PKAr, making possible the 94 disruption of the protein tetramer of PKA holoenzyme with ARCs. This result demonstrates 95 that bifunctional ligands possessing very high (picomolar) affinity towards a partner protein of 96 PPI complex can disrupt stable PPI complexes, provided that the SMI interaction overlaps with 97 the area of hot spot interactions.