Hi-JAK-ing the ubiquitin system: The design and physicochemical optimisation of JAK PROTACs

Abstract

PROTACs have recently emerged as a novel paradigm in drug discovery. They can hijack existing biological machinery to selectively degrade proteins of interest, in a catalytic fashion. Here we describe the design, optimisation and biological activity of a set of novel PROTACs targeting the Janus kinase family (JAK1, JAK2, JAK3 and TYK2) of proximal membrane-bound proteins. The JAK family proteins display membrane localisation by virtue of their association with cytoplasmic tails of cytokine receptors and there are no reports of a successful PROTAC strategy being deployed against this class of proteins. JAK PROTACs from two distinct JAK chemotypes were designed optimising the physicochemical properties for each template to enhance cell permeation. These PROTACs are capable of inducing JAK1 and JAK2 degradation, demonstrating an extension of the PROTAC methodology to an unprecedented class of protein targets. A number of the known ligase binders were explored, and it was found that PROTACs bearing an inhibitor of apoptosis protein (IAP) ligand induced significantly more JAK degradation over Von Hippel-Lindau (VHL) and Cereblon (CRBN) PROTACs. In addition, the mechanism of action of the JAK PROTACs was elucidated, and it was confirmed that JAK degradation was both IAP-and proteasome-dependant.

Current strategies for the design of PROTAC linkers: a critical review

“…Recently, an increasing number of publications have emerged where the physical properties of PROTACs are accounted for in their design, or in the rationalisation of their efficacy [121][122][123][124]. Mares et al [125], developed a potent cIAP-recruiting PROTAC (82) for the degradation of RIPK2 containing a PEG linker (pDC 50 = 9.4 in THP-1 monocytes, Figure 17).…”

From Conception to Development: Investigating PROTACs Features for Improved Cell Permeability and Successful Protein Degradation

“…In this way, the transfer of ubiquitin units within the ternary complex can lead to potent target degradation through the ubiquitin-proteasome system. Polyethylene glycol (PEG) chains are the most common type of linkers used so far due to their polarity and flexible nature (Bondeson et al, 2018;Pettersson and Crews, 2019;Sun et al, 2019), but other examples include pure lipophilic alkyl chains (Winter et al, 2015;Shah et al, 2020;Zhang et al, 2020a;. In some cases, rigidifying the linker through heterocyclic scaffolds (e.g., piperazine/piperidines) led to a stable ternary complex formation and potent protein degradation (Farnaby et al, 2019;Han et al, 2019).…”

“…This well-known "hook effect" negatively impacts PROTAC's potency in a concentrationdependent manner (Figure 3). Being an intrinsic event in PROTAC pharmacology, the hook effect is difficult to avoid and often occurs at concentrations in the range of 1-10 µM, which are typically applied to test small molecules in vitro (Chan et al, 2018;Olson et al, 2018;Anderson et al, 2020;Shah et al, 2020;Yang et al, 2020;Zhang et al, 2020b,c;. Since positive cooperativity favors stable ternary complexes rather than unproductive binary complexes, it is reasonable to assume that the hook effect may be circumvented or at least reduced by improving cooperativebinding PPIs (Figure 3) (Gadd et al, 2017;Roy et al, 2017;Buhimschi et al, 2018).…”

Proteolysis targeting chimeras (PROTACs) are emerging therapeutics for hematologic malignancies

“…Nunes et al reported a VHL-based PROTAC that was capable of degrading IRAK4 in peripheral blood mononuclear cells and dermal fibroblasts with DC 50 values of 151 nM and 36 nM, respectively [ 184 ]. In addition, PROTACs targeting tripartite motif-containing protein 24 (TRIM24), receptor-interacting protein kinase 2 (RIPK2), and the Janus kinase family enzymes for degradation may also have the potential to be used to treat certain types of hematologic malignancies but require further studies [ 185 – 187 ].…”