In a previous post, I tested hit analogues against USP5 Zf-UBD using a SPR assay. Compound AE-641/11456811, now aptly re-named XSR00035795a (an SGC global ID- since our database infrastructure is finally re-vamped), had a binding affinity of approximately 60 µM. The addition of a methyl group on the carboxylic chain of the compound increased potency 3-fold!
You can find details of this experiment on Zenodo. I crystallized the complex of USP5 Zf-UBD with XSR00035795a, and the structure was solved by Dr. Wolfram Tempel. The addition of the methyl group leads to increased hydrophobic interactions in the binding pocket with W209; the side chain of R221 also rotates out of the pocket (Figure 1).
Figure 1. a) Superimposed USP5 structures of compound DAT194 (PDB: 6NFT; cyan) and compound XSR00035795a (magenta) b) Binding pocket of co-crystal structure of USP5 Zf-UBD and XSR00035795a
Next, I tested this compound against HDAC6 Zf-UBD to see if it confers selectivity against USP5 Zf-UBD using a SPR assay (Figure 2). Compound XSR00035795a (LE=0.36) was not selective for USP5 Zf-UBD. XSR00035795a and HDAC6 Zf-UBD has a KD of 15 ± 0.4 µM (n=2) whereas USP5 Zf-UBD has an average KD of 60 ± 8 µM (n=3).
Figure 2. Representative SPR binding curves and sensograms of compound XSR00035795a with a) HDAC6 Zf-UBD (KD= 15 ± 0.4 µM; n=2) and b) USP5 Zf-UBD (KD= 60 ± 8 µM; n=3)
When co-crystal structures of HDAC6 Zf-UBD and USP5 Zf-UBD are superimposed, binding of XSR00035795a to HDAC6 Zf-UBD makes sense. Figure 3 shows the binding pocket of HDAC6 Zf-UBD is able to accommodate the methyl group of compound XSR00035795a. Replacing a valine residue in the primary pocket of HDAC6 with an alanine in USP5 generates a small cavity that may be exploited by extending the aliphatic group on the carboxylic chain to confer selectivity towards USP5.
Figure 3. Superimposed CPK representation of HDAC6 Zf-UBD (purple PDB: 6CE8) and USP5 Zf-UBD and compound XSR00035795a co-crystal structure (cyan)
Commercial compounds with extended aliphatic groups on the scaffold have been ordered but unfortunately due to vendor delays, I haven’t received them! Here’s hoping the compounds will arrive soon…
On another note, I will be doing a three month internship at the Montreal biotech, Molecular Forecaster Inc., as part of a NSERC-CREATE ChemNET grant. I will further explore computational chemistry approaches and structure-based drug design to design inhibitors against USP5 Zf-UBD and use the docking platform to dock compounds against available subfamily protein domain structures, as a predictive tool for selectivity. I’ll be heading to Montreal next week! Stay tuned for some neat computational simulations against USP5 Zf-UBD and the results of the extended aliphatic groups on the XSR00035795a scaffold.