Compound (SMILES): Cn1c(=O)c2c(nc(N3CCN(C(=O)CCOCCNc4cccc5c4C(=O)N(C4CCC(=O)NC4=O)C5=O)CC3)n2Cc2nnc(-c3ccc(Cl)c(Cl)c3)o2)n(C)c1=O
Modality: PROteolysis-TArgeting Chimera (PROTAC)
Target protein (POI): NUDT5 (UniProt Q9UKK9)
POI sequence: MESQEPTESSQNGKQYIISEELISEGKWVKLEKTTYMDPTGKTRTWESVKRTTRKEQTADGVAVIPVLQRTLHYECIVLVKQFRPPMGGYCIEFPAGLIDDGETPEAAALRELEEETGYKGDIAECSPAVCMDPGLSNCTIHIVTVTINGDDAENARPKPKPGDGEFVEVISLPKNDLLQRLDALVAEEHLTVDARVYSYALALKHANAKPFEVPFLKF
E3 ligase: CRBN (UniProt Q96SW2)
Ligase sequence: MAGEGDQQDAAHNMGNHLPLLPAESEEEDEMEVEDQDSKEAKKPNIINFDTSLPTSHTYLGADMEEFHGRTLHDDDSCQVIPVLPQVMMILIPGQTLPLQLFHPQEVSMVRNLIQKDRTFAVLAYSNVQEREAQFGTTAEIYAYREEQDFGIEIVKVKAIGRQRFKVLELRTQSDGIQQAKVQILPECVLPSTMSAVQLESLNKCQIFPSKPVSREDQCSYKWWQKYQKRKFHCANLTSWPRWLYSLYDAETLMDRIKKQLREWDENLKDDSLPSNPIDFSYRVAACLPIDDVLRIQLLKIGSAIQRLRCELDIMNKCTSLCCKQCQETEITTKNEIFSLSLCGPMAAYVNPHGYVHETLTVYKACNLNLIGRPSTEHSWFPGYAWTVAQCKICASHIGWKFTATKKDMSPQKFWGLTRSALLPTIPDTEDEISPDKVILCL
Cell line: HAP1 (Homo sapiens)
Cell line description: Has lost the second copy of chromosome 8 of parent cell line KBM-7. Retains a second copy of a fragment of chromosome 15 fused to chromosome 19.
Gene fusion; HGNC; HGNC Mutation; HGNC; HGNC
CVCL_A426 ! KBM-7
In situ; Bone marrow; 
Haploid karyotype cell line.
Cancer cell line
Assay: Western Blot
Dmax:  0 %
Assay description: To confirm that the physical presence of NUDT5 protein, rather than its enzymatic function or any downstream cellular adaptation, would explain the effect observed with the genetic knock-out, we decided to develop a small molecule tool compound for the acute chemical degradation of NUDT5. The resulting compound, dNUDT5 (Fig. 3C), is a PROTAC that induces proximity between NUDT5 and the cereblon (CRBN) E3 ligase complex. We also obtained a corresponding negative control, dNUDT5nc, by N-methylation of the imide ring to attenuate CRBN binding. Both dNUDT5 and dNUDT5nc engaged their cognate target NUDT5 in live cells as shown by NanoBRET assays (Fig. 9A). However, only dNUDT5 was able to induce proficient ternary complex formation between NUDT5 and CRBN (Fig. 9B) This translated into strong NUDT5 degradation in HEK293 and HAP-1 cells in a concentration- and time- dependent manner (DC50 = 0.5 nM and 0.3 nM, respectively, Fig. 3D, E, Fig. 9C-F). In line with the need for concomitant ligand binding of both NUDT5 and CRBN for efficient ternary complex formation, we observed that degradation efficiency is declining at concentrations beyond 300 nM, a hook effect commonly observed with PROTACs. Rescue experiments confirmed that NUDT5 degradation by dNUDT5 was E3 ligase- and proteasome-dependent (Fig 3F). Next, we performed mass spectrometry analyses of HAP-1 cells treated with dNUDT5. These data indicate dNUDT5 is highly specific for NUDT5 with no other protein being degraded in this context (Fig. 3G). A crystal structure of dNUDT5 bound to NUDT5 confirmed that the binding mode was not significantly altered compared to the parental NUDT5 ligand (Fig. 3H), consistent with the observation that TH5427 is able to prevent NUDT5 degradation by dNUDT5 (Fig. 3F). After extensively characterizing our degrader, we treated WT and MTHFD1K56R cells with dNUDT5. While the compound did not affect the viability of WT cells, it prevented adenosine toxicity in MTHFD1K56R with dose-responsiveness perfectly matching NUDT5 degradation, with maximum rescue of cell viability at 100 nM concentration (Fig. 3I). As expected, the negative control compound dNUDT5nc did not cause any effects in these assays. (Fig. 9G). The availability of a chemical tool for rapid NUDT5 degradation further enabled us to test the effect on adenosine toxicity in cells derived from MTHFD1 deficiency patients. Matching the MTHFD1K56R cell data, we show that addition of dNUDT5 also reverses adenosine-mediated toxicity, highlighting the potential role of NUDT5 in folate- related disorders (Fig. 9H).
Reference: HETEROCYCLIC COMPOUNDS TARGETING THE NUDT5 PROTEIN (patent)
Source database: TPDdb