Crystal structures of NUDT15 variants enabled by a potent inhibitor reveal the structural basis for thiopurine sensitivity

The enzyme NUDT15 efficiently hydrolyzes the active metabolites of thiopurine drugs, which are routinely used for treating cancer and inflammatory diseases. Loss-of-function variants in strongly associated with intolerance, such as leukopenia, preemptive genotyping has been clinically implemented to personalize dosing. However, understanding molecular consequences these difficult, no structural information was available proteins encoded by actionable pharmacogenetic because their inherent instability. Recently, small molecule inhibitor TH1760 shown sensitize cells thiopurines, through enhanced accumulation 6-thio-guanine DNA. Building upon this, we herein report development potent specific inhibitor, TH7755. TH7755 demonstrates a greatly improved cellular target engagement 6-thioguanine potentiation compared TH1760, while showing cytotoxicity on its own. This also stabilized NUDT15, enabling analysis X-ray crystallography. We have determined high-resolution structures relevant Arg139Cys, Arg139His, Val18Ile, V18_V19insGlyVal. These provide clear insights into basis intolerance phenotype observed patients carrying variants. findings will aid predicting effects new sequence variations yet be discovered clinic. belongs NUDIX (nucleoside diphosphates linked moiety x) superfamily proteins, characterized containing conserved box motif (GX5EX7REVXEEXGU, where U is hydrophobic residue). enzymes metabolize diverse range substrates including canonical (d)NTPs, oxidized nonnucleoside polyphosphates, capped mRNAs (1Bessman M.J. Frick D.N. O'Handley S.F. MutT or “nudix” hydrolases, family versatile, widely distributed,“housecleaning” enzymes.J. Biol. Chem. 1996; 271: 25059-25062Abstract Full Text PDF PubMed Scopus (579) Google Scholar). 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Singh Trehan Parish Smith Bhojwani Yuen L.Y.P. al.Massively parallel characterization identifies alleles toxicity.Proc. Acad. Sci. 2020; 117: 5394-5401Crossref (31) Similar TPMT, loss-of-function toxicity, increased It becoming increasingly strategies need include both identify at-risk toxicities particularly important consideration certain racial/ethnic groups more people Asian Hispanic descent Caucasians A recent study cohort 270 diagnosed ALL treated four (7Moriyama showed 74.4% 100% loss activity, relative WT enzyme. included three single point mutants c.415C>T (Arg139Cys), c.416G>A (Arg139His), c.52G>A (Val18Ile) fourth mutant c.36_37insGGAGTC (Val18_Val19insGlyVal), microsatellite expansion an in-frame insertion two (glycine valine). additional inserted segment already glycine–valine repeats extends Gly-Val repetition. Based 6-thio(d)GTP, reduces means improving NUDT15. Specifically, first selective (TH1760) recently (22Zhang S.M. N.C.K. Wallner O. Throup Almlof Loseva Lundback Axelsson Regmi al.Development chemical probe against NUDT15.Nat. 16: 1120-1128Crossref (6) increase effectiveness allowing lower doses still maintaining good therapeutic effect. In study, present novel small-molecule TH7755, substantially well TH1760. solved crystal structure complex adopts highly binding mode Thermal shift indicated significantly stability No isolated previously instability, renders them very difficult crystallize. utilized stabilizing effect determine Analysis relation provides explanations detrimental phenotypic synthesized our developed differs contains methyl groups, attached piperazine ring system nitrogen indole group (Fig. 1A). mouse NUTD15 vitro using malachite green–based assay IC50 values were 30 nM (pIC50 = 7.53 ± 0.03, n 2) 144 6.84 0.02, 2), respectively, 1, B C), indicating affinity higher isoform value reported (25 nM) To investigate ability engage cells, thermal (CETSA) method. Intact AML HL-60 incubated either dimethyl sulfoxide (DMSO) only, after heated, lysed, examined Western blot analysis. At concentration 10 μM heat-induced denaturation precipitation, 4 °C apparent aggregation temperature (Tagg) Importantly, significant stabilization suggests binds D–F). contrast, same concentration, Tagg 1 °C. potency further investigated isothermal dose–response fingerprint CETSA (ITDRFCETSA), intact increasing concentrations before constant screening temperature. consistently engaged start G H). Collectively, data show engages larger extent Loss activity/expression easily quantifiable phenotype. 6-thio-dGTPase leads hypersensitivity, exploited analyze characterize matrix (6-TG) days, viabilities resazurin reduction viability assay. agreement inhibitory 6-TG, evidenced decrease 6-TG EC50 (half maximal effective concentration), starting low 50 nM. did not measurable when applied alone up 100 2, B). data, D–F), far superior in-cell measured potentiation. synergize NUDT15-proficient NB4 effectively abrogated shRNA-mediated depletion, specifically inhibiting C D). produced saturation curves substrate, five kcat Km S1, Table S1). analyzed dependence found clearly S1B), remained unaltered S1C), indicative competitive inhibition. Ki 10.2 0.9 calculated compound. mechanism resolution 1.6 Å. overall homodimer 3A), consistent size-exclusion chromatography small-angle scattering experiments dimeric solution monomer comprised alpha-helices (α1 α2), seven beta-strands (β1–β7), 310 helices (η1–η4) 3B). individual monomers classic fold one another, RMSD 0.47 Å corresponding Cα-atoms. Each site density molecule, positioned deep within pocket 3C). coordinated extensive hydrogen bond interactions. benzoxazolone direct bonds main chain atoms Gly137 Leu138 supported interactions Leu45 Phe135. sulfonamide involving Val16 oxygen Thr94. compound exclusively Phe135, Tyr94, His49. attained chair conformation N-substituents semi-equatorial orientations. combined absolute R configuration forces adjacent 2-methyl axial orientation. amide water-mediated Arg34, itself interacts side Gln44 Val38. Finally, perpendicular π-stacking interaction Tyr90 Val38, Trp136 Comparison NUDT15-TH7755 NUDT15–TH1760 Scholar) shows superimpose well, 0.51 There differences between exception minor flexible loop regions. surrounding superimposing 3D). An interesting difference however system, flipped 180° likely whether (R139C, R139H, V18I Val18_Val19insGlyVal), technique differential scanning fluorimetry. absence (6-thio-GTP) (TH7755), all constructs less thermally stable melting temperatures (Tm) 4A). R139C R139H had decreases Tm (−9.4 −8.2 °C, respectively) Val18_Val19insGlyVal (−3.7 −1.8 respectively). Addition resulted constructs, average +5 even impressive protein, +11 performed tests published stabilizes enzyme, +11.5 addition investigating stability, tested enzymatic vitro. R139C, V18I, displayed reduced ranging 65 85% 4B). least active, followed Val18_Val19insGlyVal, lastly, V18I. Before determined, instability rendered Previous aimed elucidating mechanisms affect protein draw conclusions structures, instead actual data. By utilizing able grow high-quality crystals solve V18_V19insGV A–C). structurally NUDT15-TH7755, Cα-atoms 0.47, 0.42, 0.45, 0.44 Å, respectively. reasons stability. most 1.4 resolution. Cys139 (located α2) conformations, Leu131 η3) Leu134 connecting α2 distances 3.2 3.4 respectively 5A). Arg139 what structure. involves (at distance 2.9 Å) superimposes structures. 310-helix η3 shifted slightly closer α2, than 5D). then position 139, contrast residue 139 capable Leu134, His139 5B). As comparison 5E). 1.5 Val18 close dimer interface lies opposite solvent exposed. V18 located β1 proximity motif, beta-sheet (β4) region alpha-helix (α1). Ile18 surrounded α1 (Thr64, Ala60, Glu63), (Val20), β4 (Gly47), β5 (Leu72) (Pro46) 5C). secondary proteins. disrupts favorable packing Pro46, Gly47, Glu63, smaller valine 5F). mutation glycine GlyVal repeats, thus extending repetition 6, While Gly15 Val20 occupy Gly13 preceding being out frame 6B). mentioned abo