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SDR enzymes oxidize specific lipidic alkynylcarbinols into cytotoxic protein-reactive species

Hundreds of cytotoxic natural or synthetic lipidic compounds contain chiral alkynylcarbinol motifs, but the mechanism of action of those potential therapeutic agents remains unknown. Using a genetic screen in haploid human cells, we discovered that the enantiospecific cytotoxicity of numerous terminal alkynylcarbinols, including the highly cytotoxic dialkynylcarbinols, involves a bioactivation by HSD17B11, a short-chain dehydrogenase/reductase (SDR) known to oxidize the C-17 carbinol center of androstan-3-alpha,17-beta-diol to the corresponding ketone. A similar oxidation of dialkynylcarbinols generates dialkynylketones, that we characterize as highly protein-reactive electrophiles. We established that, once bioactivated in cells, the dialkynylcarbinols covalently modify several proteins involved in protein-quality control mechanisms, resulting in their lipoxidation on cysteines and lysines through Michael addition. For some proteins, this triggers their association to cellular membranes and results in endoplasmic reticulum stress, unfolded protein response activation, ubiquitin-proteasome system inhibition and cell death by apoptosis. Finally, as a proof-of-concept, we show that generic lipidic alkynylcarbinols can be devised to be bioactivated by other SDRs, including human RDH11 and HPGD/15-PGDH. Given that the SDR superfamily is one of the largest and most ubiquitous, this unique cytotoxic mechanism-of-action could be widely exploited to treat diseases, in particular cancer, through the design of tailored prodrugs.

Hundreds of cytotoxic natural or synthetic lipidic compounds contain chiral alkynylcarbinol 26 motifs, but the mechanism of action of those potential therapeutic agents remains 27 unknown. Using a genetic screen in haploid human cells, we discovered that the 28 enantiospecific cytotoxicity of numerous terminal alkynylcarbinols, including the highly 29 cytotoxic dialkynylcarbinols, involves a bioactivation by HSD17B11, a short-chain 30 dehydrogenase/reductase (SDR) known to oxidize the C-17 carbinol center of androstan-31 3-alpha,17-beta-diol to the corresponding ketone. A similar oxidation of dialkynylcarbinols 32 generates dialkynylketones, that we characterize as highly protein-reactive electrophiles. 33 We established that, once bioactivated in cells, the dialkynylcarbinols covalently modify    Figure 12D). Accumulation of unfolded proteins in the ER activates the Unfolded 295 Protein Response (UPR) which helps maintain ER protein homeostasis by reducing the influx of 296 proteins into the ER and by increasing the activity of ER protein quality control mechanisms. The 297 UPR is initiated by the activation of three signal transducers: IRE1, PERK and ATF6, each 298 controlling a specific aspect of the UPR (40). IRE1 is a ER-resident transmembrane protein, the 299 activation of which results from its homodimerization mediated by its luminal domain, which is 300 controlled by ER-resident chaperones including HSPA5/GRP78/BiP. IRE1 activation triggers its 301 autophosphorylation, which is used as a marker of UPR activation. In agreement with (S)-3 302 triggering unfolded proteins accumulation and UPR activation, we observed that treatment with 303 (S)-3 induced the rapid autophosphorylation of IRE1 preceding the accumulation of cytoplasmic 304 HSP70 ( Figure 4G). Ultimately, (S)-3 treatment resulted in apoptosis, as marked by PARP-1 305 cleavage (Figure 4G), in a caspase inhibitor (z-VAD-fmk)-sensitive manner ( Figure 4H). Caspase 306 inhibition blocked cell death but not the vacuolization process (Supplementary Figure 12E). 307 Altogether, these results show that (S)-3, once bioactivated by HSD17B11, covalently modifies 308 multiple proteins including critical components of the PQC, resulting in ER-stress, inhibition of the 309 UPS and activation of the UPR, ultimately resulting in apoptotic cell death. 310 Identification of new SDR-specific prodrugs. Our discovery that an SDR, HSD17B11, 311 bioactivates a secondary alcohol prodrug by oxidation into the corresponding ketone is particularly 312 interesting given that the SDR superfamily is one of the largest protein superfamilies, with over 313 500,000 members found in most forms of life. This means that this mechanism of prodrug 314 activation could be exploited to develop an extensive range of new drugs and compounds to kill 315 cells or organisms expressing specific SDRs with high selectivity. SDRs use NAD(H) or NADP(H) 316 cofactors to perform oxidoreductase, lyase or isomerase activities on a large variety of substrates 317 including steroids, retinoic acids, lipids, polyols, sugars, nucleotides and xenobiotics (41). In vitro, p.14/55 SDR enzymes can frequently catalyze both oxidation and reduction, depending on the supplied 319 co-factor. In cells, however, they show directionality, which depends on their sub-cellular 320 localization and cofactor availability. We first determined whether other SDRs with the proper in 321 cellulo directional polarity could activate other secondary alkynylcarbinol-containing compounds 322 into cytotoxic species. We found that the AAC (S)-4 (with a terminal C=CH 2 group, Figure 5A Figure 13C). In conclusion, the RDH11 gene was mutated ( Figure 5C) 335 or downregulated in all the AACR clones, implicating it as an SDR, with similar prodrug 336 bioactivation capacity as HSD17B11, that mediates AAC (S)-4 toxicity. 337 The human RDH11 gene codes for retinol dehydrogenase 11 (also called PSDR1 or SDR7C1), a 338 member of the SDR superfamily which also localizes to the ER (42) where it uses NADP+ to 339 catalyze the conversion of retinol ( Figure 5D, preferentially the 11-cis, all-trans and 9-cis isomers) 340 into retinal through oxidation of the C-15 carbinol center, with a pro-R hydrogen specificity(43). 341 Full-length RDH11 protein was not detectable in any of the AACR clones, suggesting that all the 342 identified mis-sense mutations decrease RDH11 stability ( Figure 5E). To validate the ability of p.15/55 RDH11 to mediate some of the (S)-4 cytotoxic effects, we compared its activity on U2OS, U2OS 344 KO HSD17B11 and U2OS KO HSD17B11 cells in which RDH11 was inactivated using 345 CRISPR/Cas9 (Supplementary Figure 14A). In this panel, we confirmed that, while HSD17B11 346 was responsible for the nM activity of the AAC (S)-4 (48-fold IC 50 increase as a result of HSD17B11 347 inactivation), the remaining toxicity of (S)-4 in HSD17B11 KO cells was further decreased by 348 inactivating RDH11 (6-fold IC50 increase by RDH11 inactivation, Supplementary Figure 14B).  As a proof-of-principle of the potential value of these discoveries for designing novel prodrugs with 356 controlled cytotoxic activity, we attempted to design a new prodrug whose cytotoxic effects would 357 depend more on the RDH11 SDR. We used WT U2OS or U2OS inactivated for HSD17B11, 358 RDH11 or both ( Figure 5F) to test several AAC analogues. Introduction of a second secondary 359 carbinol function in the AAC structure, generating the AlkenylAlkynylDiCarbinol 12 (AADC), 360 reduced its bioactivation by HSD17B11, while leaving intact its RDH11-dependent activation and 361 thereby generating a new prodrug equally bioactivated by HSD17B11 and RDH11 ( Figure 5G). 362 As mixtures of diastereoisomers were used for this experiment, lower IC50 can be expected with 363 selected AADC enantiomers, especially with the external carbinol center in the RDH11-preferred 364  autophosphorylation. In addition to unfolded proteins accumulation, UPR activation could also be 395 the result of HSPA5/GRP78/BiP lipoxidation and/or tethering of multiple proteins to ER 396 membranes. Finally, (S)-DACs also quickly induce mitochondrial fission and caspase-dependent 397 apoptosis (Figure 6). Of note, hundreds of known cytotoxic natural compounds have one or 398 several alkynylcarbinol motifs. The mechanism of action identified here could thus be shared in its 399 principles (enantiospecific bioactivation into protein-reactive lipids) by these cytotoxic molecules 400

(see Supplementary Note 2). 401
Our work also provides a proof-of-concept that new pro-cytotoxic agents can be designed to be 402 bioactivated through an enantiospecific oxidation catalyzed by selected SDRs. We exemplified 403 this with three different human dehydrogenases, HSD17B11, RDH11 and HPGD (Figure 6). We 404 also show here that multiple human cell lines derived from osteosarcoma, a rare pediatric cancer, 405 were particularly sensitive to (S)-DACs (Supplementary Figure 4C), suggesting that HSD17B11-406 and more generally SDR-bioactivated prodrugs could find anticancer applications, especially 407 considering that 71 different SDRs are found in humans (47). For example, HSD3B1-specific pro-408 drugs could prove useful to treat castration-resistant prostate cancers, since the stabilized N367T 409 variant of this SDR, found in ~30 % of the population (rs1047303), has been associated to 410 resistance to androgen deprivation therapies by allowing self-sufficient production of pro-411 proliferative androgens by prostate cancer cells (48-50). SDRs has revealed that among the 314 SDR subfamilies identified (51), half are specific to 417 bacteria. This offers the prospect of designing antibiotics with novel mechanisms of action.

p.18/55
Finally, our findings extend the toolbox of protein-reactive warheads with the identification of the 419 DACone motif as a potent reactive group toward the thiol and amino groups of the side chains of 420 cysteine and lysine, respectively. This reaction is quantitative, without co-product and operating 421 in aqueous buffer at room temperature, making it well suited for many applications. In our study, 422 multiple proteins could be functionalized by a terminal alkyne motif using clickable DACone 10 or 423 11 and the resulting linkage was found to be highly stable. The reactive species could also be 424 readily produced in situ by using the appropriate SDR with its co-factor (see Figure 2F). Our 425 unbiased study of the DACones reactivity using the isoDTB-ABPP approach (30) provides the first 426 insights into the proteome-wide reactivity of these novel electrophiles.  to the cell medium. After recovery, two 140 mm dishes at 10 6 cells/dish were seeded from this 502 mutagenized population and selected by treatment with 0.25 µM DAC (S)-3 for 72 h. After 503 treatment, the medium was refreshed and, after 2-3 weeks, individual clones were isolated (DACR 504 clones). To isolate AACR clones, the DACR clone #A4 at early passage was mutagenized again 505 (0.3 mg/ml EMS for 72 h). After recovery, the mutagenized DACR population was seeded into 140 506 mm dishes (10 6 cells/dish) and selected by treatment with 10 µM (S)-4 for 72 h. After treatment, 507 the medium was refreshed and, after 2-3 weeks, individual clones were isolated (AACR clones). Expression levels were computed with featureCounts (64) using Ensembl annotation. Alignments 522 were deduplicated with samtools rmdup and reads not uniquely mapped removed. Then GATK 523 v3.5 base quality score recalibration was applied (65). Indel realignment, SNP and INDEL 524 discovery were performed with HaplotypeCaller using standard hard filtering parameters 525 p.25/55 according to GATK best practices recommendations for RNAseq. Finally variants were annotated 526 using snpEff v4.3t (66). A python script was used to select protein coding variants specific to 527 resistant clones as compared to the parental HAP-1 (wild-type for DACR clones, and DACR#A4 528 for AACR clones) with a minimal allele frequency of 0.9 and a depth greater than 10 reads. Among 529 these variants, were selected the ones that resulted in frameshifts, mis-and non-sense mutations 530 as compared to the reference human genome hg38. Cytoscape v3.2.0 (67) was used to identify 531 genes found mutated in several clones and to generate a graphical overview.  Antibodies. For immunoblotting, horse-radish peroxidase-conjugated goat anti-mouse or anti-540 rabbit secondary antibodies (Jackson Immunoresearch Laboratories), or IRDye800CW-541 conjugated donkey anti-mouse or anti-rabbit secondary antibodies (LI-COR Biosciences) were 542 used, diluted at 1/10000 in PBS 0.1% Tween-20. For immunofluorescence, AlexaFluor488-or 543 AlexaFluor594-conjugated goat anti-mouse or anti-rabbit antibodies (Thermo Fisher Scientific) 544 were used diluted at 1/1000 in blocking buffer. A list of primary antibodies used in this study, 545 together with related information is provided in the  Nanododrop spectrometer (Thermo Fisher Scientific) was used to evaluate protein concentration 578 and, after adjustment with SDS-Lysis buffer, extracts were diluted by addition of equal volume of 579 SDS-Loading Buffer (5 mM Tris pH 6.8, 0.01% bromophenol blue, 0.2 M dithiothreitol). 580 Immunoblotting was performed with 25-50 µg of WCE. Proteins were separated on gradient gels 581 (BioRad 4-12% TGX pre-cast gels) and transferred onto Protran 0.45 µm nitrocellulose 582 membranes (GE Healthcare). After transfer, membranes could be scanned with an infrared imager 583 (Odyssey, LI-Cor Biosciences) to acquire the signal from AlexaFluor647 modified proteins. 584 Homogeneous loading and transfer were checked by Ponceau S staining. When necessary, 585 membranes were cut into horizontal strips to simultaneously probe for multiple proteins. For 586 immunoblotting, membranes were blocked with PBS containing 5% non-fat dry cow milk, washed 587 and incubated for 1-2 h at room temperature or 16 h at 4 °C with primary antibodies diluted in 588 p.28/55

Analysis by SDS-PAGE of proteins modified by DACs in cells.
Sub-confluent 140 mm dishes, 598 seeded two days before with U2OS or HAP-1 cells, were treated for 2 h with 2 µM of DAC. At the 599 end of treatment, cells were collected by trypsination and centrifugation (900 RPM, 4 °C, 5 min). 600 The cell pellet was washed with cold PBS before being lysed by sonication on ice (Vibracell, 601 Bioblock Scientific, ten 2s-pulses, of amplitude 30) in 400 µL of IPL buffer (20 mM Tris-HCl pH 602 7.8, 1 mM EDTA, 150 mM NaCl, 0.5% IGEPAL CA-630, HALT proteases and phosphatases 603 inhibitor cocktail (Thermo Fisher Scientific)). A centrifugation (15000 RPM, 4 °C, 4 min) was used 604 to remove insoluble material and the supernatant was used for click reactions. The click reaction 605 was performed by incubation at 20 °C for 30 h of a mix containing 240 µg of proteins (diluted in 606 10 µL IPL buffer), 4 mM CuSO 4 , 5 µM azido-AlexaFluor647 and 10 mM sodium ascorbate in IPD 607 buffer (20 mM Tris-HCl pH 7.8, 1 mM EDTA, 150 mM NaCl, 0.05% IGEPAL CA-630, HALT 608 inhibitors cocktail) to reach 80 µL. 20 µL of 5XLoading Buffer (300 mM Tris-HCl pH 6.8, 5% SDS, 609 0.025% bromophenol blue, 15% glycerol, 250 mM dithiothreitol) was added at the end of the 610 reaction, followed by incubation at 95 °C for 5 min. 24 µg of proteins were separated on SDS-611 PAGE gels (BioRad 4-15% TGX pre-cast gels), followed by transfer onto Protran 0.45 µm p.29/55 nitrocellulose membranes (GE Healthcare) which were scanned on an infrared imager (Odyssey, 613 LI-COR Biosciences). A ponceau S staining was used to control for homogeneous loading. At that stage, unclicked azido-AlexaFluor647 could be removed using BioRad MicroBioSpin P-6 655 columns equilibrated with SDS-Lysis buffer and following manufacturer's instructions. Then, 8 µL 656 of the click reaction medium were supplemented with 12 µL of SDS-Lysis buffer and 20 µL of SDS 657 Loading buffer, incubated 5 min at 95 °C and separated on gradient gels (BioRad 4-12% TGX pre-658 cast gels). The modified proteins could be detected with an infrared imager (Odyssey, LI-COR 659 Biosciences) directly in the gel or after transfer onto a nitrocellulose membrane. Total proteins in 660 the gel or on the membrane were visualized using Coomassie (InstantBlue, Sigma-Aldrich, 661 scanned with the Odyssey or BioRad Chemidoc imagers) or Ponceau S staining, respectively.

p.31/55
In vitro DAC bioactivation assays. HSD17B11 was immunopurified from UO2S KO HSD17B11 663 complemented either with HSD17B11-GFP wild-type or the S172L mutant. After extensive washes 664 with a buffer containing 500 mM NaCl, the magnetic beads were used as a source of HSD17B11 665 enzyme. HSD17B11 is a membrane-anchored protein (17) and its activity required maintaining a 666