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Advancing ASMS with LC‐MS/MS for the discovery of novel PDCL2 ligands from DNA‐encoded chemical library selections

Advancing ASMS with LC‐MS/MS for the discovery of novel PDCL2 ligands from DNA‐encoded chemical... INTRODUCTIONPhosducin‐like 2 (PDCL2) is encoded by the phosducin‐II subgroup (PhLP2) of the phosducin (Pdc) gene family.1 It has been found that PDCL2 forms a complex with heat shock protein Hsp90,2 functions as chaperone, and is essential for cell growth in unicellular organisms.3,4,5 More importantly, PDCL2 is a testis‐specific phosphoprotein in mice and humans.6,7 Previously, we discovered that CRISPR/Cas9‐mediated PDCL2 mutant male mice are sterile due to globozoospermia caused by impaired sperm head formation (accompanying paper submitted to Andrology). This finding motivated our search for small molecule PDCL2 ligands that could disrupt sperm head formation and thereby be useful clinical candidates for nonhormonal contraception in men.To identify and discover a small molecule ligand for PDCL2, we employed the screening of DNA‐encoded chemical libraries (DECLs), which contain billions of chemically unique DNA‐barcoded compounds. These DECLs are generated through individual sequences of reactions and different combinations of functionalized building blocks (Figure 1).8‐11 To screen the library compounds, an affinity selection experiment to PDCL2 was applied to the library pool. The affinity selection is a binding‐based screen that retains those DECL molecules that bind with reasonably high affinity to PDCL2 that is immobilized on a solid support while removing nonbinding DECL molecules by washing. The structures of the PDCL2 binders are proposed based on sequencing analysis12 of the DNA barcodes that are covalently linked to each individual DECL compound (Figure 1). Although DECL screening analysis can generate several chemical series enriched with certain structure‐enrichment relationships (SER), such statistical results cannot exactly represent the binding activity of the corresponding chemical structure. In addition, because PDCL2 is not an active enzyme, traditional enzyme assays are not available for screening and measuring small molecule inhibition with PDCL2. To overcome this limitation, we developed a unique affinity selection mass spectrometry (ASMS) strategy to evaluate the PDCL2 binding affinity of small molecules identified by DECL.1FIGUREWorkflow of DNA‐encoded chemical library (DECL) selection assay of PDCL2 and billions of DECL molecules. The DECL compounds are incubated with immobilized PDCL2, treated with selection washes, processed by protein removal, and subjected to DNA sequencing. Compounds with high sequencing counts are selected as hit molecules.The ASMS assay13‐15 developed in this paper is based on a liquid chromatography with tandem mass spectrometry (LC‐MS/MS) technique (Figure 2). To the best of our knowledge, a systematic method development to incorporate LC‐MS/MS in a DECL/ASMS approach for protein ligand discovery has not been reported. By taking advantage of the well‐developed LC‐MS/MS technique, ligand signal was detected with high selectivity and sensitivity, which allows the detection and quantification of small molecule with high accuracy at picomolar concentration. The basic workflow of the ASMS binding affinity assay starts with incubating the small molecule hits with PDCL2 in buffer solution. For an active compound, the ligand‐PDCL2 complex was eluted and collected after passing through a size‐exclusion column (SEC), and the amount of the bound ligands was detected by LC‐MS/MS in the following procedures. For nonbinding compounds, the free small molecule is adsorbed by the SEC column material, leaving the PDCL2 protein alone to be eluted out from the SEC column (Figure 2A). The absence of small molecule in the nonbinder SEC eluant will show zero signal intensity in LC‐MS/MS data. The collected eluant containing either ligand‐PDCL2 complex (binder compound presence) or PDCL2 alone (nonbinder compound presence) was then denatured by heating to allow any small molecule binder, in the case when a ligand‐PDCL2 complex is formed, to be released and detected by mass spectrometry. Importantly, the ASMS method extensively reproduces the exact binding conditions as the DECL screening condition, making it straightforward to compare the binding affinity between the on‐DNA compound and the corresponding off‐DNA compound. Moreover, the binding affinity in ASMS is evaluated by the actual amount of molecules that bound to the protein, rendering an unambiguous result for structure‐activity relationship (SAR) analysis and studies.2FIGUREMechanism of affinity selection mass spectrometry (ASMS) assay developed based on liquid chromatography with tandem mass spectrometry (LC‐MS/MS) technique. Figure 2A shows the protein and ligand‐protein complex are separated from salts and small organic molecule impurities. Figure 2B shows the function of each quadrupole in the LC‐MS/MS system, which allows the highly sensitive and reproducible measurement of the ligand concentration.Samples obtained from SEC contain organic buffer molecules, organic detergent molecules, denatured protein residues, and other impurities, which could have an overlapping signal (retention time and mass) with the ligand signal and interfere with the quantification results. Rather than using conventional LC/MS to analyze the complex milieu collected from SEC, we applied LC‐MS/MS, a highly sensitive and reproducible technique,16–19 to quantify the ligand and evaluate the binding affinity of the small molecules. To decrease the background noise and improve the signal intensity of the small molecule ligand, we developed a selected reaction monitoring mass spectrometry method with a triple quadrupole (QqQ) MS instrument operating in the product‐ion scan mode (Figure 2B). In the QqQ instrument, the first quadrupole (Q1, Figure 2B) is set to remove background ions and isolate the ligand precursor ion, which is then fragmented in the second quadrupole (q2, Figure 2B). The generated fragment ions are then transferred to the third quadrupole (Q3, Figure 2B) for mass analysis and signal intensity measurement (Figure 2B). We measure the binding affinity based on the signal intensity detected by the LC‐MS/MS system. This paper describes our efforts in identifying a novel PDCL2 ligand from DECLs and improvement of the ASMS assay using a LC‐MS/MS technique.RESULTS AND DISCUSSIONDECL screening results with PDCL2To identify small molecules that bind to PDCL2, DECLs (from HitGen) cumulatively containing >440 billion compounds were pooled together for parallel screening of PDCL2 at 5 μM, 1 μM, and 0.2 μM concentrations. Blank control affinity selection was performed in parallel without protein to identify any nonspecific bead binders. Bioinformatics sequencing analysis of the amplified DNA barcodes was processed and presented as count number to approximate the relative binding affinity of on‐DNA molecules to the PDCL2 protein during the screening process. The sequencing data identified a series of bis‐substituted pyrimidine candidates enriched with good SER from one of HitGen's DECL libraries (DEL 1113, Table 1).12,20 As shown in Table 1, the selection counts in this series of hit candidates positively relate to the PDCL2 concentration in the selection screenings indicating a reliable DEL screening result. The enriched hit series contains a pyrimidine core from the Cycle 1 (C1) building block (blue, Table 1), a heterocyclic Cycle 2 building block (C2, red, Table 1) with a primary amine group enabling C‐N bond attachment to the C1 pyrimidine core, and a phenyl borylating reagent as the Cycle 3 building block (C3, gray, Table 1) for Suzuki coupling with the C1 core. Based on the SER analysis, we selected three candidates shown in Table 1 to investigate further by performing their off‐DNA organic syntheses.1TABLEDNA‐encoded chemical library (DECL) enrichment results for PDCL2 at three different concentrations. Three hit candidates with the same Cycle 1 building block were selected from a tri‐synthon DECL library (DEL 1113). The building blocks for cycles 1, 2, and 3 are presented in blue, red, and black, respectivelyCycle 2 building block112Cycle 3 building block345Sequence count at 5 µM205727Sequence count at 1 µM118520Sequence count at 0.2 µM1463Sequence count in blank (0 µM)000Validation and synthesis of PDCL2 selection hitsCandidate hit molecules were synthesized by truncating the DNA barcode linkage down to a methyl amide (Table 1 and Scheme 1). The synthesis route was redesigned from the on‐DNA library synthesis as shown in Scheme 1. To prepare the tri‐synthon parent compounds (CDD‐1923, CDD‐1835, and CDD‐2364), we commenced with the C1 building block bromo‐dichloropyrimidine (6) and installed the amine C2 building block (1, or 2) via an SNAr reaction under basic conditions to produce the intermediates 7a and 7b, respectively. The chloro group in 7a and 7b was further eliminated and substituted with methyl amine to yield the bis‐amine intermediates (8a and 8b). The methyl amine group is a surrogate for the DNA attachment point of the DECL on‐DNA compounds and maintains the binding affinity. The bromo intermediates (8a and 8b) were further reacted with the borylated reagents (3 and 5) using Suzuki–Miyaura reaction conditions to afford the final products CDD‐1923 and CDD‐1835, respectively. During the transformation of 8a to 9, the resulting mass ion peak for the product in the LC‐MS analysis was 1 mass units greater than the expected (m/z was determined to be 396.1 instead of 395.1 for 9). Extensive characterization of the product by 1H NMR, 13C NMR, HSQC, HMBC, HRMS, and IR analysis confirmed it to be phenanthridinone CDD‐2364 (Scheme 1C and shown in Supporting Information). This is rationalized on grounds that product 9 is initially formed but undergoes subsequent reactions. The nitrile group in 9 is activated by the presence of the ortho‐pyrimidine and para‐fluoro substituents—this favors its intramolecular attack by the amine to form an amidine intermediate, which hydrolyzes into the phenanthridinone product (Figure 3). Based on this observation and literature precedent,21–30 we believe that the same reaction mechanism occurs during the DECL synthesis to generate the phenanthridinone structure as the major product on DNA. Inspired by the unexpected phenanthridinone formation, we prepared CDD‐2377 (Scheme 1B) as the C2 truncated version of CDD‐2364 in a similar manner for structural activity relationship studies.1SCHEMESynthesis of CDD‐1923, 1835, 2364, and 2377a. aReagents and conditions: (a) Et3N (3.0 equiv.), 1 (1.3 equiv.), MeCN, 23°C, 18 h, 32%; (b) Et3N (2.0 equiv.), 2 (1.3 equiv.), MeCN, 23°C, 18 h, 34%; (c) Et3N (2.0 equiv.), MeNH2 (3.0 equiv.), EtOH, 80°C, 18 h, 64%; (d) Et3N (2.0 equiv.), MeNH2 (3.0 equiv.), EtOH, 80°C, 18 h, 34%; (e) Pd2(dba)3 (0.05 equiv.), cataCXium A (0.1 equiv.), K3PO4 (2.0 equiv.), 3 (1.2 equiv.), dioxane:H2O (5:1, v/v), MW (110°C), 30 min, 64%; (f) Pd2(dba)3 (0.05 equiv.), cataCXium A (0.1 equiv.), K3PO4 (2.0 equiv.), 5 (1.2 equiv.), dioxane:H2O (5:1, v/v), MW (110°C), 30 min, 68%; (g) Pd2(dba)3 (0.05 equiv.), cataCXium A (0.1 equiv.), K3PO4 (2.0 equiv.), 4 (1.2 equiv.), dioxane:H2O (5:1, v/v), MW (110°C), 30 min, 74% from 8a; (h) MeNH2•HCl (6.0 equiv.), DIPEA:DMA (2:1, v/v), 110°C, 18 h, 70%; (i) Pd2(dba)3 (0.05 equiv.), cataCXium A (0.1 equiv.), K3PO4 (2.0 equiv.), 2 (1.2 equiv.), dioxane:H2O (5:1, v/v), MW (110°C), 30 min, 72%. Pd2(dba)3 = tris(dibenzylideneacetone)dipalladium(0), cataCXium A = di(1‐adamantyl)‐n‐butylphosphine, DIPEA = N,N‐diisopropylethylamine, DMA = N,N‐dimethylacetamide, HMBC = heteronuclear multiple bond correlation, IR = infrared spectroscopy. The color in the CDD compound structure represents functional groups derived from C1, C2, and C3 building block respectively (C1 in blue, C2 in red, and C3 in black).3FIGUREReaction mechanism for the formation of CDD‐2364 from 9. Once 9 is generated from the Suzuki reaction; the amine nitrogen atom attacks the nitrile carbon 5‐bond away and forms a six‐membered ring with an imine structure (11). 11 underwent a deprotonation process to form 12, which is hydrolyzed into CDD‐2364 through 13.ASMS resultsThe binding affinity of each CDD compounds to PDCL2 was determined by the ASMS assay. The ASMS assay for each CDD compound included a nontarget control (NTC) sample and a PCDL2 incubated sample. The NTC sample served as a blank control and was prepared by the exact same procedure as the PDCL2 incubated sample without adding the PDCL2 aliquot. Both the NTC sample solution and the PDCL2 containing sample solution were incubated at 25°C for 45 min, passed through the SEC column, processed by heat elution condition, and centrifuged to obtain the supernatant for LC‐MS/MS experiment. The difference in the CDD compound peak area between the NTC sample and the PDCL2 incubated sample reveals the binding affinity of the CDD compound. The affinity value was calculated based on LC‐MS/MS peak areas using Equations 1 and 2 (also shown in Figure 4A,B).31 The peak area of the CDD compound in the PDCL2 incubated sample and the NTC sample was first normalized by the corresponding internal standard peak area (Equation 1). With the normalized peak area, the affinity value was determined by calculating the fold enrichment of the CDD compounds in the PDCL2 incubated sample over the parallel NTC sample (Equation 2). Among the three tri‐synthon compounds (CDD‐1835, CDD‐1923, and CDD‐2364), only the phenanthridinone CDD‐2364 showed a 10‐fold enrichment in the PDCL2 incubated sample over the corresponding NTC sample. To study the SAR of the C2 structure in CDD‐2364, we prepared CDD‐2377 as the truncated version (Scheme 1B), and the affinity value of CDD‐2377 was decreased by 3‐fold relative to CDD‐2364 (Figure 4B). The binding affinity of CDD‐2364 and CDD‐2377 was further confirmed in a thermal shift assay (TSA), and both compounds gave an approximately one‐degree stabilizing effect to the PDCL2 protein.1Normalized peak area of each sample=CDD compound peak areaInternal standard peak area$$\begin{equation} \text{Normalized peak area of each sample}=\frac{\text{CDD compound peak area}}{\text{Internal standard peak area}} \end{equation}$$2AffinityvalueofCDDcompounds=NormalizedpeakareainthePDCL2incubatedsampleNormalizedpeakareainNTC$$\begin{eqnarray}&&{\rm{Affinity\;value\;of\;CDD\;compounds\;}}\nonumber\\ &=& \frac{{{\rm{Normalized\;peak\;area\;in\;the\;PDCL}}2{\rm{\;incubated\;sample}}}}{{{\rm{Normalized\;peak\;area\;in\;NTC}}}}\; \end{eqnarray}$$4FIGUREBinding assay results of CDD‐1835, CDD‐1923, CDD‐2364, and CDD‐2377. Panel A shows the normalized peak area of nontarget control (NTC) sample, and PDCL2 incubated sample for each CDD compounds. Affinity enrichment was only observed with CDD‐2364 and CDD‐2377 compounds. Panel B calculates the affinity value based on the equation shown at the bottom of the figure. Panel B also shows the thermal shift assay (TSA) results of CDD‐2364 and CDD‐2377, and the TSA data are consistent with the affinity selection mass spectrometry (ASMS) results.CONCLUSIONSIn conclusion, we have screened PDCL2 with a DECL collection containing >440 billion on‐DNA compounds, generated three rational structures to perform organic synthesis, synthesized, and characterized four small molecule compounds, and identified two promising compounds (CDD‐2364 and CDD‐2377) showing binding affinities in our ASMS assay. The ASMS results of the two compounds were further validated by TSA, warranting further SAR studies featuring analogs with modifications on the C2 portion of the CDD‐2364 structure. The functional effects of the optimized lead compounds will be tested in wildtype mice and our recently generated Pdcl2 knockout mouse model (also being published as part of this special issue).32The flexibility of our DECL and ASMS screening strategy could pave the way for the systematic studies of other nonenzymatic targets, which are challenging due to the lack of biochemical assays. Furthermore, the current work is expected to foster future utilization of the sensitivity and reproducibility of the LC‐MS/MS technique to perform semi‐quantification ASMS experiments for apparent inhibition constant (Ki,app) evaluation.AUTHOR CONTRIBUTIONSQY, ZY, YF, and MMM designed research. QY, HB, YW, ZY, MP, and JYL performed research. QY, YW, ZY, MP, KRM, and MMM analyzed data. QY, YW, SAK, MT, DWY, YF, and MMM wrote the paper. HB, YW, and ZY contributed equally to this work.ACKNOWLEDGMENTSThis work is supported by the Eunice Kennedy Shriver National Institute of Child Health and Human Development (P01HD087157 and R01HD088412), the Bill & Melinda Gates Foundation (INV‐001902), the Welch Foundation (H‐Q‐0042), and a Core Facility Support Award from the Cancer Prevention Research Institute of Texas (RP160805). HitGen DEL screening was conducted with the support from the Bill & Melinda Gates Foundation under a contract to HitGen (INV‐020850). MT receives research funding (RR220012) from the Cancer Prevention and Research Institute of Texas (CPRIT). MP is supported by NIH (R03CA259664) and the Cancer Prevention Research Institute of Texas (RP220524). YF is supported by Japan Society for the Promotion of Science (JSPS) KAKENHI grant (JP20KK0155). The authors thank Dr. Xuan Qin and Professor Feng Li for mass spectrometric assistance. We are thankful to Drs. Kurt M. Bohren, Ramana Sreenivasa Rao, and Ravikumar Jimmidi for the valuable assistance in data analysis.REFERENCESWillardson BM, Howlett AC. Function of phosducin‐like proteins in G protein signaling and chaperone‐assisted protein folding. Cell. Signal. 2007;19(12):2417‐2427.Krzemien‐Ojak L, Goral A, Joachimiak E, Filipek A, Fabczak H. Interaction of a novel chaperone PhLP2A with the heat shock protein Hsp90. J. Cell. Biochem. 2017;118(2):420‐429.Bregier C, Krzemien‐Ojak L, Wloga D, et al. PHLP2 is essential and plays a role in ciliogenesis and microtubule assembly in Tetrahymena thermophila. J. Cell. Physiol. 2013;228(11):2175‐2189.Blaauw M, Knol JC, Kortholt A, et al. Phosducin‐like proteins in Dictyostelium discoideum: implications for the phosducin family of proteins. EMBO J. 2003;22(19):5047‐5057.Flanary PL, DiBello PR, Estrada P, Dohlman HG. Functional analysis of Plp1 and Plp2, two homologues of phosducin in yeast. J. Biol. Chem. 2000;275(24):18462‐18469.Uhlen M, Fagerberg L, Hallstrom BM, et al. Proteomics. Tissue‐based map of the human proteome. Science 2015;347(6220):1260419.Jamin SP, Hikmet F, Mathieu R, et al. Combined RNA/tissue profiling identifies novel cancer/testis genes. Mol. Oncol. 2021;347(6220):1260419.Takamiya M, Yagi G, Tanoue A, et al. Mortality rate in schizophrenia with tardive dyskinesia. Seishin shinkeigaku zasshi = Psychiatria et neurologia Japonica 1988;90(7):559‐569.Chen YC, Faver JC, Ku AF, et al. C‐N coupling of DNA‐Conjugated (Hetero)aryl bromides and chlorides for DNA‐encoded chemical library synthesis. Bioconjugate Chem. 2020;31(3):770‐780.Li JY, Miklossy G, Modukuri RK, et al. Palladium‐catalyzed hydroxycarbonylation of (Hetero)aryl halides for DNA‐encoded chemical library synthesis. Bioconjugate Chem. 2019;30(8):2209‐2215.Chamakuri S, Lu S, Ucisik MN, et al. DNA‐encoded chemistry technology yields expedient access to SARS‐CoV‐2 M(pro) inhibitors. Proc. Natl Acad. Sci. USA. 2021;118(36):e2111172118.Chen Q, Hall J, Foley TL, Wan J, Li Y, Israel DI. A method for estimating binding affinity from primary DEL selection data. Biochem. Biophys. Res. Commun. 2020;533(2):249‐255.Motoyaji T. Revolution of small molecule drug discovery by affinity selection‐mass spectrometry technology. Chem. Pharmaceut. Bull. 2020;68(3):191‐193.Prudent R, Annis DA, Dandliker PJ, Ortholand J‐Y, Roche D. Exploring new targets and chemical space with affinity selection‐mass spectrometry. Nat. Rev. Chem. 2021;5(1):62‐71.Annis DA, Nickbarg E, Yang X, Ziebell MR, Whitehurst CE. Affinity selection‐mass spectrometry screening techniques for small molecule drug discovery. Curr. Opin. Chem. Biol. 2007;11(5):518‐526.Yost RA, Enke CG. Triple quadrupole mass spectrometry for direct mixture analysis and structure elucidation. Anal. Chem. 1979;51(12):1251‐1264.Hossain M. Selected reaction monitoring mass mass spectrometry (MS) spectrometry. 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The chemistry of indoles. XLIV. Synthetic study directed toward 3,4,5,6‐tetrahydro‐1H‐azepino[5,4,3‐cd]indoles. Chem. Pharm. Bull. 1988;36(3):1162.Uesugi S, Li Z, Yazaki R, Ohshima T. Chemoselective catalytic conjugate addition of alcohols over amines. Angew Chem. Int. Ed. 2014;53(6):1611‐1615.Yan C, Fraga‐Dubreuil J, Garcia‐Verdugo E, et al. The continuous synthesis of ε‐caprolactam from 6‐aminocapronitrile in high‐temperature water. Green Chem. 2008;10(1):98‐103.Schwenker G, Metz G. The conversion of 3‐acetyl‐3‐arylhexanedinitriles into hexahydroindoles and 1H‐pyrrolo[2,3‐b]pyridines. J. Chem. Res. Synop. 1985;16(4). https://doi.org/10.1002/chin.198537187Joshi BK, Ramsey B, Johnson B, et al. Elucidating the pathways of degradation of denagliptin. J. Pharm. Sci. 2010;99(7):3030‐3040.Weber K, Ohnmacht U, Gmeiner P. Enantiopure 4‐ and 5‐aminopiperidin‐2‐ones: regiocontrolled synthesis and conformational characterization as bioactive β‐turn mimetics. J. Org. 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Andrology 2022, Resubmitted, ANDR‐2022‐0434. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Andrology Wiley

Advancing ASMS with LC‐MS/MS for the discovery of novel PDCL2 ligands from DNA‐encoded chemical library selections

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Abstract

INTRODUCTIONPhosducin‐like 2 (PDCL2) is encoded by the phosducin‐II subgroup (PhLP2) of the phosducin (Pdc) gene family.1 It has been found that PDCL2 forms a complex with heat shock protein Hsp90,2 functions as chaperone, and is essential for cell growth in unicellular organisms.3,4,5 More importantly, PDCL2 is a testis‐specific phosphoprotein in mice and humans.6,7 Previously, we discovered that CRISPR/Cas9‐mediated PDCL2 mutant male mice are sterile due to globozoospermia caused by impaired sperm head formation (accompanying paper submitted to Andrology). This finding motivated our search for small molecule PDCL2 ligands that could disrupt sperm head formation and thereby be useful clinical candidates for nonhormonal contraception in men.To identify and discover a small molecule ligand for PDCL2, we employed the screening of DNA‐encoded chemical libraries (DECLs), which contain billions of chemically unique DNA‐barcoded compounds. These DECLs are generated through individual sequences of reactions and different combinations of functionalized building blocks (Figure 1).8‐11 To screen the library compounds, an affinity selection experiment to PDCL2 was applied to the library pool. The affinity selection is a binding‐based screen that retains those DECL molecules that bind with reasonably high affinity to PDCL2 that is immobilized on a solid support while removing nonbinding DECL molecules by washing. The structures of the PDCL2 binders are proposed based on sequencing analysis12 of the DNA barcodes that are covalently linked to each individual DECL compound (Figure 1). Although DECL screening analysis can generate several chemical series enriched with certain structure‐enrichment relationships (SER), such statistical results cannot exactly represent the binding activity of the corresponding chemical structure. In addition, because PDCL2 is not an active enzyme, traditional enzyme assays are not available for screening and measuring small molecule inhibition with PDCL2. To overcome this limitation, we developed a unique affinity selection mass spectrometry (ASMS) strategy to evaluate the PDCL2 binding affinity of small molecules identified by DECL.1FIGUREWorkflow of DNA‐encoded chemical library (DECL) selection assay of PDCL2 and billions of DECL molecules. The DECL compounds are incubated with immobilized PDCL2, treated with selection washes, processed by protein removal, and subjected to DNA sequencing. Compounds with high sequencing counts are selected as hit molecules.The ASMS assay13‐15 developed in this paper is based on a liquid chromatography with tandem mass spectrometry (LC‐MS/MS) technique (Figure 2). To the best of our knowledge, a systematic method development to incorporate LC‐MS/MS in a DECL/ASMS approach for protein ligand discovery has not been reported. By taking advantage of the well‐developed LC‐MS/MS technique, ligand signal was detected with high selectivity and sensitivity, which allows the detection and quantification of small molecule with high accuracy at picomolar concentration. The basic workflow of the ASMS binding affinity assay starts with incubating the small molecule hits with PDCL2 in buffer solution. For an active compound, the ligand‐PDCL2 complex was eluted and collected after passing through a size‐exclusion column (SEC), and the amount of the bound ligands was detected by LC‐MS/MS in the following procedures. For nonbinding compounds, the free small molecule is adsorbed by the SEC column material, leaving the PDCL2 protein alone to be eluted out from the SEC column (Figure 2A). The absence of small molecule in the nonbinder SEC eluant will show zero signal intensity in LC‐MS/MS data. The collected eluant containing either ligand‐PDCL2 complex (binder compound presence) or PDCL2 alone (nonbinder compound presence) was then denatured by heating to allow any small molecule binder, in the case when a ligand‐PDCL2 complex is formed, to be released and detected by mass spectrometry. Importantly, the ASMS method extensively reproduces the exact binding conditions as the DECL screening condition, making it straightforward to compare the binding affinity between the on‐DNA compound and the corresponding off‐DNA compound. Moreover, the binding affinity in ASMS is evaluated by the actual amount of molecules that bound to the protein, rendering an unambiguous result for structure‐activity relationship (SAR) analysis and studies.2FIGUREMechanism of affinity selection mass spectrometry (ASMS) assay developed based on liquid chromatography with tandem mass spectrometry (LC‐MS/MS) technique. Figure 2A shows the protein and ligand‐protein complex are separated from salts and small organic molecule impurities. Figure 2B shows the function of each quadrupole in the LC‐MS/MS system, which allows the highly sensitive and reproducible measurement of the ligand concentration.Samples obtained from SEC contain organic buffer molecules, organic detergent molecules, denatured protein residues, and other impurities, which could have an overlapping signal (retention time and mass) with the ligand signal and interfere with the quantification results. Rather than using conventional LC/MS to analyze the complex milieu collected from SEC, we applied LC‐MS/MS, a highly sensitive and reproducible technique,16–19 to quantify the ligand and evaluate the binding affinity of the small molecules. To decrease the background noise and improve the signal intensity of the small molecule ligand, we developed a selected reaction monitoring mass spectrometry method with a triple quadrupole (QqQ) MS instrument operating in the product‐ion scan mode (Figure 2B). In the QqQ instrument, the first quadrupole (Q1, Figure 2B) is set to remove background ions and isolate the ligand precursor ion, which is then fragmented in the second quadrupole (q2, Figure 2B). The generated fragment ions are then transferred to the third quadrupole (Q3, Figure 2B) for mass analysis and signal intensity measurement (Figure 2B). We measure the binding affinity based on the signal intensity detected by the LC‐MS/MS system. This paper describes our efforts in identifying a novel PDCL2 ligand from DECLs and improvement of the ASMS assay using a LC‐MS/MS technique.RESULTS AND DISCUSSIONDECL screening results with PDCL2To identify small molecules that bind to PDCL2, DECLs (from HitGen) cumulatively containing >440 billion compounds were pooled together for parallel screening of PDCL2 at 5 μM, 1 μM, and 0.2 μM concentrations. Blank control affinity selection was performed in parallel without protein to identify any nonspecific bead binders. Bioinformatics sequencing analysis of the amplified DNA barcodes was processed and presented as count number to approximate the relative binding affinity of on‐DNA molecules to the PDCL2 protein during the screening process. The sequencing data identified a series of bis‐substituted pyrimidine candidates enriched with good SER from one of HitGen's DECL libraries (DEL 1113, Table 1).12,20 As shown in Table 1, the selection counts in this series of hit candidates positively relate to the PDCL2 concentration in the selection screenings indicating a reliable DEL screening result. The enriched hit series contains a pyrimidine core from the Cycle 1 (C1) building block (blue, Table 1), a heterocyclic Cycle 2 building block (C2, red, Table 1) with a primary amine group enabling C‐N bond attachment to the C1 pyrimidine core, and a phenyl borylating reagent as the Cycle 3 building block (C3, gray, Table 1) for Suzuki coupling with the C1 core. Based on the SER analysis, we selected three candidates shown in Table 1 to investigate further by performing their off‐DNA organic syntheses.1TABLEDNA‐encoded chemical library (DECL) enrichment results for PDCL2 at three different concentrations. Three hit candidates with the same Cycle 1 building block were selected from a tri‐synthon DECL library (DEL 1113). The building blocks for cycles 1, 2, and 3 are presented in blue, red, and black, respectivelyCycle 2 building block112Cycle 3 building block345Sequence count at 5 µM205727Sequence count at 1 µM118520Sequence count at 0.2 µM1463Sequence count in blank (0 µM)000Validation and synthesis of PDCL2 selection hitsCandidate hit molecules were synthesized by truncating the DNA barcode linkage down to a methyl amide (Table 1 and Scheme 1). The synthesis route was redesigned from the on‐DNA library synthesis as shown in Scheme 1. To prepare the tri‐synthon parent compounds (CDD‐1923, CDD‐1835, and CDD‐2364), we commenced with the C1 building block bromo‐dichloropyrimidine (6) and installed the amine C2 building block (1, or 2) via an SNAr reaction under basic conditions to produce the intermediates 7a and 7b, respectively. The chloro group in 7a and 7b was further eliminated and substituted with methyl amine to yield the bis‐amine intermediates (8a and 8b). The methyl amine group is a surrogate for the DNA attachment point of the DECL on‐DNA compounds and maintains the binding affinity. The bromo intermediates (8a and 8b) were further reacted with the borylated reagents (3 and 5) using Suzuki–Miyaura reaction conditions to afford the final products CDD‐1923 and CDD‐1835, respectively. During the transformation of 8a to 9, the resulting mass ion peak for the product in the LC‐MS analysis was 1 mass units greater than the expected (m/z was determined to be 396.1 instead of 395.1 for 9). Extensive characterization of the product by 1H NMR, 13C NMR, HSQC, HMBC, HRMS, and IR analysis confirmed it to be phenanthridinone CDD‐2364 (Scheme 1C and shown in Supporting Information). This is rationalized on grounds that product 9 is initially formed but undergoes subsequent reactions. The nitrile group in 9 is activated by the presence of the ortho‐pyrimidine and para‐fluoro substituents—this favors its intramolecular attack by the amine to form an amidine intermediate, which hydrolyzes into the phenanthridinone product (Figure 3). Based on this observation and literature precedent,21–30 we believe that the same reaction mechanism occurs during the DECL synthesis to generate the phenanthridinone structure as the major product on DNA. Inspired by the unexpected phenanthridinone formation, we prepared CDD‐2377 (Scheme 1B) as the C2 truncated version of CDD‐2364 in a similar manner for structural activity relationship studies.1SCHEMESynthesis of CDD‐1923, 1835, 2364, and 2377a. aReagents and conditions: (a) Et3N (3.0 equiv.), 1 (1.3 equiv.), MeCN, 23°C, 18 h, 32%; (b) Et3N (2.0 equiv.), 2 (1.3 equiv.), MeCN, 23°C, 18 h, 34%; (c) Et3N (2.0 equiv.), MeNH2 (3.0 equiv.), EtOH, 80°C, 18 h, 64%; (d) Et3N (2.0 equiv.), MeNH2 (3.0 equiv.), EtOH, 80°C, 18 h, 34%; (e) Pd2(dba)3 (0.05 equiv.), cataCXium A (0.1 equiv.), K3PO4 (2.0 equiv.), 3 (1.2 equiv.), dioxane:H2O (5:1, v/v), MW (110°C), 30 min, 64%; (f) Pd2(dba)3 (0.05 equiv.), cataCXium A (0.1 equiv.), K3PO4 (2.0 equiv.), 5 (1.2 equiv.), dioxane:H2O (5:1, v/v), MW (110°C), 30 min, 68%; (g) Pd2(dba)3 (0.05 equiv.), cataCXium A (0.1 equiv.), K3PO4 (2.0 equiv.), 4 (1.2 equiv.), dioxane:H2O (5:1, v/v), MW (110°C), 30 min, 74% from 8a; (h) MeNH2•HCl (6.0 equiv.), DIPEA:DMA (2:1, v/v), 110°C, 18 h, 70%; (i) Pd2(dba)3 (0.05 equiv.), cataCXium A (0.1 equiv.), K3PO4 (2.0 equiv.), 2 (1.2 equiv.), dioxane:H2O (5:1, v/v), MW (110°C), 30 min, 72%. Pd2(dba)3 = tris(dibenzylideneacetone)dipalladium(0), cataCXium A = di(1‐adamantyl)‐n‐butylphosphine, DIPEA = N,N‐diisopropylethylamine, DMA = N,N‐dimethylacetamide, HMBC = heteronuclear multiple bond correlation, IR = infrared spectroscopy. The color in the CDD compound structure represents functional groups derived from C1, C2, and C3 building block respectively (C1 in blue, C2 in red, and C3 in black).3FIGUREReaction mechanism for the formation of CDD‐2364 from 9. Once 9 is generated from the Suzuki reaction; the amine nitrogen atom attacks the nitrile carbon 5‐bond away and forms a six‐membered ring with an imine structure (11). 11 underwent a deprotonation process to form 12, which is hydrolyzed into CDD‐2364 through 13.ASMS resultsThe binding affinity of each CDD compounds to PDCL2 was determined by the ASMS assay. The ASMS assay for each CDD compound included a nontarget control (NTC) sample and a PCDL2 incubated sample. The NTC sample served as a blank control and was prepared by the exact same procedure as the PDCL2 incubated sample without adding the PDCL2 aliquot. Both the NTC sample solution and the PDCL2 containing sample solution were incubated at 25°C for 45 min, passed through the SEC column, processed by heat elution condition, and centrifuged to obtain the supernatant for LC‐MS/MS experiment. The difference in the CDD compound peak area between the NTC sample and the PDCL2 incubated sample reveals the binding affinity of the CDD compound. The affinity value was calculated based on LC‐MS/MS peak areas using Equations 1 and 2 (also shown in Figure 4A,B).31 The peak area of the CDD compound in the PDCL2 incubated sample and the NTC sample was first normalized by the corresponding internal standard peak area (Equation 1). With the normalized peak area, the affinity value was determined by calculating the fold enrichment of the CDD compounds in the PDCL2 incubated sample over the parallel NTC sample (Equation 2). Among the three tri‐synthon compounds (CDD‐1835, CDD‐1923, and CDD‐2364), only the phenanthridinone CDD‐2364 showed a 10‐fold enrichment in the PDCL2 incubated sample over the corresponding NTC sample. To study the SAR of the C2 structure in CDD‐2364, we prepared CDD‐2377 as the truncated version (Scheme 1B), and the affinity value of CDD‐2377 was decreased by 3‐fold relative to CDD‐2364 (Figure 4B). The binding affinity of CDD‐2364 and CDD‐2377 was further confirmed in a thermal shift assay (TSA), and both compounds gave an approximately one‐degree stabilizing effect to the PDCL2 protein.1Normalized peak area of each sample=CDD compound peak areaInternal standard peak area$$\begin{equation} \text{Normalized peak area of each sample}=\frac{\text{CDD compound peak area}}{\text{Internal standard peak area}} \end{equation}$$2AffinityvalueofCDDcompounds=NormalizedpeakareainthePDCL2incubatedsampleNormalizedpeakareainNTC$$\begin{eqnarray}&&{\rm{Affinity\;value\;of\;CDD\;compounds\;}}\nonumber\\ &=& \frac{{{\rm{Normalized\;peak\;area\;in\;the\;PDCL}}2{\rm{\;incubated\;sample}}}}{{{\rm{Normalized\;peak\;area\;in\;NTC}}}}\; \end{eqnarray}$$4FIGUREBinding assay results of CDD‐1835, CDD‐1923, CDD‐2364, and CDD‐2377. Panel A shows the normalized peak area of nontarget control (NTC) sample, and PDCL2 incubated sample for each CDD compounds. Affinity enrichment was only observed with CDD‐2364 and CDD‐2377 compounds. Panel B calculates the affinity value based on the equation shown at the bottom of the figure. Panel B also shows the thermal shift assay (TSA) results of CDD‐2364 and CDD‐2377, and the TSA data are consistent with the affinity selection mass spectrometry (ASMS) results.CONCLUSIONSIn conclusion, we have screened PDCL2 with a DECL collection containing >440 billion on‐DNA compounds, generated three rational structures to perform organic synthesis, synthesized, and characterized four small molecule compounds, and identified two promising compounds (CDD‐2364 and CDD‐2377) showing binding affinities in our ASMS assay. The ASMS results of the two compounds were further validated by TSA, warranting further SAR studies featuring analogs with modifications on the C2 portion of the CDD‐2364 structure. The functional effects of the optimized lead compounds will be tested in wildtype mice and our recently generated Pdcl2 knockout mouse model (also being published as part of this special issue).32The flexibility of our DECL and ASMS screening strategy could pave the way for the systematic studies of other nonenzymatic targets, which are challenging due to the lack of biochemical assays. Furthermore, the current work is expected to foster future utilization of the sensitivity and reproducibility of the LC‐MS/MS technique to perform semi‐quantification ASMS experiments for apparent inhibition constant (Ki,app) evaluation.AUTHOR CONTRIBUTIONSQY, ZY, YF, and MMM designed research. QY, HB, YW, ZY, MP, and JYL performed research. QY, YW, ZY, MP, KRM, and MMM analyzed data. QY, YW, SAK, MT, DWY, YF, and MMM wrote the paper. HB, YW, and ZY contributed equally to this work.ACKNOWLEDGMENTSThis work is supported by the Eunice Kennedy Shriver National Institute of Child Health and Human Development (P01HD087157 and R01HD088412), the Bill & Melinda Gates Foundation (INV‐001902), the Welch Foundation (H‐Q‐0042), and a Core Facility Support Award from the Cancer Prevention Research Institute of Texas (RP160805). HitGen DEL screening was conducted with the support from the Bill & Melinda Gates Foundation under a contract to HitGen (INV‐020850). MT receives research funding (RR220012) from the Cancer Prevention and Research Institute of Texas (CPRIT). MP is supported by NIH (R03CA259664) and the Cancer Prevention Research Institute of Texas (RP220524). YF is supported by Japan Society for the Promotion of Science (JSPS) KAKENHI grant (JP20KK0155). The authors thank Dr. Xuan Qin and Professor Feng Li for mass spectrometric assistance. We are thankful to Drs. Kurt M. Bohren, Ramana Sreenivasa Rao, and Ravikumar Jimmidi for the valuable assistance in data analysis.REFERENCESWillardson BM, Howlett AC. Function of phosducin‐like proteins in G protein signaling and chaperone‐assisted protein folding. Cell. Signal. 2007;19(12):2417‐2427.Krzemien‐Ojak L, Goral A, Joachimiak E, Filipek A, Fabczak H. Interaction of a novel chaperone PhLP2A with the heat shock protein Hsp90. J. Cell. Biochem. 2017;118(2):420‐429.Bregier C, Krzemien‐Ojak L, Wloga D, et al. PHLP2 is essential and plays a role in ciliogenesis and microtubule assembly in Tetrahymena thermophila. J. Cell. Physiol. 2013;228(11):2175‐2189.Blaauw M, Knol JC, Kortholt A, et al. Phosducin‐like proteins in Dictyostelium discoideum: implications for the phosducin family of proteins. EMBO J. 2003;22(19):5047‐5057.Flanary PL, DiBello PR, Estrada P, Dohlman HG. Functional analysis of Plp1 and Plp2, two homologues of phosducin in yeast. J. Biol. 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Journal

Andrology – Wiley

Published: Jul 1, 2023

Keywords: affinity selection mass spectrometry; DNA‐encoded chemical libraries; medicinal chemistry

References

  • Function of phosducin‐like proteins in G protein signaling and chaperone‐assisted protein folding
    Willardson, BM; Howlett, AC
  • Interaction of a novel chaperone PhLP2A with the heat shock protein Hsp90
    Krzemien‐Ojak, L; Goral, A; Joachimiak, E; Filipek, A; Fabczak, H
  • PHLP2 is essential and plays a role in ciliogenesis and microtubule assembly in Tetrahymena thermophila
    Bregier, C; Krzemien‐Ojak, L; Wloga, D
  • Phosducin‐like proteins in Dictyostelium discoideum: implications for the phosducin family of proteins
    Blaauw, M; Knol, JC; Kortholt, A
  • Functional analysis of Plp1 and Plp2, two homologues of phosducin in yeast
    Flanary, PL; DiBello, PR; Estrada, P; Dohlman, HG
  • Proteomics. Tissue‐based map of the human proteome
    Uhlen, M; Fagerberg, L; Hallstrom, BM
  • Combined RNA/tissue profiling identifies novel cancer/testis genes
    Jamin, SP; Hikmet, F; Mathieu, R
  • Mortality rate in schizophrenia with tardive dyskinesia
    Takamiya, M; Yagi, G; Tanoue, A
  • C‐N coupling of DNA‐Conjugated (Hetero)aryl bromides and chlorides for DNA‐encoded chemical library synthesis
    Chen, YC; Faver, JC; Ku, AF
  • Palladium‐catalyzed hydroxycarbonylation of (Hetero)aryl halides for DNA‐encoded chemical library synthesis
    Li, JY; Miklossy, G; Modukuri, RK
  • DNA‐encoded chemistry technology yields expedient access to SARS‐CoV‐2 M(pro) inhibitors
    Chamakuri, S; Lu, S; Ucisik, MN
  • A method for estimating binding affinity from primary DEL selection data
    Chen, Q; Hall, J; Foley, TL; Wan, J; Li, Y; Israel, DI
  • Revolution of small molecule drug discovery by affinity selection‐mass spectrometry technology
    Motoyaji, T
  • Exploring new targets and chemical space with affinity selection‐mass spectrometry
    Prudent, R; Annis, DA; Dandliker, PJ; Ortholand, J‐Y; Roche, D
  • Affinity selection‐mass spectrometry screening techniques for small molecule drug discovery
    Annis, DA; Nickbarg, E; Yang, X; Ziebell, MR; Whitehurst, CE
  • Triple quadrupole mass spectrometry for direct mixture analysis and structure elucidation
    Yost, RA; Enke, CG
  • Selected Reaction Monitoring Mass Spectrometry (SRM‐MS) in Proteomics: A Comprehensive View
    Hossain, M
  • Targeted liquid chromatography‐tandem mass spectrometry analysis of proteins: basic principles, applications, and perspectives
    Kulyyassov, A; Fresnais, M; Longuespee, R
  • Quantitative high performance liquid chromatography/tandem mass spectrometric analysis of the four classes of F(2)‐isoprostanes in human urine
    Li, H; Lawson, JA; Reilly, M
  • Selecting approaches for hit identification and increasing options by building the efficient discovery of actionable chemical matter from DNA‐encoded libraries
    Foley, TL; Burchett, W; Chen, Q
  • Asymmetric synthesis of the protoberberine alkaloid (S)‐(‐)‐xylopinine using enantiopure sulfinimines
    Davis, FA; Mohanty, PK
  • The chemistry of indoles. XLIV. Synthetic study directed toward 3,4,5,6‐tetrahydro‐1H‐azepino[5,4,3‐cd]indoles
    Somei, M; Wakida, M; Ohta, T
  • Chemoselective catalytic conjugate addition of alcohols over amines
    Uesugi, S; Li, Z; Yazaki, R; Ohshima, T
  • The continuous synthesis of ε‐caprolactam from 6‐aminocapronitrile in high‐temperature water
    Yan, C; Fraga‐Dubreuil, J; Garcia‐Verdugo, E
  • The conversion of 3‐acetyl‐3‐arylhexanedinitriles into hexahydroindoles and 1H‐pyrrolo[2,3‐b]pyridines
    Schwenker, G; Metz, G
  • Elucidating the pathways of degradation of denagliptin
    Joshi, BK; Ramsey, B; Johnson, B
  • Enantiopure 4‐ and 5‐aminopiperidin‐2‐ones: regiocontrolled synthesis and conformational characterization as bioactive β‐turn mimetics
    Weber, K; Ohnmacht, U; Gmeiner, P
  • A general and efficient heterogeneous gold‐catalyzed hydration of nitriles in neat water under mild atmospheric conditions
    Liu, Y‐M; He, L; Wang, M‐M; Cao, Y; He, H‐Y; Fan, K‐N
  • Ruthenium‐catalyzed amidation of nitriles with amines. A novel, facile route to amides and polyamides
    Murahashi, S; Naota, T; Saito, E
  • Total synthesis of (S)‐(‐)‐stepholidine using (S)‐tert‐butanesulfinylimine
    Cheng, J; Fu, L; Ling, C; Yang, Y
  • Toward the assembly and characterization of an encoded library hit confirmation platform: Bead‐Assisted Ligand Isolation Mass Spectrometry (BALI‐MS)
    Ratnayake, AS; Flanagan, ME; Foley, TL
  • PDCL2 is essential for sperm acrosome formation and male fertility in mice
    Yoshitaka Fujihara, KK; Abbasi, F; Endo, T; Yu, Z; Ikawa, M; Matzuk, MM