pharmaceutics

Article Novel Non-Congeneric Derivatives of the Alpha Inhibitor ICL-CCIC-0019

Ning Wang 1, Diana Brickute 1, Marta Braga 1 , Chris Barnes 1, Haonan Lu 1 , Louis Allott 1,2,* and Eric O. Aboagye 1,*

1 Comprehensive Cancer Imaging Centre, Department of Surgery and Cancer, Faculty of Medicine, Imperial College London, Hammersmith Hospital, London W12 0NN, UK; [email protected] (N.W.); [email protected] (D.B.); [email protected] (M.B.); [email protected] (C.B.); [email protected] (H.L.) 2 Positron Emission Tomography Research Centre, Faculty of Health Sciences, University of Hull, Kingston upon Hull HU6 7RX, UK * Correspondence: [email protected] (L.A.); [email protected] (E.O.A.)

Abstract: alpha (CHKA) is a promising target for the development of cancer therapeu- tics. We have previously reported ICL-CCIC-0019, a potent CHKA inhibitor with high cellular activity but with some unfavorable pharmacological properties. In this work, we present an active analogue of ICL-CCIC-0019 bearing a piperazine handle (CK146) to facilitate further structural elaboration of the pharmacophore and thus improve the biological profile. Two different strategies were evaluated in this study: (1) a prodrug approach whereby selective CHKA inhibition could be achieved through   modulating the activity of CK146, via the incorporation of an ε-(Ac) Lys motif, cleavable by elevated levels of histone deacetylase (HDAC) and cathepsin L (CTSL) in tumour cells; (2) a prostate-specific Citation: Wang, N.; Brickute, D.; membrane antigen (PSMA) receptor targeted delivery strategy. Prodrug (CK145) and PSMA-targeted Braga, M.; Barnes, C.; Lu, H.; Allott, (CK147) derivatives were successfully synthesized and evaluated in vitro. While the exploitation L.; Aboagye, E.O. Novel Non-Congeneric Derivatives of the of CK146 in those two strategies did not deliver the expected results, important and informative Choline Kinase Alpha Inhibitor structure-activity relationships were observed and have been reported. ICL-CCIC-0019. Pharmaceutics 2021, 13, 1078. https://doi.org/10.3390/ Keywords: choline kinase alpha (CHKA) inhibitor; ICL-CCIC-0019; prodrug; PSMA; targeted drug pharmaceutics13071078 delivery; PIK4CB

Academic Editor: Luisa Carlota Lopez-Cara 1. Introduction Received: 22 June 2021 Abnormal lipid metabolism is a common feature observed in many cancers [1]. En- Accepted: 9 July 2021 hanced de novo synthesis of lipids in tumorigenesis supports the rapid proliferation of Published: 14 July 2021 cancer cells, cell signaling and tumor survival [2]. The cholinic phenotype, characterized by the overexpression of choline kinase alpha isoform (CHKA) and increased Publisher’s Note: MDPI stays neutral (PCho) levels, is one of the aberrant lipid metabolism pathways revealed in cancer [3]. with regard to jurisdictional claims in Choline kinase is a class of responsible for generating PCho which is involved in published maps and institutional affil- the of , a major lipid component of cell membranes [4]. iations. Choline kinase exists in at least three isoforms, CHKA1, CHKA2 and CHKB [4], of which the A, but not the B isoform, has been implicated in cancer development [3,5]. Overexpression of CHKA has been reported in a wide range of solid tumors [3,5–12], and correlates with advanced histological tumor grade, poor prognosis and reduced survival rates in breast Copyright: © 2021 by the authors. and non-small-cell lung cancers [9,13]. In prostate cancer (PCa), CHKA is a co-chaperone Licensee MDPI, Basel, Switzerland. for the androgen receptor (AR), thus maintains AR signaling [14]. Taken together, CHKA This article is an open access article is a prognostic marker and a potential therapeutic target for cancer treatment. distributed under the terms and A number of small-molecule CHKA inhibitors have been developed as novel anti- conditions of the Creative Commons Attribution (CC BY) license (https:// cancer strategies for CHKA inhibition [15]. Hemicholinium-3 (HC-3) was the first CHKA creativecommons.org/licenses/by/ inhibitor built around a bis-oxazonium pharmacophore (Figure1)[ 16–18], but exhibited 4.0/). off-target effects on choline transporters, acetyltransferase and acetylcholinesterase [19].

Pharmaceutics 2021, 13, 1078. https://doi.org/10.3390/pharmaceutics13071078 https://www.mdpi.com/journal/pharmaceutics Pharmaceutics 2021, 13, x FOR PEER REVIEW 2 of 17 Pharmaceutics 2021, 13, 1078 2 of 17

off-target effects on choline transporters, acetyltransferase and acetylcholinesterase [19]. MN58BMN58B waswas subsequently subsequently designed designed by by structural structural modifications modifications of HC-3 of HC-3 and demonstratedand demon- goodstrated antiproliferative good antiproliferative potencies potenciesin vitro and in vitroin vivo and(Figure in vivo1)[ (Figure20–22]. The1) [20–22]. replacement The re- of pyridiniumsplacement of inpyridiniums MN58B by in quinoliniums MN58B by quinolin as cationiciums head as cationic groups head elicited groups the bestelicited results the inbest vitro resultsand in vivovitro, and in gave vivo, rise and to RSM-932Agave rise to (Figure RSM-932A1), the (Figure first CHKA 1), the inhibitorfirst CHKA to enterinhibitor Phase to Ienter clinical Phase trials I clinical [23,24 ].trials [23,24].

FigureFigure 1. 1.Chemical Chemical structuresstructures andand pharmacologicalpharmacological datadata of correspondingcorresponding CHKA inhibitors: HC-3 [18,25], [18,25], MN58B MN58B [20,21], [20,21], RSM-932ARSM-932A [ 23[23],], ICL-CCIC-0019 ICL-CCIC-0019 [[26,27]26,27] andand CK14CK14 [[26].26].

InIn recentrecent years, the the x-ray x-ray crystal crystal structur structuree of of CHKA, CHKA, together together with with computational computational in insilico silico techniques techniques have have boosted boosted the the developmen developmentt of CHKA of CHKA inhibitors inhibitors (the (the crystal crystal structure struc- tureof human of human CHKA, CHKA, pdb code: pdb 2CKO) code: 2CKO)[18,20,28–36]. [18,20 ,A28 –series36]. of A symmetrical series of symmetrical N,N-dimethyl-N,N- dimethylaminopyridineaminopyridine (DMAP) (DMAP) derivatives derivatives bearing bearing alkyl linkers alkyl linkers of varying of varying lengths lengths were were de- designedsigned by by Trousil Trousil et et al. al. as as CHKA CHKA inhibitors inhibitors (Figure (Figure 1),1), where DMAP mimicked cationiccationic cholinecholine moietiesmoieties [[26];26]; fromfrom thisthis study, ICL-CCIC-0019ICL-CCIC-0019 was selected for further biological evaluationsevaluations and displayed displayed great great potential potential in in preclinical preclinical studies studies [27]. [27 ICL-CCIC-0019]. ICL-CCIC-0019 selec- se- lectivelytively inhibited inhibited CHKA CHKA activity activity with with potent potent antiproliferative antiproliferative activities activities against against a a broad spectrumspectrum of human cancer cancer cell cell lines lines (IC (IC50:50 0.27: 0.27 ± 0.06± 0.06 μM;µ meanM; mean GI50 of GI 850 cancerof 8 cancer cell lines: cell lines:1.09 μ 1.09M, range:µM, range: 0.38–2.70 0.38–2.70 μM; GIµ50M; range GI50 ofrange 2 normal of 2 normal cell lines: cell 30–120 lines: 30–120μM); ICL-CCIC-µM); ICL- CCIC-00190019 caused caused rapid and rapid sustained and sustained lipid synthesi lipid synthesiss inhibition inhibition by acting by actingon CDP-choline on CDP-choline path- pathwayway and andalso alsoshowed showed potent potent in vivoin vivo anti-tanti-tumorumor activity. activity. The Thecompound, compound, however, however, in- inducedduced mitochondrial mitochondrial changes changes and and slight slight reduct reductionion in inbody body weight weight (not (not below below the the stand- stan- dardard 15–20%) 15–20%) observed observed in in the the rodent rodent model model bear bearinging HCT116 HCT116 xenografts; xenografts; implying implying adverse adverse pharmacologicalpharmacological effectseffects which which precluded precluded clinical clinical translation translation [25 [25].]. Modulating Modulating the the unfavor- unfa- ablevorable properties properties of ICL-CCIC-0019 of ICL-CCIC-0019 was was likely likely to be to challengingbe challenging for for two two reasons: reasons: firstly, firstly, the intrinsicthe intrinsic toxicity toxicity of quaternary of quaternary ammonium ammonium CHKA CHKA inhibitors inhibitors is difficult is difficult to overcome to overcome since theirsince structurestheir structures mimic mimic choline choline and the and inhibition the inhibition of choline of transporterscholine transporters was observed was ob- for theseserved molecules for these [molecules26]. Secondly, [26]. the Secondly, limited th structurale limited diversity structural of diversity ICL-CCIC-0019 of ICL-CCIC- makes direct0019 makes modification direct modification of the molecule of the less molecule feasible. less While feasible. the quaternary While the ammonium quaternary moieties ammo- arenium essential moieties to are the activityessential and to the could activity not be and modified, could not we be hypothesized modified, we that hypothesized controlling thethat release controlling or delivery the release of potent or delivery CHKA inhibitors of potent inCHKA target inhibitors tissue may in reduce target off-target tissue may ac- cumulationreduce off-target and toxicity. accumulation These strategies and toxicity. have Th beenese strategies exemplified have for been a range exemplified of compounds for a and biological targets, using either a prodrug strategy, whereby compound potency is masked in off-target tissue and selectively released in target tissue, or a receptor-targeted

Pharmaceutics 2021, 13, 1078 3 of 17

delivery strategy where the drug concentration in target tissue is controlled based on receptor expression [37,38]. This study evaluated two strategies: (1) Enzymatic prodrug strategy whereby an ε-(Ac)Lys peptide motif is cleaved stepwise by upregulated histone deacetylase (HDAC) and endogenous protease cathepsin L (CTSL) in malignant cells [39–42]; (2) Peptide-receptor targeted drug delivery strategy targeting prostate specific membrane antigen (PSMA), a transmembrane receptor overexpressed (100–1000 fold higher) in PCa [43,44]. To exploit these non-congeneric strategies for our purposes, a scaffold based around ICL-CCIC-0019 with comparable CHKA activity would be required to bear a reactive handle for further structural elaboration. We report the synthesis of a novel potent asym- metrical CHKA active analogue of ICL-CCIC-0019 for evaluation in the aforementioned strategies, and two novel drug conjugates evaluated in vitro.

2. Materials and Methods 2.1. Chemical Synthesis The chemical synthesis procedures of ICL-CCIC-0019, CK14, CK145 (5), CK146 (4), CK147 (8) and CK148 (7) are described in the Supplementary Information. The radiosyn- thesis of [18F]D4-FCH has been reported elsewhere [45].

2.2. Cell Culture HCT-116, A549, 22Rv1, C4-2B, LNCap, R1-AD1, R1-D567, PC3 and PNT1A were cultured with RPMI 1640 media (Sigma Life Science, Gillingham, UK). HepG2 and Caco-2 were cultured with DMEM media (BioWhittaker®, Lonza, Basel, Switzerland). All the media were supplemented with 10% FCS (Fetal Calf Serum, First Link UK Ltd., Wolver- hampton, UK) and 10% L-glutamine (gibco®, Life Technologies, Paisley, UK; 200 mmol, 100 mL). Caco-2 cell line was maintained in DMEM media (BioWhittaker®, Lonza, Basel, Switzerland) containing 20% FCS (Fetal Calf Serum, First Link UK Ltd., Wolverhampton, UK) and 10% L-glutamine (gibco®, Life Technologies, Paisley, UK; 200 mmol, 100 mL). ◦ Cells were cultured at 37 C in a humidified atmosphere containing 5% CO2. Cell lines were authenticated by provider (Human colorectal adenocarcinoma cell line, ATCC). No additional authentication of cells was performed.

2.3. Anti-Proliferative Assay (Sulforhodamine B Assay)

Half-maximal growth inhibitory concentrations (GI50) were determined using sul- forhodamine B (SRB) assay as described elsewhere [46]. In brief, cells were seeded in 96-wells at respective cell densities determined by their cell growth curves. After 24 h, the cells were treated in six replicates with medium or different concentrations of CHKA inhibitors in medium and incubated for another 72 h. On completion of incubation, the cells were fixed with 10% trichloroacetic acid (TCA) and stained with 0.4% SRB in 1% acetic acid. The stained was solubilized with 10 mM Tris base and optical density values were measured at 540 nm using a Multiskan® EX Micro plate photometer (Thermo Scientific, Loughborough, UK). Growth inhibition curves were plotted as percentage of the control groups and GI50 data were determined by least squares fitting method using GraphPad Prism 7.0. All the chemicals used in this experiment were obtained from Sigma-Aldrich (Gillingham, UK).

2.4. Lipid Kinase Screening The lipid kinase screening for ICL-CCIC-0019, CK14, CK145 (5), CK146 (4) and CK147 (8) (testing concentration for all compounds: 10 µM) was performed by MRC PPU In- ternational Centre for Kinase Profiling (Dundee, UK). The lipid kinase screening panel includes 15 human recombinant lipid and the tests used Promega ADP GloTM High Throughput Assay kit (Promega, Southampton, UK). For each assay, reference compounds Pharmaceutics 2021, 13, 1078 4 of 17

were used as quality control. Finally, mean percentage activity inhibited by compounds and standard deviation for all the repeats were calculated.

2.5. In Vitro ‘Absorption, Distribution, Metabolism and Excretion’ (ADME) Study The ADME study for ICL-CCIC-0019, CK14, CK145 (5), CK146 (4) and CK147 (8) was performed by Charles River Drug Discovery Services (Essex, UK).

2.6. Immunoblotting Analysis (General) Cell pellets were prepared and lysed with radioimmunoprecipitation assay (RIPA) buffer (R0278, Sigma-Aldrich, Gillingham, UK) supplemented with protease and phos- phatase inhibitors (Thermo Fisher, Loughborough, UK). Equal amounts of protein (10 µg) were resolved on 4–15% mini-protein TGX gels (456-1086 Bio-Rad) and transferred to PVDF membranes (Trans-Blot Turbo Transfer Packs, Bio-Rad, Watford, UK). Membranes were blocked with 5% milk in phosphate buffered saline (PBS) containing 0.1% v/v tween-20 and subsequently incubated with the following primary antibodies at 4 ◦C overnight: HDAC1 (10E2), 1:8000, #5356s, Cell Signaling Technology, London, UK; Calnexin, 1:5000, #ADI- SPA-860-D, ENZO life sciences, Exeter, UK; CHKA, 1:1000, #HPA024153, Sigma-Aldrich, Gillingham, UK; PSMA (D78E), 1:1000, #12815, Cell Signaling Technology, London, UK; GAPDH (D16H11), 1:10,000, #5174, Cell Signaling Technology, London, UK. Secondary HRP-conjugated mouse and rabbit antibodies (1:8000 and 1:5000; #7076P2 and #sc-2005; Cell Signaling Technology, London, UK and Santa Cruz Biotechnology, California, USA, respectively) were used for 1 h at room temperature. Signals were visualized by chemi- luminescence detection using Amersham ECL Western Blotting Detection Reagent (GE Healthcare, Amersham, UK) and Amersham Hyperfilm (GE Healthcare, Amersham, UK).

2.7. Conjugate Stability Test of CK147 (8) To assess stability of CK147 in medium, 5 mL of medium containing 10% FCS and 1% L-glutamine was added sterilely to a six-well plate. Next, 1.7 µL of 300 mM CK147 in DMSO or 1.7 µL DMSO was added into wells and the plate was gently shaken for 5 s ◦ before being incubated at 37 C in a humidified atmosphere containing 5% CO2. After 0, 2, 4, 6, 24, 48 and 72 h, 200 µL aliquots of medium from each well were transferred into a 1.5-mL tube containing 198.4 µL acetonitrile and 1.6 µL of 2.5 mg/mL ICL-CCIC- 0019 as an internal standard (IS) (20 µg/mL). After a brief vortex, the Eppendorf tube was placed at 4 ◦C for 30 min for protein precipitation. Fifty microliters of the resulting supernatant from centrifuge (4 ◦C, 15,000× g) was taken to supplement with 70 µL of water:acetonitrile:formic acid (95:5:0.2, v/v/v) and sonicated for 1 min. One hundred microliters of the solution were injected into the HPLC system described in Supplementary HPLC method D. Three repeats were performed for each sample.

2.8. Cellular Uptake and Metabolism Study of CK145 (5) HCT-116 colorectal cancer cells were seeded into cell culture dishes (150 × 25 mm) at a density of 7 × 106 per dish. The cells were allowed to settle and grow for another 48 h before being treated with or without ICL-CCIC-0019 or CK145 (50 µM of 5 mL RPMI medium with 10% FCS and 1% L-glutamine) for 1 h and 2 h in the incubator. Cells were scraped, washed with ice-cold PBS three times and collected by centrifugation (4 ◦C, 4000× g). The cell pellets were resuspended in 1 mL water: acetonitrile: formic acid (95:5:0.2, v/v/v) and disrupted by an ultrasonic cell disruptor (Soniprep 150, MSE Ltd., London, UK) for 1 min on ice. One hundred microliters of the filtered supernatant from centrifuge (4 ◦C, 15,000× g) were analyzed by HPLC. Three injections were performed for each sample. Intracellular drug was normalized to cellular protein of each plate using bicinchoninic acid (BCA) assay. The HPLC method used in this study is described in Supplementary HPLC method C. Pharmaceutics 2021, 13, 1078 5 of 17

2.9. Cellular Uptake and Metabolism Study of ICL-CCIC-0019, CK146 (4), CK147 (8) and CK148 (7) in Presence and Absence of Serum Cell lysate sample preparation: C4-2B prostate cancer cells were seeded into cell culture dishes (150 × 25 mm) at a density of 7 × 106 per dish. The cells were allowed to settle and grow for another 48 h. The cells were treated with ICL-CCIC-0019, CK146, CK147 and CK148 (50 µM of 7 mL RPMI medium with or without 10% FCS) for 4 h in the incubator. After 4-h incubation, the aliquots of medium were collected (see the medium sample preparation section). Cells were scraped, washed with ice-cold PBS three times and collected by centrifugation (4 ◦C, 4000× g). The cell pellets were resuspended in 500 µL of chilled acetonitrile:PBS (50:50, v/v) for cell disruption and homogenization. The cell disruption was performed by a Precellys® 24 homogenizer (Bertin Technologies, Montigny- le-Bretonneux, France) at 4 ◦C. After acetonitrile evaporation at room temperature, 50 µL of cell lysis was taken for protein analysis. Another 100 µL of the supernatant obtained after centrifuge (4 ◦C, 15,000× g) was resuspended in 100 µL formic acid:water (0.2%, v/v). Next, 100 µL of the filtered solution was analyzed by HPLC system described in Supplementary HPLC method D. Three injections were performed for each sample. Intracellular drug was normalized to cellular protein of each plate using BCA assay. Medium sample preparation for HPLC analysis: 250-µL aliquots of medium (with or without 10% FCS) were transferred into 1.5-mL tube containing 250 µL acetonitrile. After a brief vortex, the tube was placed at 4 ◦C for 30 min for protein precipitation. One hundred microliters of the resulting supernatant from centrifuge (4 ◦C, 15,000× g) were resuspended in 100 µL formic acid: water (0.2%, v/v). One hundred microliters of the filtered sample were analyzed by HPLC system described in Supplementary HPLC method D. Three injections were performed for each sample.

2.10. [18F]D4-FCH Uptake In Vitro Study HCT-116 and C4-2B cells were seeded at a density of 5 × 105 cells per well into 6-well plates and incubated overnight. Cells were treated with 1 or 10 µM CHKA inhibitors (CK14, CK145, CK146 and CK147) for 1 h and pulsed with [18F]D4-FCH (740 kBq) in presence of inhibitors in 1 mL for an additional hour at 37 ◦C. Cells were washed with ice-cold PBS three times and lysed in 1 mL RIPA buffer. The radioactivity of 800 µL lysate from each well was counted on a gamma counter (Perkin Elmer, Beaconsfield, UK) and normalized to protein content as determined by BCA assay.

2.11. Statistical Analysis Data were represented as mean values ± standard deviation (SD). Unpaired two-tailed t-tests from GraphPad Prism 7.0 were used to determine the significance in the experiments. Statistic differences were significant at 0.01 < p < 0.05, very significant at 0.001 < p < 0.01 and extremely significant at 0.0001 ≤ p < 0.001 and p < 0.0001.

3. Results 3.1. Synthesis of Novel CHKA Inhibitors The synthesis of CK146 from building blocks 1 and 2 is described in Scheme1A. The nonsymmetric tetradecyl linker (1) was accessed by reacting 1,14-bromotetradecane with DMAP to afford the product as a white precipitate [26]. Compound 2 was synthesized from 1-(pyridin-4-yl) piperazine by boc-protection of the secondary amine in an excellent yield (94%). Quaternization of the pyridinyl ring of 2 by coupling with 1 produced compound 3 in a near quantitative yield (97%) which was subsequently deprotected under acidic conditions to give CK146 (4) in a yield of 93%. Pharmaceutics 2021, 13, x FOR PEER REVIEW 6 of 17

Pharmaceutics 2021, 13, 1078 yield (94%). Quaternization of the pyridinyl ring of 2 by coupling with 1 produced 6com- of 17 pound 3 in a near quantitative yield (97%) which was subsequently deprotected under acidic conditions to give CK146 (4) in a yield of 93%.

Scheme 1. The modificationsmodifications of CK146 via the prodrug strategy and drug delivery strategy. ( A)) Synthesis of of CK146 (4); (4); reaction conditions: (i) 1,14-bromotetradecane, 2-butanone, 110 °C, 3 h; (ii) Di-tert-butyl decarbonate, DMAP, DCM, RT, reaction conditions: (i) 1,14-bromotetradecane, 2-butanone, 110 ◦C, 3 h; (ii) Di-tert-butyl decarbonate, DMAP, DCM, RT, 16 h; (iii) MeCN, 110 °C, 72 h; (iv) TFA, MeCN, RT, 16 h; (B) Synthesis of CK145 (5) and CK147 (8); reaction conditions: (v) 16 h; (iii) MeCN, 110 ◦C, 72 h; (iv) TFA, MeCN, RT, 16 h; (B) Synthesis of CK145 (5) and CK147 (8); reaction conditions: α-Boc-Lys (ε-Ac)-OH, EDC HCl, HOBt, DIEA, DMF, RT, 24 h; (vi) Dithiodiglycolic acid, EDC HCl, HOBt, DIPEA, DMF, (v) α-Boc-Lys (ε-Ac)-OH, EDC HCl, HOBt, DIEA, DMF, RT, 24 h; (vi) Dithiodiglycolic acid, EDC HCl, HOBt, DIPEA, DMF, RT, 72 h; (vii) TCEP, Tris-HCl, NaOH, PSMA-Maleimide (9), DMF, N2, RT, 24 h; (viii) TFA, DCM, RT, 16 h. RT, 72 h; (vii) TCEP, Tris-HCl, NaOH, PSMA-Maleimide (9), DMF, N2, RT, 24 h; (viii) TFA, DCM, RT, 16 h.

The synthesis of CK145 (5) was performed by contract synthesis (Creative Chemistry Ltd., Hampton, UK) following a literature procedure [47]. In brief, compound 4 was coupled with commercially available α-Boc-Lys (ε-Ac)-OH in the presence of EDC HCl, HOBt and DIEA in DMF at room temperature (Scheme1B, prodrug strategy). CK145 was purified by semi-prep HPLC which produced pure product (>98%) in a combined yield of Pharmaceutics 2021, 13, x FOR PEER REVIEW 7 of 17

The synthesis of CK145 (5) was performed by contract synthesis (Creative Chemistry

Pharmaceutics 2021, 13, 1078 Ltd., Hampton, UK) following a literature procedure [47]. In brief, compound 4 was7 of cou- 17 pled with commercially available α-Boc-Lys (ε-Ac)-OH in the presence of EDCHCl, HOBt and DIEA in DMF at room temperature (Scheme 1B, prodrug strategy). CK145 was puri- fied by semi-prep HPLC which produced pure product (>98%) in a combined yield of 37.5%37.5% ready ready for for biological biological evaluation evaluation (see (see Supplementary, Supplementary, HPLC HPLC method method B). TheB). The synthesis synthe- ofsis CK148 of CK148 (7) is (7) shown is shown in Scheme in Scheme1B (drug 1B (d deliveryrug delivery strategy). strategy). Dimer Dimer 6 was 6 was synthesized synthesized byby peptide peptide coupling coupling with with dithiodiglycolic dithiodiglycolic acid acid which which was was reduced reduced to to a monomera monomer in in the the presencepresence of of TCEP TCEP hydrochloridehydrochloride in degassed degassed Tris-HCl Tris-HCl buffer buffer (pH (pH 7) followed 7) followed by the by drop- the dropwisewise addition addition of 9 of ((OtBu) 9 ((OtBu)3 PSMA-maleimide,3 PSMA-maleimide, Creative Creative Chemistry Chemistry Ltd., Ltd., Hampton, Hampton, UK). UK).This This produced produced 7 in 7 a in moderate a moderate yield yield of 56% of 56% which which was was deprotected deprotected to produce to produce the thefinal finalcompound compound CK147 CK147 (8) ready (8) ready for biological for biological evaluation evaluation without without further further purification purification (purity: (purity:95% determined 95% determined by HPLC; by HPLC;Figure S31). Figure All S31). compounds All compounds were characterized were characterized by NMR byand NMRMass and Spectrometry Mass Spectrometry (spectra (spectrashown in shown the Supplementary, in the Supplementary, Figures S7–S30). FiguresS7–S30). 3.2. Conjugate Stability Test of CK147 (8) 3.2. Conjugate Stability Test of CK147 (8) The conjugate stability of CK147 was assessed by HPLC, where ICL-CCIC-0019 served The conjugate stability of CK147 was assessed by HPLC, where ICL-CCIC-0019 as an internal standard. At the early time points, CK147 remained stable in medium at served as an internal standard. At the early time points, CK147 remained stable in me- 37 ◦C (93.0 ± 0.8% intact parent at 2 h and 89.7 ± 0.3% intact parent at 4 h). After 72 h, the dium at 37 °C (93.0 ± 0.8% intact parent at 2 h and 89.7 ± 0.3% intact parent at 4 h). After concentration of CK147 in medium reduced to 60.0 ± 0.3% (21.7 ± 0.2 µg/mL) (Figure2). 72 h, the concentration of CK147 in medium reduced to 60.0 ± 0.3% (21.7 ± 0.2 μg/mL) Chemical decomposition was not detected by HPLC/MS analysis. A hypothesis for this (Figure 2). Chemical decomposition was not detected by HPLC/MS analysis. A hypothesis phenomenon is further elaborated in the Discussion. for this phenomenon is further elaborated in the Discussion.

FigureFigure 2. 2.PSMA-targeted PSMA-targeted CHKA CHKA inhibitor inhibitor (CK147) (CK147) concentration concentration trend trend (72 (72 h) h) in in medium medium supple- supple- mented with 10% FCS (60 ± 0.3% intact parent at 72 h); n = 3, mean ± SD. mented with 10% FCS (60 ± 0.3% intact parent at 72 h); n = 3, mean ± SD.

3.3.3.3. Antiproliferative Antiproliferative Activity Activity Assays Assays AppropriateAppropriate cell cell lines lines were were selected selected on on the the basis basis of of mRNA mRNA levels levels of of HDAC HDAC I classI class andand CTSL CTSL using using Cancer Cancer Cell Cell Line Line Encyclopedia Encyclopedia RNA RNA sequencing sequencing database database (Table (Table S1). S1). The The HDACHDAC 1 subtype1 subtype was was found found highly highly expressed expressed in in all all four four selected selected cell cell lines lines as as determined determined byby immunoblotting immunoblotting (Figure (Figure3a). 3a). The The antiproliferative antiproliferative activities activities (GI (GI50)50 of) of ICL-CCIC-0019, ICL-CCIC-0019, CK146CK146 and and CK145 CK145 were were evaluatedevaluated inin these four cell cell lines lines by by SRB SRB assay. assay. It Itwas was shown shown that thatall allthe the three three compounds compounds potently potently inhibited inhibited the theproliferation proliferation of cancer of cancer cells cells at low at low sub- sub-micromolarmicromolar concentrations concentrations (Figure (Figure 3b,3 b,Table Table S2). S2). ICL-CCIC-0019 ICL-CCIC-0019 exhibited exhibited the the best best anti- antiproliferativeproliferative potency potency (mean (mean GI50 GI in50 fourin four cancer cancer cell lines: cell lines: 0.5 ± 0.50.02± μM);0.02 CK146µM); CK146still dis- stillplayed displayed potent potent antiproliferative antiproliferative activity activity (mean (mean GI50 against GI50 against four cancer four cancercell lines: cell 2.5 lines: ± 0.3 2.5μM)± 0.3 withµM HepG2) with HepG2being most being sensitive most sensitive to the treatment to the treatment (0.21 ± 0.01 (0.21 μM).± The0.01 overallµM). The anti- overallproliferative antiproliferative activity of activity CK145 ofwas CK145 similar was to similar CK146 to(mean CK146 GI (mean50 against GI50 fouragainst cancer four cell cancerlines: cell5.0 ± lines: 0.3 μM), 5.0 but± 0.3 it resultedµM), but in it less resulted growth in inhibition less growth in HepG2 inhibition and in Caco-2 HepG2 cells. and All Caco-2three cells.compounds All three displayed compounds weaker displayed antiprolifer weakeration antiproliferation in Caco-2 cells—a in Caco-2 less tumorigenic cells—a lesscolon tumorigenic cancer cell colon line—com cancerpared cell line—compared to HCT-116 cells. to HCT-116 cells. In addition, the antiproliferative activity of CK147 was assessed in PSMA-positive (22Rv1, C4-2B, LNCap) and PSMA-negative cell lines (PC3, PNT1A, R1-AD1, R1-D567 and HCT-116). The expression of CHKA and PSMA in prostate cell lines was determined by immunoblotting (Figure4a). ICL-CCIC-0019 and CK146 showed potent antiproliferative ac- tivities, with mean GI50 across eight cell lines of 0.6 ± 0.1 µM and 3.8 ± 0.9 µM respectively

(Figure4b). However, all the tested cell lines were not sensitive towards CK147 (mean GI 50: 66.7 ± 7.2 µM) and the correlation of GI50 with PSMA expression was not observed. Pharmaceutics 2021, 13, x FOR PEER REVIEW 8 of 17

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(a) (b) Figure 3. Antiproliferative activity characterization of ICL-CCIC-0019, CK146 and CK145. (a) Im- munoblotting analysis of HDAC1 in HepG2, HCT-116, A549 and Caco-2 cell lines (n = 2); (b) Com- parison of growth inhibitory activity among ICL-CCIC-0019, CK146 and CK145 in HCT-116, A549, HepG2 and Caco-2 cells (n = 6, mean ± SD).

In addition, the antiproliferative activity of CK147 was assessed in PSMA-positive (22Rv1, C4-2B, LNCap) and PSMA-negative cell lines (PC3, PNT1A, R1-AD1, R1-D567 and HCT-116). The expression of CHKA and PSMA in prostate cell lines was determined (a) (b) by immunoblotting (Figure 4a). ICL-CCIC-0019 and CK146 showed potent antiprolifera- FigureFiguretive activities, 3.3. AntiproliferativeAntiproliferative with mean activityactivity GI50 across characterizationcharacterization eight cell ofoflines ICL-CCIC-0019,ICL-CCIC-0019, of 0.6 ± 0.1 μ CK146M and andand 3.8 CK145.CK145. ± 0.9 μ (aM) Im-Im-re- munoblotting analysis of HDAC1 in HepG2, HCT-116, A549 and Caco-2 cell lines (n = 2); (b) Com- munoblottingspectively (Figure analysis 4b). of However, HDAC1 in HepG2,all the tested HCT-116, cell A549lines andwere Caco-2 not sensitive cell lines towards (n = 2); (b CK147) Com- parison of growth inhibitory activity among ICL-CCIC-0019, CK146 and CK145 in HCT-116, A549, parison(mean GI of growth50: 66.7 inhibitory± 7.2 μM) activityand the among correlation ICL-CCIC-0019, of GI50 with CK146 PSMA and CK145expression in HCT-116, was not A549, ob- HepG2 and Caco-2 cells (n = 6, mean ± SD). HepG2served. and Caco-2 cells (n = 6, mean ± SD). In addition, the antiproliferative activity of CK147 was assessed in PSMA-positive (22Rv1, C4-2B, LNCap) and PSMA-negative cell lines (PC3, PNT1A, R1-AD1, R1-D567 and HCT-116). The expression of CHKA and PSMA in prostate cell lines was determined by immunoblotting (Figure 4a). ICL-CCIC-0019 and CK146 showed potent antiprolifera- tive activities, with mean GI50 across eight cell lines of 0.6 ± 0.1 μM and 3.8 ± 0.9 μM re- spectively (Figure 4b). However, all the tested cell lines were not sensitive towards CK147 (mean GI50: 66.7 ± 7.2 μM) and the correlation of GI50 with PSMA expression was not ob- served.

(a) (b)

FigureFigure 4.4. AntiproliferativeAntiproliferative activityactivity characterizationcharacterization ofof ICL-CCIC-0019,ICL-CCIC-0019, CK146 andand CK147.CK147. (a) Im-Im- munoblottingmunoblotting analysis of of PSMA PSMA and and CHKA CHKA in in 22R 22Rv1,v1, C4-2B, C4-2B, LNCap, LNCap, PC3, PC3, PNT1A, PNT1A, R1-AD1 R1-AD1 and and R1- D567 cell lines (n = 2); (b) Growth inhibitory activity comparison among ICL-CCIC-0019, CK146 and R1-D567 cell lines (n = 2); (b) Growth inhibitory activity comparison among ICL-CCIC-0019, CK146 CK147 in 22Rv1, C4-2B, LNCap, PC3, PNT1A, R1-AD1, R1-D567 and HCT-116 cell lines (n = 6, mean and CK147 in 22Rv1, C4-2B, LNCap, PC3, PNT1A, R1-AD1, R1-D567 and HCT-116 cell lines (n = 6, ± SD).

mean ± SD). 3.4. Cellular Uptake and In Vitro Metabolism 3.4. Cellular Uptake and In Vitro Metabolism Uptake of ICL-CCIC-0019 and CK145 in HCT-116 cells increased with incubation (a) (b) time Uptake(Figure of5). ICL-CCIC-0019 The cellular uptake and CK145 of ICL-CCIC-0019 in HCT-116 cellswas approximately increased with incubationtwo-fold higher time (FigurethanFigure CK145 4.5). Antiproliferative The at cellular 1 h and uptake 2 activityh post-incubation. of ICL-CCIC-0019characterization However, of was ICL-CCIC-0019, approximately in vitro conversion CK146 two-fold and ofCK147. higher CK145 (a than )was Im- CK145notmunoblotting observed at 1 h analysis and(Figures 2 hof post-incubation. PSMAS32 and and S33); CHKA CK146 in However, 22R orv1, intermediate C4-2B,in vitro LNCap,conversion species PC3, PNT1A, were of CK145 not R1-AD1 detected was and not R1- in observedtheD567 media, cell lines (Figures which (n = 2); could S32 (b) Growth and arise S33); from inhibitory CK146 cellular activity or intermediateacti vecomp effluxarison (Figure species among S34). wereICL-CCIC-0019, The not cellular detected CK146 concen- in theand CK147 in 22Rv1, C4-2B, LNCap, PC3, PNT1A, R1-AD1, R1-D567 and HCT-116 cell lines (n = 6, mean media,tration whichof converted could arise CK145 from products cellular activewas found efflux to (Figure be lower S34). than The cellularthe limit concentration of detection ± SD). of(LOD) converted of the CK145HPLC method products used was (1.382 found μ tog/L). be lower than the limit of detection (LOD) of the HPLC method used (1.382 µg/L). 3.4. CellularAn in vitro Uptakeuptake and studyIn Vitro using Metabolism C4-2B cell line was conducted to investigate the cellular uptakeUptake and metabolism of ICL-CCIC-0019 of CK147. and The CK145 study in comprised HCT-116 twocells experimentalincreased with conditions incubation to evaluatetime (Figure the effects5). The of cellular serum uptake on drug of uptake: ICL-CCIC-0019 one group was included approximately medium two-fold supplemented higher withthan 10%CK145 FCS at (FCS1 h and positive 2 h post-incubation. group) and the However, other was in FCS-free vitro conversion medium (FCSof CK145 negative was group).not observed ICL-CCIC-0019, (Figures S32 CK146 and andS33); CK148 CK146 ((OtBu) or intermediate3-protected species CK147) were were includednot detected in the in experimentthe media, which for comparison. could arise In from both cellular experiment active groups, efflux ICL-CCIC-0019(Figure S34). The (tR cellular: 7.36 min: concen- sec), CK146tration (tofR :converted 6.30 min: sec)CK145 and products CK148 (t wasR: 11.30 found min: to sec)be lower were detectedthan the inlimit the of cell detection lysates by(LOD) HPLC of the following HPLC method 4 h incubation, used (1.382 while μg/L). uptake of CK147 was not seen (Figure S35). ICL-CCIC-0019 and CK146 had similar mean uptake of 24.9 ± 1.4% µg−1 protein and 34.1 ± 0.6% µg−1 protein for FCS positive and negative groups respectively (Figure6). Significant increase in the cellular uptake in both experiment groups was seen in CK148 (mean ± SD of two experiment groups: 42.5 ± 11.5% µg−1 protein) compared to CK147 (Figure6). Drug uptake was independent to the presence of FCS.

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Figure 5. Percentage drug uptake in HCT-116 cell line determined by HPLC at selected time points (1-h and 2-h) (n = 3, mean ± SD). The data were calculated by the drug amount in cells compared to the initial drug amount in media %; the values represent the percentage of drugs taken into cells.

Pharmaceutics 2021, 13, 1078 An in vitro uptake study using C4-2B cell line was conducted to investigate9 of 17 the cel- Pharmaceutics 2021, 13, x FOR PEER REVIEWlular uptake and metabolism of CK147. The study comprised two experimental9 of conditions 17

to evaluate the effects of serum on drug uptake: one group included medium supple- mented with 10% FCS (FCS positive group) and the other was FCS-free medium (FCS negative group). ICL-CCIC-0019, CK146 and CK148 ((OtBu)3-protected CK147) were in- cluded in the experiment for comparison. In both experiment groups, ICL-CCIC-0019 (tR: 7.36 min: sec), CK146 (tR: 6.30 min: sec) and CK148 (tR: 11.30 min: sec) were detected in the cell lysates by HPLC following 4 h incubation, while uptake of CK147 was not seen (Figure S35). ICL-CCIC-0019 and CK146 had similar mean uptake of 24.9 ± 1.4% μg−1 protein and 34.1 ± 0.6% μg−1 protein for FCS positive and negative groups respectively (Figure 6). Sig- nificant increase in the cellular uptake in both experiment groups was seen in CK148 (mean ± SD of two experiment groups: 42.5 ± 11.5% μg−1 protein) compared to CK147 (Fig-

ure 6). Drug uptake was independent to the presence of FCS. FigureFigureHPLC/MS 5. 5.Percentage Percentage analysis drug drug uptake uptake of incell in HCT-116 HCT-116 lysates cell celland line line the determined de terminedmedium by by HPLCafter HPLC 4 at ath selected selectedincubation time time points pointswith CHKA (1-h(1-h and and 2-h) 2-h) (n (n= = 3, 3, mean mean± ±SD). SD). The The data data were were calculated calculated by by the the drug drug amount amount in in cells cells compared compared to to inhibitorsthe initial drug confirmed amount in the media stability %; the valuesof all rethpresente compounds the percentage in cells of drugs and takenmedium. into cells. Decomposi- thetion initial or interconversion drug amount in media was %; not the valuesobserved represent from the the percentage HPLC/MS of drugs (Figures taken intoS36–S45). cells. An in vitro uptake study using C4-2B cell line was conducted to investigate the cel- lular uptake and metabolism of CK147. The study comprised two experimental conditions to evaluate the effects of serum on drug uptake: one group included medium supple- mented with 10% FCS (FCS positive group) and the other was FCS-free medium (FCS negative group). ICL-CCIC-0019, CK146 and CK148 ((OtBu)3-protected CK147) were in- cluded in the experiment for comparison. In both experiment groups, ICL-CCIC-0019 (tR: 7.36 min: sec), CK146 (tR: 6.30 min: sec) and CK148 (tR: 11.30 min: sec) were detected in the cell lysates by HPLC following 4 h incubation, while uptake of CK147 was not seen (Figure S35). ICL-CCIC-0019 and CK146 had similar mean uptake of 24.9 ± 1.4% μg−1 protein and 34.1 ± 0.6% μg−1 protein for FCS positive and negative groups respectively (Figure 6). Sig- nificant increase in the cellular uptake in both experiment groups was seen in CK148 (mean ± SD of two experiment groups: 42.5 ± 11.5% μg−1 protein) compared to CK147 (Fig- ure 6). Drug uptake was independent to the presence of FCS. Figure 6. Drug uptake percentage following 4-h incubation with ICL-CCIC-0019, CK146, CK147 and FigureHPLC/MS 6. Drug uptake analysis percentage of cell followinglysates and 4-h the incubation medium with after ICL-CCIC-0019, 4 h incubation CK146, with CK147CHKA CK148 in medium with or without FCS (n = 3, mean ± SD). The data were calculated from chroma- andinhibitors CK148 inconfirmed mediumwith the stability or without of FCSall th (ne =compounds 3, mean ± SD). in cells The and data medium. were calculated Decomposi- from tographic peak integration normalized to protein amount in cell lysates. p Values via unpaired two- chromatographiction or interconversion peak integration was not normalized observed to from protein the amount HPLC/MS in cell lysates.(Figuresp ValuesS36–S45). via unpaired two-tailedtailed t-test:t-test: **** **** p

3.5. HPLC/MSADME Study analysis of cell lysates and the medium after 4 h incubation with CHKA inhibitors confirmed the stability of all the compounds in cells and medium. Decomposition or interconversionThe predicted was in not vitro observed pharmacokinetic from the HPLC/MS characteristics (Figures of S36–S45). compounds were evaluated by four ADME assays, namely kinetic solubility assay, metabolic stability assay using hu- 3.5.man ADME liver Studymicrosome S9, permeability and efflux study using the Caco-2 cell line, and matrixThe stability predicted assayin vitro usingpharmacokinetic fasted state simula characteristicsted gastric of compounds fluid (FaSSGF). were evaluated All tested CHKA byinhibitors four ADME were assays, highly namely soluble kinetic (>150 solubility μM) in 0.1 assay, M PBS metabolic (2% DMSO) stability (Table assay using1). All CHKA humaninhibitors liver except microsome for CK145 S9, permeability were stable and effluxin the study metabolic using thestudy; Caco-2 ICL-CCIC-0019, cell line, and CK14, matrixCK145 stability and CK146 assay usinghad long fasted biological state simulated half-lives gastric (>100 fluid min) (FaSSGF). and therefore All tested lower CHKA clearance inhibitors were highly soluble (>150 µM) in 0.1 M PBS (2% DMSO) (Table1). All CHKA inhibitorsFigure 6. Drug except uptake for percentage CK145 were following stable 4-h in theincubation metabolic with study;ICL-CCIC-0019, ICL-CCIC-0019, CK146, CK147 CK14, and CK145CK148 andin medium CK146 with had or long without biological FCS (n half-lives = 3, mean(>100 ± SD). min)The data and were therefore calculated lower from clearance chroma- valuestographic (<14 peakµL/min/mg) integration normalized (Table1). Theto proteinin vivo amountclearance in cell lysates. was calculated p Values via to unpaired predict thetwo-

actualtailed clearancet-test: **** p in < humans0.0001, ** byp < scaling0.01. the clearance values, which takes into account liver weight, microsomes per liver and liver blood flow. By contrast, CK145 had a short half- life3.5. value ADME (7.4 Study min) and a predicted clearance of 19 mL/min/kg, giving a value for the percentageThe predicted of compound in vitro removed pharmacokinetic compared characteristics to liver blood flowof compounds (PCT_LBF%) were up evaluated to 92%. Inby Caco-2 four ADME cell permeability assays, namely assay, kinetic drug solubility permeability assay, was metabolic investigated stability and assay presented using ashu- apparentman liver permeability microsome coefficientsS9, permeability (Papp) and in Aefflux to B (A-B,study apicalusing tothe basolateral Caco-2 cell direction) line, and andmatrix B to stability A (B-A, assay basolateral using tofasted apical state direction). simulated All gastric the tested fluid compounds(FaSSGF). All exhibited tested CHKA low −6 intestinalinhibitors permeability were highly classification soluble (>150 (Papp μM) A-Bin 0.1 < 5M× PBS10 (2%cm/s). DMSO) Notably, (Table CK14 1). All was CHKA the mostinhibitors subject except to efflux for with CK145 an effluxwere ratiostable of in 87; the the metabolic permeability study; of CK147 ICL-CCIC-0019, was too poor CK14, to permitCK145 calculation and CK146 of had the long efflux biological ratio. half-lives (>100 min) and therefore lower clearance

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Table 1. Pharmacokinetic parameters of ICL-CCIC-0019, CK14, CK145, CK146 and CK147 from four ADME assays: kinetic solubility, human microsomal metabolic stability, permeability and efflux, and matrix stability.

Parameters ICL-CCIC-0019 CK14 CK146 CK145 CK147 Kinetic Solubility Solubility (µM) 161 154 158 178 186 Half-life (min) >100 >100 >100 7.4 >100 Clint (µL/min/mg) <14 <14 <14 188 <14 Human Microsomal Predicted in vivo clearance <10 <10 <10 19 <10 Metabolic Stability 1 (mL/min/kg) PCT_LBF % <45 <45 <45 92 <45 Remaining % 92 101 127 2 105 Papp A-B (×10−6 cm/s) <0.1 <0.1 <0.1 <0.1 <0.1 Permeability and Papp B-A (×10−6 cm/s) 0.9 8.7 0.3 0.3 <0.1 Efflux Efflux ratio (Papp B-A/Papp A-B) >8.6 >87 >2.6 >2.8 No data 1 FaSSGF stability Half-life (h) >10 >10 >10 9.2 >10 Matrix Stability Remaining % 117 95 92 74 97 1 Poorly permeable compound which prevented the calculation of an efflux ratio.

3.6. Lipid Kinase Screening The selectivity of ICL-CCIC-0019, CK14, CK145, CK146 and CK147 against 15 human lipid kinases was assessed using an ADP-GloTM Kinase Assay. This assay measured generated ADP as a function of the kinase activity via a luciferase reaction (Figure7). Four of the CHKA inhibitors displayed good inhibitory activities against CHKA (53–77%) while not affecting CHKB at the tested concentration (10 µM), except for CK147. The tetradecyl linker exhibited superior CHKA affinity compared to the dodecyl linker, which supports our previous structure-activity relationship (SAR) data [26]. CK14 was the most active compound and inhibited 77% of CHKA activity. CK146 exhibited higher CHKA activity compared to ICL-CCIC-0019 (69% and 53%, respectively). Intriguingly, CK146 and CK145 Pharmaceutics 2021, 13, x FOR PEER REVIEW 11 of 17 displayed effective inhibition against phosphatidylinositol 4-kinase beta (PIK4CB) with 47% and 51% inhibition respectively. CK147 was inactive in the test panel.

− − − − − − − − −

− − − − − − − − − − − − −

− − − −

− − − − − − − −

FigureFigure 7. 7.The The heatmap heatmap of lipid of lipid kinase kinase selectivity selectivity screening screening against 15against lipid kinases15 lipid using kinases ICL-CCIC- using ICL- 0019,CCIC-0019, CK14, CK145, CK14, CK146 CK145, and CK146 CK147. and The CK147. inhibition The % inhibi of CHKAtion and % of PIK4CB CHKA are and boxed PIK4CB in white are boxed in andwhite dash-line and dash-line rectangles rectangles respectively. respectively. The data in The percentage data in percentage represent the represent mean kinase the activitymean kinase inhibitedactivity byinhibited drug molecules. by drug Tested molecules. concentration Tested ofconc theentration CHKA inhibitors of the CHKA in the assay:inhibitors 10 µM. in the assay: 10 μM.

3.7. [18F]D4-FCH Uptake In Vitro Study In C4-2B, a PSMA expressing cell line, CK14, CK145 and CK146 inhibited uptake of [18F]D4-FCH in a dose-dependent manner (Figure 8). At the dose of 1 μM, the compounds inhibited radio tracer uptake by approximately half (mean inhibition% by CK14, CK145 and CK146: 46 ± 1%). CK14 exhibited the best inhibitory potency in this study, inhibiting radiotracer uptake by 93 ± 5% when a higher dose was used (10 μM). In HCT-116 cells (PSMA negative), a similar inhibition trend by test compounds (10 μM) was observed (Figure 8). The average radiotracer uptake inhibition by 10 μM CK147 was 59 ± 7% across C4-2B and HCT-116 cell lines.

Figure 8. Uptake % of [18F]D4-FCH (0.74 MBq) in C4-2B and HCT-116 cells following 1-h incubation in the absence (Control; DMSO) or presence of CHKA inhibitors at 1 or 10 μM (n = 6; mean ± SD); p values via unpaired two-tailed t-test: *** p = 0.0001, **** p < 0.0001.

4. Discussion Novel asymmetrical CHKA inhibitor CK146 was developed with the aim of main- taining the activity of ICL-CCIC-0019 pharmacophore but with the addition of a pipera- zine “reactive handle” (Figure 9), to allow further structural modifications. Previously de- scribed SAR of symmetrical CHKA inhibitors highlighted that the length of alkyl linker chain between the DMAP moieties impacted the potency of these molecules. A 14-carbon

Pharmaceutics 2021, 13, x FOR PEER REVIEW 11 of 17

− − − − − − − − −

− − − − − − − − − − − − −

− − − −

− − − − − − − −

Figure 7. The heatmap of lipid kinase selectivity screening against 15 lipid kinases using ICL- CCIC-0019, CK14, CK145, CK146 and CK147. The inhibition % of CHKA and PIK4CB are boxed in white and dash-line rectangles respectively. The data in percentage represent the mean kinase activity inhibited by drug molecules. Tested concentration of the CHKA inhibitors in the assay: 10 μM.

Pharmaceutics 2021, 13, 1078 11 of 17 3.7. [18F]D4-FCH Uptake In Vitro Study In C4-2B, a PSMA expressing cell line, CK14, CK145 and CK146 inhibited uptake of 3.7.[18 [F]D4-FCH18F]D4-FCH Uptake in a dose-dependent In Vitro Study manner (Figure 8). At the dose of 1 μM, the compounds inhibitedIn C4-2B, radio a PSMA tracer expressing uptake cell line, by CK14, approximat CK145 andely CK146 half inhibited (mean uptakeinhibition% of by CK14, CK145 [18F]D4-FCH in a dose-dependent manner (Figure8). At the dose of 1 µM, the compounds inhibitedand CK146: radio tracer 46 ± uptake 1%). byCK14 approximately exhibited half the (mean best inhibition% inhibitory by CK14,potency CK145 in this study, inhibiting andradiotracer CK146: 46 ± uptake1%). CK14 by exhibited 93 ± 5% the bestwhen inhibitory a higher potency dose in this was study, used inhibiting (10 μM). In HCT-116 cells radiotracer(PSMA uptakenegative), by 93 ±a 5%similar when ainhibition higher dose wastrend used by (10 testµM). compounds In HCT-116 cells (10 μM) was observed (PSMA negative), a similar inhibition trend by test compounds (10 µM) was observed (Figure(Figure8). The 8). average The average radiotracer radiotracer uptake inhibition uptake by 10 inhibitionµM CK147 was by 5910± μ7%M across CK147 was 59 ± 7% across C4-2BC4-2B and and HCT-116 HCT-116 cell lines. cell lines.

FigureFigure 8. Uptake 8. Uptake % of [18 %F]D4-FCH of [18F]D4-FCH (0.74 MBq) in (0.74 C4-2B MBq) and HCT-116 in C4-2B cells and following HC 1-hT-116 incubation cells following 1-h incubation inin the the absence absence (Control; (Control; DMSO) or DMSO) presence or of CHKApresence inhibitors of CHKA at 1 or 10inhibitorsµM(n = 6; meanat 1 or± SD);10 μM (n = 6; mean ± SD); p p valuesvalues via via unpaired unpaired two-tailed two-tailedt-test: *** pt-test:= 0.0001, *** **** p =p <0.0001, 0.0001. **** p < 0.0001. 4. Discussion 4. NovelDiscussion asymmetrical CHKA inhibitor CK146 was developed with the aim of maintain- ing the activityNovel of asymmetrical ICL-CCIC-0019 pharmacophoreCHKA inhibitor but with CK146 the addition was ofdeveloped a piperazine with the aim of main- “reactive handle” (Figure9), to allow further structural modifications. Previously described SARtaining of symmetrical the activity CHKA of inhibitors ICL-CCIC-0019 highlighted pharmacophore that the length of alkyl but linker with chain the addition of a pipera- betweenzine “reactive the DMAP moietieshandle” impacted (Figure the 9), potency to allow of these further molecules. structural A 14-carbon modifications. alkyl Previously de- linkerscribed was thusSAR incorporated of symmetrical into the structureCHKA ofinhibitors CK146 in an highlighted attempt to maintain that the the length of alkyl linker high potency of CK14 (IC50 against recombinant CHKA2: ICL-CCIC-0019: 270 nM; CK14: 150chain nM) [ 26between]. Compound the CK146 DMAP was moieties successfully impacted synthesized th ine 4 potency steps in an overallof these yield molecules. A 14-carbon of 54%. Despite the structural deviation of CK146 from ICL-CCIC-0019, good inhibitory activity against CHKA was maintained, essential for the successful development of a CHKA inhibitor with tuneable biological properties by further structural elaboration in prodrug and drug delivery strategies (Figure9).

4.1. Prodrug Strategy The upregulation of HDAC and CTSL enzymes in cancer has previously been ex- ploited in a prodrug strategy whereby an ε-acetylated lysine linkage was selectively cleaved to release an active drug in a stepwise manner (Figure9a) [ 47]. This strategy was adopted in this work and compound CK145, containing the ε-acetylated lysine linkage, was synthe- sized and evaluated as a prodrug of CK146. High expression of HDAC I class, including HDAC 1, 2, 3 and 8 subtypes, was thought to be necessary for converting CK145 into its active form. HDAC I subtype 1 may contribute the most to the first deacetylation step, activating the complete conversion of CK145 [47]. The enzymatic conversion of CK145 was Pharmaceutics 2021, 13, 1078 12 of 17

Pharmaceutics 2021, 13, x FOR PEER REVIEW 12 of 17

analyzed by HPLC after incubation with HCT-116 cells. Despite HCT-116 cells showing high HDAC1 expression, CK146 and corresponding intermediates were not detected, indi- catingalkyl thatlinker the was intracellular thus incorporated conversion into of CK145the structure into CK146 of CK146 was notin an initiated; attempt this to maintain led to a differencethe high inpotency GI50 between of CK14 the (IC active50 against scaffold recombinant (CK146) versus CHKA prodrug2: ICL-CCIC-0019: (CK145) in HepG2270 nM; andCK14: Caco-2 150 nM) cells. [26]. HPLC Compound analysis CK146 of cell lysateswas successfully and SRB assaysynthesized indicated in 4 thatsteps these in an com- over- poundsall yield can of be54%. taken Despite up into the cells, structural likely deviation via active of internalization CK146 from ICL-CCIC-0019, by choline transporters, good in- despitehibitory their activity low-permeability against CHKA revealed was maintained, in the ADME essential studies. for the Rapid successful decomposition development of CK145of a CHKA was observed inhibitor in with the tuneable FaSSGF assaybiological and metabolicproperties stability by further assay structural due to Bocelaboration group deprotectionin prodrug and at low drug pH delivery (1.6) and strategies peptide degradation(Figure 9). mediated by liver microsomes.

FigureFigure 9. 9. DesignDesign of two drug drug development development strategies strategies based based on on CK146. CK146. (a) (εa-(Ac)) ε-(Ac) Lys Lysprodrug prodrug strat- egy; (b) PSMA-targeted drug delivery strategy. strategy; (b) PSMA-targeted drug delivery strategy.

4.1. ToProdrug assess Strategy inhibitory activity against CHKA, compounds were screened against a panelThe of 15 upregulation human lipid of kinases, HDAC where and CTSL CK14 enzymes displayed in the cancer best has CHKA previously activity been (CK14: ex- 77%ploited inhibition in a prodrug of CHKA); strategy CK146 whereby displayed an a betterε-acetylated CHKA lysine activity linkage than ICL-CCIC-0019 was selectively (ICL-CCIC-0019:cleaved to release 53% an inhibition active drug of CHKA;in a stepwi CK146:se manner 69% inhibition (Figure 9a) of CHKA).[47]. This These strategy results was indicatedadopted thein this advantage work and of compound using a 14-carbon CK145, alkylcontaining linker the to achieveε-acetylated effective lysine potency linkage, againstwas synthesized CHKA, which and is evaluated in agreement as a withprodrug the SARof CK146. of linker High length expression documented of HDAC by Trousil I class, etincluding al. (IC50 HDACagainst 1, recombinant 2, 3 and 8 subtypes, CHKA2: was ICL-CCIC-0019: thought to be necessary 270 nM; CK14:for converting 150 nM) CK145 [26]. CK145into its offered active similarform. HDAC CHKA I inhibitionsubtype 1 may to CK146 contribute (63% andthe most 69%, to respectively), the first deacetylation and this suggestedstep, activating that the the prodrug complete strategy, conversion whereby of aCK145 bulky [47].ε-(Ac)Lys The enzymatic structural modificationconversion of hadCK145 been was made, analyzed was not by ableHPLC to maskafter incubation the intrinsic with activity HCT-116 of CK146. cells. Despite Another HCT-116 interesting cells observationshowing high in this HDAC1 study wasexpression, the additional CK146kinase and corresponding activity towards intermediates PIK4CB (IC50 were: ~10 notµM de-), atected, potential indicating antimalarial that the and intracellular antiviral target, conver elicitedsion of byCK145 the modificationinto CK146 was of ournot initiated; CHKA inhibitorsthis led to with a difference a piperazine in GI50 moietybetween [ 48the,49 active]. In scaffold this regard, (CK146) several versus CHKA prodrug inhibitors (CK145) havein HepG2 been reported and Caco-2 as capable cells. HPLC of inhibiting analysisthe of cell growth lysates of parasiteand SRB species assay indicatedP. falciparum that (P.f.these) through compounds multiple can mechanisms, be taken up includinginto cells, thelikely inhibition via active of P.f. internalizationcholine kinase by and cholineP.f. ethanolaminetransporters, kinase despite [15 their,50]. low-permeability CK146 was less selective revealed to in choline the ADME kinase. studies. Kinase Rapid promiscuity decom- canposition lower of compound CK145 was activity observed towards in the the FaSSGF desired assay target and and metabolic induce adverse stability effects, assay due both to ofBoc which group are deprotection undesirable characteristicsat low pH (1.6) for and cancer peptide therapeutics; degradation however, mediated it isby noteworthy liver micro- thatsomes. the structure (CK146) presented here may have potential to be further modified into anti-parasite or antiviral drugs with a dual target-hitting PIK4CB and CHKA alike. This To assess inhibitory activity against CHKA, compounds were screened against a may warrant further investigation by researchers in the field, perhaps further supported panel of 15 human lipid kinases, where CK14 displayed the best CHKA activity (CK14: by in silico docking studies against P. falciparum CHKA (pdb code: 6YXS) [51,52]. 77% inhibition of CHKA); CK146 displayed a better CHKA activity than ICL-CCIC-0019 A 18F-radiolabeled choline analogue [18F]D4-FCH was utilized in this work to predict (ICL-CCIC-0019: 53% inhibition of CHKA; CK146: 69% inhibition of CHKA). These results the in vitro choline uptake inhibition by compounds of interest. Parallel to the lipid kinase indicated the advantage of using a 14-carbon alkyl linker to achieve effective potency against CHKA, which is in agreement with the SAR of linker length documented by Trou- sil et al. (IC50 against recombinant CHKA2: ICL-CCIC-0019: 270 nM; CK14: 150 nM) [26].

Pharmaceutics 2021, 13, 1078 13 of 17

screening results, CK145 inhibited the uptake of [18F]D4-FCH in a similar trend to CK146, which suggested that the intact CK145 harbored CHKA activity.

4.2. Drug Delivery Strategy A small-molecule peptide (Glu-Urea-Lys) with high affinity towards PSMA was conjugated to CK146 via a non-degradable linker, which gave rise to CK147, a proposed ligand-drug conjugate (LDC) for targeted PCa treatment (Figure9b) [ 53]. CK147 was expected to bind to the PSMA receptor and internalize, followed by intracellular lysosomal degradation to release CK146; therefore, several PSMA-expressing prostate cell lines and PSMA-negative prostate cell lines were used in the following evaluation of the strategy [54]. The conjugate stability of CK147 in serum-containing medium was firstly investigated by HPLC over a period of 72 h which is in line with the incubation time used in the SRB proliferation assay (60% intact parent at 72 h). The antiproliferative activity (GI50) of CK147, in 7 prostate cell lines with differential PSMA expression and the HCT-116 colon cell line, was unrelated to PSMA expression; furthermore, all the tested cell lines were not sensitive to CK147. The observed poor antiproliferative activity of CK147 relative to CK146 implied that the two mechanisms driving the cellular uptake of CK147, (1) PSMA-driven internalization and (2) choline transporter-driven internalization, were not as efficient as expected. This led to subsequent experiments to investigate cellular uptake efficiency and in vitro metabolism by HPLC analysis of cell lysates and [18F]D4-FCH competitive study in the whole cells. At physiological pH, CK147 contains two positive charges from the quaternary am- monium moieties, and three negative charges from the carboxylates of the Glu-Urea-Lys peptide. Although not zwitterionic due to the charge imbalance, we propose a putative mechanism where the positive quaternary ammonium charges may be masked by inter- or intramolecular electrostatic interactions which neutralize the charges and form unfavorable structural configurations capable of inhibiting both PSMA-binding and CHKA activity (Figure 10)[55]. The inactivity of CK147 observed in kinase screening was hypothesized to be related to the electrostatic neutralization; likely due to ionization of three carboxylate anions and two quaternary ammonium moieties at physiological pH. This hypothesis can also be supported by the significant increase in cellular uptake observed in the in vitro uptake study with CK148, the (OtBu)3-protected form of CK147. Electrostatic interactions may also explain the concentration reduction of CK147 observed in the stability study whilst no chemical decomposition was detected. The compound may have formed com- Pharmaceutics 2021, 13, x FOR PEER REVIEWplexes with over the time and then have been removed in the next protein14 of 17

precipitation step before MS analysis.

Figure 10.10. ProposedProposed ( A(A) intramolecular) intramolecular and and (B )(B intermolecular) intermolecular electrostatic electrostatic interactions interactions (blue (blue dashed dashed line) forline) CK147 for CK147 which which inhibit activity towards the PSMA receptor and CHKA. inhibit activity towards the PSMA receptor and CHKA. 5. Conclusions A novel derivative of ICL-CCIC-0019 bearing a piperazine handle (CK146) was suc- cessfully synthesized and retained biological activity towards CHKA; our work opens up the possibility for further structural elaboration of CK146, to explore the chemical space around this moiety and build up SAR to modulate biological and pharmacokinetic prop- erties. Interestingly, the introduction of the piperazine structure to the scaffold seems to offer CHKA inhibitors additional activities towards PIK4CB, a potential antimalarial and antiviral target. In this work, we presented the successful transformation of CK146 into two drug development programs to produce targeted cancer therapies via prodrug strat- egy (CK145) and drug delivery strategy (CK147), although conversion to active compo- nents or targeted delivery was not achieved. Regardless, we have demonstrated the po- tential for modulating CHKA activity via piperazine functionalization and work in our laboratory will continue, including molecular modelling studies to understand the inter- actions between these molecules and the of interest, to develop a suitable candidate for clinical translation.

Supplementary Materials: The following are available online at www.mdpi.com/xxx/s1, Figures S1–S30: NMR and MS data of ICL-CCIC-0019, CK14 and compound 1–8, Figure S31: The purity check of CK147 by HPLC, Figure S32: The HPLC chromatograms of HCT-116 cell extracts after 1 h and 2 h incubation with/without CK145, Figure S33: MS characterization of the HCT-116 cell ex- tracts, Figure S34: The HPLC chromatograms of CK146 detection in medium after treatment, Figure S35: Cellular uptake study of ICL-CCIC-0019, CK146, CK147 and CK148 in C4-2B cells using the medium with or without FCS, Figure S36–45: MS characterization of the medium after treatment with ICL-CCIC-0019, CK146, CK147 and CK148 in C4-2B cells using the medium with or without FCS, Table S1: mRNA expression of HCT-116, HepG2, A549 and Caco-2, extracted from Cancer Cell Line Encyclopedia RNA sequencing database, Table S2: Inhibitory activity against HCT-116, A549, HepG2 and Caco-2 cancer cell lines (GI50) with ICL-CCIC-0019, CK146 and CK145, Table S3: Inhib- itory activity against human 22Rv1, C4-2B, LNCap, R1AD1, R1-D567, PC3, PNT1A and HCT-116 cell lines (GI50) with ICL-CCIC-0019, CK146 and CK147. Author Contributions: Conceptualization and methodology, N.W., D.B., L.A., E.O.A.; synthesis and data acquisition, N.W., M.B., C.B., H.L.; writing—original draft preparation, N.W.; writing—review and editing, D.B., L.A., E.O.A.; supervision, D.B., L.A., E.O.A.; funding acquisition, E.O.A.; All au- thors have read and agreed to the published version of the manuscript. Funding: PhD scholarship for N.W. was funded by the scholarship program of Imperial College London and China Scholarship Council. E.O.A. acknowledges funding from the Medical Research Council Grant (MR/M015858/1), Imperial College NIHR Biomedical Research Centre award

Pharmaceutics 2021, 13, 1078 14 of 17

5. Conclusions A novel derivative of ICL-CCIC-0019 bearing a piperazine handle (CK146) was suc- cessfully synthesized and retained biological activity towards CHKA; our work opens up the possibility for further structural elaboration of CK146, to explore the chemical space around this moiety and build up SAR to modulate biological and pharmacokinetic properties. Interestingly, the introduction of the piperazine structure to the scaffold seems to offer CHKA inhibitors additional activities towards PIK4CB, a potential antimalarial and antiviral target. In this work, we presented the successful transformation of CK146 into two drug development programs to produce targeted cancer therapies via prodrug strategy (CK145) and drug delivery strategy (CK147), although conversion to active components or targeted delivery was not achieved. Regardless, we have demonstrated the potential for modulating CHKA activity via piperazine functionalization and work in our laboratory will continue, including molecular modelling studies to understand the interactions be- tween these molecules and the binding site of interest, to develop a suitable candidate for clinical translation.

Supplementary Materials: The following are available online at https://www.mdpi.com/article/ 10.3390/pharmaceutics13071078/s1, Figures S1–S30: NMR and MS data of ICL-CCIC-0019, CK14 and compound 1–8, Figure S31: The purity check of CK147 by HPLC, Figure S32: The HPLC chromatograms of HCT-116 cell extracts after 1 h and 2 h incubation with/without CK145, Figure S33: MS characterization of the HCT-116 cell extracts, Figure S34: The HPLC chromatograms of CK146 detection in medium after treatment, Figure S35: Cellular uptake study of ICL-CCIC-0019, CK146, CK147 and CK148 in C4-2B cells using the medium with or without FCS, Figure S36–S45: MS characterization of the medium after treatment with ICL-CCIC-0019, CK146, CK147 and CK148 in C4-2B cells using the medium with or without FCS, Table S1: mRNA expression of HCT-116, HepG2, A549 and Caco-2, extracted from Cancer Cell Line Encyclopedia RNA sequencing database, Table S2: Inhibitory activity against HCT-116, A549, HepG2 and Caco-2 cancer cell lines (GI50) with ICL-CCIC-0019, CK146 and CK145, Table S3: Inhibitory activity against human 22Rv1, C4-2B, LNCap, R1AD1, R1-D567, PC3, PNT1A and HCT-116 cell lines (GI50) with ICL-CCIC-0019, CK146 and CK147. Author Contributions: Conceptualization and methodology, N.W., D.B., L.A., E.O.A.; synthesis and data acquisition, N.W., M.B., C.B., H.L.; writing—original draft preparation, N.W.; writing—review and editing, D.B., L.A., E.O.A.; supervision, D.B., L.A., E.O.A.; funding acquisition, E.O.A.; All authors have read and agreed to the published version of the manuscript. Funding: PhD scholarship for N.W. was funded by the scholarship program of Imperial Col- lege London and China Scholarship Council. E.O.A. acknowledges funding from the Medical Research Council Grant (MR/M015858/1), Imperial College NIHR Biomedical Research Centre award (WSCC_P62585), Imperial College Experimental Cancer Medicines award (C1312/A25149) and National Cancer Imaging Translational Accelerator (C2536/A28680). Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. Data Availability Statement: Data available in a publicly accessible repository. The mRNA expres- sion data presented in this study are openly available at Cancer Cell Line Encyclopedia (CCLE) Consortium, and Genomics of Drug Sensitivity in Cancer Consortium. 2015. Pharmacogenomic Agreement between Two Cancer Cell Line Data Sets. Nature 528 (7580):84–87. https://doi.org/ 10.1038/nature15736. The RNA-sequencing data utilized by CCLE are openly available on https: //depmap.org/portal/download/. Acknowledgments: The authors would like to thank Ala Amgheib for interesting scientific discussion. Conflicts of Interest: The authors declare no conflict of interest.

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