X-Ray Crystallography of Epacadostat in Adduct with Carbonic Anhydrase IX
Andrea Angeli, Thomas S. Peat, Silvia Selleri, Abdulmalik Saleh Alfawaz Altamimi, Claudiu T. Supuran, Fabrizio Carta
PII: S0045-2068(20)30171-1
DOI: https://doi.org/10.1016/j.bioorg.2020.103669
Reference: YBIOO 103669

To appear in: Bioorganic Chemistry

Received Date: 22 January 2020
Revised Date: 11 February 2020
Accepted Date: 13 February 2020

Please cite this article as: A. Angeli, T.S. Peat, S. Selleri, A. Saleh Alfawaz Altamimi, C.T. Supuran, F. Carta, X- Ray Crystallography of Epacadostat in Adduct with Carbonic Anhydrase IX, Bioorganic Chemistry (2020), doi: https://doi.org/10.1016/j.bioorg.2020.103669

This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

© 2020 Published by Elsevier Inc.

X- Ray Crystallography of Epacadostat in Adduct with Carbonic Anhydrase IX

Andrea Angeli,a,b Thomas S. Peat,c Silvia Selleri,a Abdulmalik Saleh Alfawaz Altamimi,d Claudiu
T. Supurana* and Fabrizio Cartaa*
a University of Florence, NEUROFARBA Dept., Pharmaceutical and Nutraceutical section, Via Ugo Schiff 6, 50019 Sesto Fiorentino, Florence, Italy
b Centre of Advanced Research in Bionanoconjugates and Biopolymers Department,“Petru Poni” Institute of Macromolecular Chemistry, Iasi, Romania
c CSIRO, 343 Royal Parade, Parkville, Victoria 3052, Australia
d Department of Pharmaceutical Chemistry, College of Pharmacy, Prince Sattam Bin Abdulaziz University, P.O. Box 173, Alkharj 11942, Saudi Arabia.

Abstract. Epacadostat (EPA), a new and promising anti-cancer small molecule is firmly established as selective inhibitor of the enzyme indoleamine 2,3-dioxygenase 1 (IDO1). The X-Ray structure of the human CA IX mimic in complex with EPA is investigated here for the first time and compared to previously reported EPA-CA II adduct. The structural information obtained are all in agreement with the in vitro kinetic data which accounted for a selective inhibition of the CA IX over the CA II isoform.

Keywords: Carbonic Anhydrases IX; Epacadostat; Metalloenzymes; Check point inhibitor; Carbonic Anhydrase Inhibitor

Corresponding authors. e-mail: [email protected]; [email protected]


The first-in-class, orally active inhibitor of the immune checkpoint enzyme indoleamine 2,3- dioxygenase 1 (IDO-1, EC Epacadostat (EPA) currently is under investigation in various clinical trials for the management of tumors [1]. IDO-1 catalyses the initial and rate-limiting step of the Tryptophan (Trp) catabolism through the kynurenine pathway. [2,3] Plenty of biological processes such as aging, immunity as well as diseases of the neurodegenerative type or cancers are clearly associated to its modulation. [4,5] Recently, new biological aspects of the IDO-1 have been revealed such as its enzymatic regulation, [6] the nitrite reductase activity in hypoxic tissues, [7] and its involvement in specific red-ox signalling pathways. [8] All such aspects clarify the intricate metabolic control achieved in immune responses by modulating IDO-1. These new findings further refine the physiological role of IDO-1 within various pathways and contribute to better understanding such processes in perspective to achieve clinical benefits.
The presence of the zinc (II) binder moiety of the sulfamide type within the structure of EPA in Figure 1 lead some of us to investigate whether such compound may act as inhibitor of the human expressed Carbonic Anhydrase (CAs, EC enzymes too. [9]
O H Br



Epacadostat (EPA)

Figure 1: Chemical structure of Epacadostat.

Besides the mere structural consideration, our hypothesis was proposed in light of the CA IX (and marginally XII) validation as druggable target for the management of hypoxic tumors. [10-14] The inhibition of such isoform necessarily determines alterations to the tumoral pH microenvironment and repression of the cancer progression. [10-13] The in vitro kinetic results in

Table 1 supported our hypothesis, and quite interestingly the tumor associated CAs IX and XII were strongly inhibited by EPA with KI values of 3.0 and 6.2 nM respectively. In addition EPA showed exceptional selectivity against these isoforms when compared to the remaining human expressed ones. [9]

Table 1: Inhibition data of hCA I, II, IX and XII isoforms with EPA and AAZ by a stopped flow CO2 hydrase assay. [9, 15] Selectivity ratios for the inhibition of the human isoform hCA II versus hCA IX and XII.

KI (nM)* Selectivity Ratioa

EPA 8262 917.4 3.0 6.2 305.8 147.9
AAZ 250.0 12.1 25.8 5.7 0.47 2.12
* Mean from 3 different assays, by a stopped flow technique (errors were in the range of  5-10 % of the reported values). a Selectivity as determined by the ratio of KI for hCA II relative to hCA IX and hCA XII.

Although our observations were very preliminary, they allowed us to reasonably speculate that the antitumor activity of EPA may be exerted both by disruptions of the cellular Trp catabolism and tumor pH microenvironment. [9] A connection between tissue acidification and immune evasion mechanisms was reported in 2017. [16] Shortly after our report the group of Dedhar et al. supported our study by demonstrating that the inhibition of the CA IX activity by means of the small molecule SLC-0111, significantly enhanced the immune checkpoint blockade and thus leading to improvement of the immune activity. [17]
As a follow up of our preliminary work [9], here we report for the first time EPA in complex within the CA IX mimic active site and we decipher its binding mode of by means of X-ray crystallographic experiments at atomic level resolution.

Protein X-ray crystal structures.

We engineered the CA II protein to mimic the CA IX isoform active site. This approach has been extensively used in order to facilitate structural biology investigations on proteins recalcitrant to crystallization techniques. [18] Specifically the 10 aminoacidic substitutions within the CA II sequence were: A65S, N67Q, E69T, I91L, F131V, G132D, V135L, K170E, L204A and C206G. [19]
First inspection of the |Fo−Fc| electron maps showed clear densities for the sulfamide moiety of EPA adjacent to the zinc atom, of the oxazole ring and the connecting alkyl chain after the initial rounds of refinement (Figure 2).

Figure 2: hCA IX mimic−EPA complex (PDB: 6VKG). The molecular surface of hydrophobic and hydrophilic halves of the CA IX mimic are coloured in red and blue, respectively. Epacadostat is showed as σA-weighted |Fo−Fc| density map at 2.0 σ.

The sulfamide moiety interacted directly with the zinc (II) ion and showed the typical hydrogen bond coordination cluster which involves the Thr199 residue and thus further stabilizing the complex as outlined in Figure 3A.

Figure 3: A) Active site region of the hCA IX mimic−EPA complex (PDB: 6VKG). Hydrogen bonds (red), van der Waals interactions (blue) are also shown. B) Structural superposition between EPA- hCAII (green) and EPA-hCA IX mimic (magenta) bound to the active site of protein. Residues of hCA IX mimic are coloured in pink.

The tail ending benzene ring of EPA showed poorer density when compared to the other sections. It is worth considering that multiple potential conformations of this moiety may take place thus explaining any lack of density. The benzene ring moiety sits in a hydrophobic pocket made up of Val131, Asp131, Leu135 and Pro202 at 3.5−3.8 Å distances. The nitrogen of the hydroxylamine group is within hydrogen bonding distance (2.9 Å) to the Gln92 side chain and the hydroxyl of the same moiety is potential hydrogen bond donor to both hetero atoms of Gln67. (Figure 3A).
Taking into account the high selectivity of EPA in inhibiting the CA IX over the other isoforms [9] we performed a structural superposition between EPA-CA II (already reported by some of us [9]) and the EPA-CAIX mimic complexes (Figure 3B). The major difference is the orientation of the inhibitor aromatic rings within the active sites. The V131F mutation in CA II forced the EPA phenyl ring down

to the hydrophobic cavity in close proximity to the V135. In addition, the T-shaped π stacking conformation between F131 and the aromatic moiety ensured stabilization. However, the EPA aromatic ring allocation within the CA II is less favourable when compared to the CA IX since better and stronger hydrophobic bonds are established in the latter (i.e. V131 and L135 in Figure 3A and 3B).
Superpositions in Figure 3A revealed that the EPA sp3 carbon acts as a hinge since no sensible variations at this point were observed as result of the ligand conformational changes. When the V131F mutation is considered, the phenyl ring movement determines through this carbon the shifting of the oxazole ring in opposite direction and towards the N67 residue.


The new and promising anti-cancer small molecule IDO-1 and CA IX/XII inhibitor EPA was investigated in adduct with the CA IX mimic enzyme by means of X-ray crystallography to determine its binding mode. In particular the F131V mutation in CA IX allowed the inhibitor to assume a more favourable energetic conformation and to engage a series of hydrophobic bonds. The high stability of the complex was in agreement with the potent kinetic inhibition data against the CA IX isoform previously reported by us [9] and will further contribute to elucidate the mechanisms underpinning its anticancer activity.

Experimental Part General
Solvents and all the reagents were purchased from Sigma- Aldrich (India), Alfa Aesar (India) and TCI (India).
Protein X-ray Crystallography

The CA-IX mimic protein was crystallized in sitting drop plates using 200 nL of protein at 5.9 mg/mL plus 200 nL of reservoir solution over 50 μL reservoirs for the CA-IX mimic. The crystallization plates were incubated at 20 °C, and large plate-like crystals were found in optimized ammonium sulfate conditions (2.6−2.8 M ammonium sulfate, 100 mM Tris, pH 8.0 to 9.0). Crystals were harvested using nylon loops with the addition of glycerol as a cryoprotectant (20% final concentration). Data were collected at the Australian Synchrotron MX2 beamline to obtain 360° of data. The data were indexed using XDS [20] and scaled using Aimless.[21] Molecular replacement was done using Phaser26 [22] using 5G03 as the initial starting model for the CA IX-mimic; the protein chain was manually built into the structure using Coot [23] and refined using Refmac. [24] The initial placement of Epacadostat and the dictionary (cif) file were generated with eLBOW [25] from the Phenix software suite and further refined using Refmac. [24] The structure and structure factors were deposited in the PDB with accession codes 6VKG.


We thank the Australian Synchrotron and the beamline scientists for their help with data collection. This research was undertaken in part using the MX2 beamline at the Australian Synchrotron and made use of the ACRF detector. We also thank the C3 Crystallization Centre for all crystallization experiments. This work was supported by a grant of the Romanian Ministry of Research and Innovation, CNCS–UEFISCDI, project number PN-III-P4-ID-PCCF-2016–0050, within PNCDI II. This project was partially supported by the Deanship of Scientific Research at Prince Sattam bin Abdulaziz University under the research project number 2016/03/6824.

Conflict of Interests.

The authors state no conflict of interests.

Supplementary Material.

Supplementary data to this article can be found online at XXX


1. https://www.cancer.gov/about-cancer/treatment/clinical-trials/intervention/epacadostat (Last access 19/01/2020)
2. Koblish H.K., Hansbury M.J., Bowman K.J., Yang G., Neilan C.L., Haley P.J., Burn T.C., Waeltz P., Sparks R.B., Yue E.W., Combs A.P., Scherle P.A., Vaddi K., Fridman J.S. Hydroxyamidine inhibitors of indoleamine-2,3-dioxygenase potently suppress systemic tryptophan catabolism and the growth of IDO-expressing tumors. Mol. Cancer Ther., 2010, 9, 489-498.
3. Liu X., Shin N., Koblish H.K., Yang G., Wang Q., Wang K., Leffet L., Hansbury M.J., Thomas B., Rupar M., Waeltz P., Bowman K.J., Polam P., Sparks R.B., Yue E.W., Li Y., Wynn R., Fridman J.S., Burn T.C., Combs A.P., Newton R.C., Scherle P.A. Selective inhibition of IDO1 effectively regulates mediators of antitumor immunity. Blood, 2010, 115, 3520-3530.
4. Badawy AA. Kynurenine pathway of tryptophan metabolism: regulatory and functional aspects. Int J Tryptophan Res. 2017, 10, 1178646917691938.

5. Platten M., Nollen E.A.A., Röhrig U.F., Fallarino F., Opitz C.A. Tryptophan metabolism as a common therapeutic target in cancer, neurodegeneration and beyond. Nat. Rev. Drug Discovery 2019, 18, 379−401.
6. Nelp M.T., Kates P.A., Hunt J.T., Newitt J.A., Balog A., Maley D., Zhu X., Abell L., Allentoff A., Borzilleri R., Lewis H.A., Lin Z., Seitz S.P., Yan C., Groves J.T. Immune-modulating enzyme indoleamine 2,3-dioxygenase is effectively inhibited by targeting its apo-form. Proc. Natl. Acad. Sci. U.S.A. 2018, 115, 3249−3254.
7. Lim Y.J., Foo T.C., Yeung A.W.S., Tu X., Ma Y., Hawkins C.L., Witting P.K., Jameson G.N.L., Terentis A.C., Thomas S.R. Human indoleamine 2,3-dioxygenase 1 is an efficient mammalian nitrite reductase. Biochemistry, 2019, 58, 974−986.
8. Stanley C.P., Maghzal G.J., Ayer A., Talib J., Giltrap A.M., Shengule S., Wolhuter K., Wang Y., Chadha P., Suarna C., Prysyazhna O., Scotcher J., Dunn L.L., Prado F.M., Nguyen N., Odiba J.O., Baell J.B., Stasch J.-P., Yamamoto Y., Di Mascio P., Eaton P., Payne R.J., Stocker
R. Singlet molecular oxygen regulates vascular tone and blood pressure in inflammation.

Nature, 2019, 566, 548−552.

9. Angeli A., Ferraroni M., Nocentini A., Selleri S., Gratteri P., Supuran C.T., Carta F. Polypharmacology of epacadostat: a potent and selective inhibitor of the tumor associated carbonic anhydrases IX and XII. Chem Commun (Camb). 2019, 55, 5720-5723.
10. Supuran C.T., Alterio V., Di Fiore A., D’ Ambrosio K., Carta F., Monti S.M., De Simone G. Inhibition of carbonic anhydrase IX targets primary tumors, metastases, and cancer stem cells: Three for the price of one. Med. Res. Rev. 2018, 38, 1799-1836.
11. Pacchiano F., Carta F., McDonald P.C., Lou Y., Vullo D., Scozzafava A., Dedhar S., Supuran

C.T. Ureido-substituted benzenesulfonamides potently inhibit carbonic anhydrase IX and show antimetastatic activity in a model of breast cancer metastasis. J. Med. Chem. 2011, 54, 1896-1902.

12. Lou Y., McDonald P.C., Oloumi A., Chia S., Ostlund C., Ahmadi A., Kyle A., Auf dem Keller U., Leung S., Huntsman D., Clarke B., Sutherland B.W., Waterhouse D., Bally M., Roskelley C., Overall C.M., Minchinton A., Pacchiano F., Carta F., Scozzafava A., Touisni N., Winum J.Y., Supuran C.T., Dedhar S. Targeting tumor hypoxia: suppression of breast tumor growth and metastasis by novel carbonic anhydrase IX inhibitors. Cancer Res. 2011, 71, 3364-3376.
13. Pastorekova S., Gillies R.J. The role of carbonic anhydrase IX in cancer development: links to hypoxia, acidosis, and beyond. Cancer Metastasis Rev. 2019, 38, 65-77.
14. Chafe S.C., McDonald P.C., Saberi S., Nemirovsky O., Venkateswaran G., Burugu S., Gao D., Delaidelli A., Kyle A.H., Baker J.H.E., Gillespie J.A., Bashashati A., Minchinton A.I., Zhou Y., Shah S.P., Dedhar S. Targeting hypoxia-induced carbonic anhydrase IX enhances immune-checkpoint blockade locally and systemically. Cancer Immunol. Res. 2019, 7, 1064- 1078.
15. Khalifah R.G. The carbon dioxide hydration activity of carbonic anhydrase I. Stop flow kinetic studies on the native human isoenzymes B and C. J. Biol. Chem. 1971, 246, 2561- 2573.
16. Kachler K., Bailer M., Heim L., Schumacher F., Reichel M., Holzinger C.D., Trump S., Mittler S., Monti J., Trufa D.I., Rieker R.J., Hartmann A., Sirbu H., Kleuser B., Kornhuber J., Finotto S. Enhanced acid sphingomyelinase activity drives immune evasion and tumor growth in non-small cell lung carcinoma. Cancer Res. 2017, 77, 5963-5976.
17. McDonald P.C., Chafe S.C., Brown W.S., Saberi S., Swayampakula M., Venkateswaran G., Nemirovsky O., Gillespie J.A., Karasinska J.M., Kalloger S.E., Supuran C.T., Schaeffer D.F., Bashashati A., Shah S.P., Topham J.T., Yapp D.T., Li J., Renouf D.J., Stanger B.Z., Dedhar
S. Regulation of pH by carbonic anhydrase 9 mediates survival of pancreatic cancer cells with activated KRAS in response to hypoxia. Gastroenterology. 2019, 157, 823-837.

18. Pinard M.A., Boone C.D., Rife B.D., Supuran C.T., McKenna R. Structural study of interaction between brinzolamide and dorzolamide inhibition of human carbonic anhydrases. Bioorg. Med. Chem. 2013, 21, 7210−7215.
19. Mujumdar P., Teruya K., Tonissen K.F., Vullo D., Supuran C.T., Peat T.S., Poulsen S.A. An Unusual Natural Product Primary Sulfonamide: Synthesis, carbonic anhydrase inhibition, and protein X-ray structures of Psammaplin C. J. Med. Chem. 2016, 59, 5462-5470.
20. Kabsch W. XDS. Acta Crystallogr., Sect. D: Biol. Crystallogr. 2010, 66, 125−132.

21. Evans P.R. An introduction to data reduction: space-group determination, scaling and intensity statistics. Acta Crystallogr., Sect. D: Biol. Crystallogr. 2011, 67, 282−292.
22. McCoy A.J., Grosse-Kunstleve R.W., Adams P.D., Winn, M.D., Storoni L.C., Read R.J. Phaser crystallographic software. J. Appl. Crystallogr. 2007, 40, 658−674.
23. Emsley P., Lohkamp B., Scott W.G., Cowtan K. Features and development of Coot. Acta Crystallogr., Sect. D: Biol. Crystallogr. 2010, 66, 486−501.
24. Murshudov G.N., Skubák P., Lebedev A.A., Pannu N.S., Steiner R.A., Nicholls R.A., Winn M.D., Long F., Vagin A.A. REFMAC5 for the refinement of macromolecular crystal structures. Acta Crystallogr., Sect. D: Biol. Crystallogr. 2011, 67, 355−367.
25. Moriarty N.W., Grosse-Kunstleve R.W., Adams P.D. electronic Ligand Builder and Optimization Workbench (eLBOW): a tool for ligand coordinate and restraint generation. Acta Crystallogr., Sect. D: Biol. Crystallogr. 2009, 65, 1074-1080.

Declaration of interests

☒ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Table of Contents graphic (TOC)


• The X-ray crystallography of EPA-hCA IX mimic adduct is reported

• The binding mode of EPA within the hCA IX mimic is deciphered