Available online at www.sciencedirect.com

ScienceDirect Targeting carbonic anhydrase IX with small organic ligands Moreno Wichert and Nikolaus Krall1 Carbonic anhydrase IX (CAIX) is expressed in many solid tumors in response to hypoxia and plays an important role in tumor acid–base homeostasis under these conditions. It is also constitutively expressed in the majority of renal cell carcinoma. Its functional inhibition with small molecules has recently been shown to retard tumor growth in murine models of cancer, reduce metastasis and tumor stem cell expansion. Additionally, CAIX is a promising antigen for targeted drug delivery approaches. Initially validated with anti-CAIX antibodies, the tumor-homing capacity of high-affinity small-molecule ligands of CAIX has recently been demonstrated. Indeed, conjugates formed of CAIX ligands and potent cytotoxic drugs could eradicate CAIX-expressing solid tumors in mice. These results suggest that CAIX is a promising target for the development of novel therapies for the treatment of solid tumors. Address ETH Zurich, Department of Chemistry and Applied Biosciences, Institute of Pharmaceutical Sciences, 8093 Zurich, Switzerland Corresponding author: Krall, Nikolaus ([email protected], [email protected]) 1 Present address: CeMM Research Center for Molecular Medicine of the Austrian Academy of Sciences, Lazarettgasse 14, AKH BT 25.3, 1090 Vienna, Austria.

Current Opinion in Chemical Biology 2015, 26:48–54 This review comes from a themed issue on Next generation therapeutics Edited by Dario Neri and Jo¨rg Scheuermann

http://dx.doi.org/10.1016/j.cbpa.2015.02.005 1367-5931/# 2015 Elsevier Ltd. All rights reserved.

Introduction Carbonic anhydrases (CAs) are a large family of zinccontaining metalloenzymes that catalyze the reversible hydration of carbon dioxide to hydrogen carbonate and H+ (CO2 + H2O fi H+ + HCO3 ) [1]. In humans, 15 CA isoforms differing in tissue expression patterns, kinetic properties and subcellular localization are currently known [2]. Their physiological roles are typically associated with acid–base homeostasis and the transport of CO2 and hydrogen carbonate [3,4]. Current Opinion in Chemical Biology 2015, 26:48–54

Carbonic anhydrase IX (CAIX) is a 459 (including a signal peptide) amino acid transmembrane protein with an extracellular active site. Full-length CAIX consists of four domains: an N-terminal proteoglycan-like domain (PG), a CA catalytic domain (CA), a single-pass helical trans-membrane region and a short intracellular tail [5] (Figure 1a). In its native form it exists as a disulfide-linked dimer [6] and is expressed in a very limited number of healthy tissues (mainly in the gastrointestinal tract, on the bile duct epithelium and in the gall bladder) [7,8]. In many solid tumors, on the other hand, CAIX levels are strongly elevated in response to hypoxia (i.e., lack of oxygen supply for example due to limited blood perfusion [9]) or mutations in the von Hippel-Lindau tumor suppressor protein (pVHL) [7]. The latter is particularly frequent in renal cell carcinoma of the clear cell subtype (ccRCC) and >90% of ccRCCs are strongly positive for CAIX [10]. Functionally, CAIX plays an important role in tumor acid–base homeostasis under hypoxic conditions (Figure 1b). In the absence of O2, tumor cells shift their metabolism from a low rate of glycolysis followed by oxidative phosphorylation of pyruvate in the mitochondria towards a high rate of glycolysis with subsequent lactic acid fermentation in the cytosol [11–14]. The subsequent accumulation of acidic metabolites has to be counteracted by their export and extrusion of H+ from the cytosol to keep intracellular pH in a range of 7.2–7.4 permissive for growth [11,14–16]. CAIX is believed to act in concert with intracellular CAs that convert hydrogen carbonate and H+ to CO2 and H2O. CO2 can then freely diffuse across the membrane, where it is hydrated by CAIX to give carbonic acid resulting in the net export of H+ to the extracellular space [17,18,19]. Similarly, CAIX has been proposed to facilitate the diffusion of H+ through entire tumor tissues [17,18,19]. The export of H+ from cancer cells leads to a marked acidification of extracellular space (pH 6.5–7.0). A low extracellular pH in turn may promote extracellular matrix breakdown [20] and favor invasion, suppress immune surveillance [21] and promote multidrug resistance [22]. Overall, these effects can lead to a more aggressive and treatment resistant tumor phenotype. Indeed, expression of CAIX in response to hypoxia is often associated with poor prognosis in the clinic [23,24]. In this article, we would like to highlight how the function and expression of CAIX inside solid tumors can be www.sciencedirect.com

Targeting CAIX with small organic ligands Wichert and Krall 49

Figure 1

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HCO3– + H+ HCO3– Na+ Cl–

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Schematic representation of CAIX’s structure and its pH regulatory function in a tumor cell. (a) The zinc-containing transmembrane protein CAIX has an extracellular active site (grey). Full-length CAIX consists of four domains: an N-terminal proteoglycan-like domain (orange), a catalytic domain (grey), a single-pass helical trans-membrane region and a short intracellular tail (blue) [5]. In its native form it exists as a disulfide-linked dimer (PDB: 3IAI) [6]. (b) CAIX plays an important role in tumor acid–base homeostasis under hypoxic conditions. In the absence of O2, due to limited blood perfusion, tumor cells shift their metabolism from a low rate of glycolysis to a high rate of glycolysis with subsequent lactic acid fermentation in the cytosol [9,11–14]. The subsequent accumulation of acidic metabolites has to be counteracted by their export and extrusion of H+ to keep intracellular pH in a range permissive for growth [11,14–16]. Under such hypoxic conditions, CA9 and numerous other hypoxiaregulated genes are activated by the hypoxia-inducible factor 1 (HIF-1). The protein CAIX (blue) is believed to act in concert with other intracellular CAs (e.g. CAII, blue) that convert hydrogen carbonate and H+ to CO2 and H2O. CO2 can then freely diffuse across the membrane, where it is hydrated by CAIX to give carbonic acid resulting in the net export of H+ to the extracellular space. A multitude of other proteins are involved in this tightly connected interplay of pH regulation within tumor cells: anion exchangers; Na+/HCO co-transporters; Vacuolar-type H+-ATPase; Na+/ H+ exchangers; monocarboxylate transporters, which transport lactic acid and other monocarboxylates formed by the glycolytic degradation of glucose. Glucose transporters are also upregulated in most tumor cells. As a result, the intracellular tumor pH (pHi) is slightly alkaline (pH 7.2–7.4), whereas the extracellular pH (pHe) is slightly acidic (pH 6.5–7.0) [14].

exploited to develop new innovative therapeutics for the treatment of cancer. We will discuss progress in this field from the recent five years and give an outlook towards clinical translation.

Inhibition of CAIX with small molecules Given the important role of CAIX in tumor acid–base homeostasis in response to hypoxia, inhibition of CAIX with small molecules has been proposed as a novel anticancer strategy [12]. Silencing of CAIX together with CAXII in LS147T colorectal cancer xenografts using shRNA resulted in a significant reduction in tumor growth [19]. In the 4T1 orthotopic model of mouse breast cancer, depletion of CAIX led to tumor regression and a reduction of metastasis to the lung [25]. Dedhar, Supuran and coworkers then demonstrated that inhibition of CAIX with aromatic sulfonamide (CAI17, U-104, Figure 2) and coumarin derivatives (GC-204) could retard tumor growth in mice in vivo [26]. The coumarin CAIX inhibitors (GC-204 and GC-205, Figure 2) furthermore inhibited metastasis of 4T1 breast cancer cells to the lung [25]. Finally, GC-205 www.sciencedirect.com

and the sulfonamide U-104 was suggested to deplete cancer stem cells in breast cancer in mice [27]. The most widely studied class of CA inhibitors are the aromatic sulfonamides (e.g., U-104, Figure 2). The sulfonamide nitrogen atom reversibly coordinates the CA’s catalytic Zn2+ and blocks the active site [28]. Additionally, other chemotypes such as the aforementioned coumarin suicide inhibitors have been described [29]. A major challenge associated with ligand-based inhibition of CAIX still lies in the discovery of ligands with good selectivity for CAIX over other CAs with important roles in healthy tissues. There are currently 15 known CA isoforms in humans with often very high sequence homology [30]. Initial attempts to obtain selectivity for CAIX exploited the fact that many CAs are intracellular whereas the active site of CAIX is located extracellularly. CA binders were conjugated with bulky moieties to restrict their movement across cell membranes. Amino phenyl sulfonamide was for example reacted with fluorescein isothiocyanate (FITC) to produce a ligand with some selectivity for CAIX [31]. Neri Current Opinion in Chemical Biology 2015, 26:48–54

50 Next generation therapeutics

Figure 2

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Structures of potent CAIX inhibitors used in in vivo imaging or therapy studies. (a) Inhibitors of the phenyl sulfonamide type: CAI17: Ki (CAIX) = 24 nM. Selectivity for CAIX over CAI (x 54), CAII (x 1.9), CAXII (x 0.2) [28]; U-104: Ki (CAIX) = 45.1 nM. Selectivity for CAIX over CAI (x 113), CAII (x 214), CAXII (x 0.1) [28]. (b) Inhibitors of the acetazolamide (AAZ) type: AAZ: Ki (CAIX) = 25 nM. Selectivity for CAIX over CAI (x 10), CAII (x 0.5), CAXII (x 0.2) [28]. (c) Alternative chemotypes: GC-204: Ki (CAIX) = 9.2 nM. Selectivity for CAIX over CAI (x > 10,870), CAII (x > 10,870), CAXII (x 4.7) [26]; GC-205: Ki (CAIX) = 201 nM. Selectivity for CAIX over CAI (x > 498), CAII (x > 498), CAXII (x 0.9) [26]; Glyco-sulfamate conjugate: Ki (CAIX) = 2 nM. Selectivity for CAIX over CAI (x 1.200), CAII (x 3.200), CAXII (x 11) [33].

and co-workers conjugated the pan-CA inhibitor acetazolamide with a portable albumin binder isolated from a DNA-encoded chemical library to restrict the molecule’s movement across cell membranes [32] (Figure 2). The resulting small molecules exhibited a statistically significant antitumor effect in xenograft models in mice. Interestingly, the strongest antitumor effect was observed in the SK-RC-52 model of pVHL inactive renal cell carcinoma that constitutively expresses high levels of CAIX even in the absence of hypoxia and not in the LS147T model, where CAIX is expressed in response to hypoxia. More recently, adducts of sulfonamides and sugars as selective CAIX inhibitors have been described. Whilst the sugar moiety may restrict diffusion across membranes, the resulting ligands also had some intrinsic binding selectivity for CAIX over other CAs [33]. Current Opinion in Chemical Biology 2015, 26:48–54

These results support the notion that a selective functional inhibition of CAIX in solid tumors with molecules is possible and may indeed be a promising new anticancer strategy. Whilst single-agent therapies with CAIX inhibitors typically only gave rise to moderate antitumor effects, experiments in preclinical mouse models of cancer suggest that combination therapies may be a fruitful avenue for further development [27,32].

CAIX as a target for drug delivery As an alternative to direct inhibition approaches, the highaffinity interaction of CAIX ligands with their cognate target can be exploited for the targeted delivery of potent cytotoxic drugs into solid tumors [34,35]. Clinically used cytotoxic drugs in their ‘naked’ form often do not reach solid tumors in high quantities but accumulate in healthy organs [36], where they can cause severe side effects. By www.sciencedirect.com

Targeting CAIX with small organic ligands Wichert and Krall 51

Figure 3

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CAIX as an antigen for targeted drug delivery approaches. (a) The G250 anti-CAIX antibody was radio-labeled with 125-Iodine (left) and injected into mice bearing CAIX-expressing SK-RC-52 tumors. Tumor accumulation ranged between 3% and 20% ID g 1 after 72 h and was dependent on the injected dose (middle). The authors suggested that the dose-dependence might be due to rapid processing of the antibody inside the tumor. The data series marked with an asterisk is from [38], all other data were taken from [39]. Tumor uptake but also uptake into lungs and thyroid were observed 72 h post injection using micro-SPECT imaging (right). This image was originally published in the Journal of Nuclear Medicine. Muselaers et al. Optical Imaging of Renal Cell Carcinoma with Anti-Carbonic Anhydrase IX Monoclonal Antibody Girentuximab. J Nucl Med. 2014; 55: 1035–1040. # by the Society of Nuclear Medicine and Molecular Imaging, Inc. (b) A peptide scaffold bearing two copies of a derivative of the CAIX ligand acetazolamide (AAZ) was labeled with the near-infrared dye IRDye1 750 (left). The bivalent ligand–dye conjugate strongly accumulated inside CAIX-expressing SK-RC-52 tumors in mice 24 h after injection and outperformed a monovalent control compound in terms of total tumor uptake and selectivity (middle). A strong tumor uptake was observed in near-infrared imaging but also uptake in the kidneys and lungs is visible (right). (c) A bivalent CAIX ligand–drug conjugate for the treatment of CAIX-expressing solid tumor. The bivalent ligand makes sure that the active drug (here the maytansinoid DM1) is transported into the tumor. There, the disulfide linker is broken and the drug released. The conjugate exhibited a potent antitumor effect in mice bearing CAIX-expressing SK-RC-52 tumors and durably cured one third of all animals.

using a ligand of an accessible tumor antigen to selectively transport toxins into antigen-expressing neoplastic lesions, the therapeutic efficacy of cell-killing agents can be increased whilst reducing their side effects [34,35]. www.sciencedirect.com

Some monoclonal antibodies against extracellular epitopes of CAIX have the striking ability to strongly accumulate inside CAIX-expressing solid tumors (Figure 3a) and played a key role in validating CAIX as an antigen for Current Opinion in Chemical Biology 2015, 26:48–54

52 Next generation therapeutics

targeted drug delivery applications [37,38]. The G250 antibody was first raised by Boermann and co-workers using hybridoma technology. In the NU-12 model of CAIX-expressing renal cell carcinoma it can accumulate at concentrations of >200% of the injected dose per gram of tumor (% ID g 1) and attain a good selectivity for the tumor over healthy organs in mice [38]. In other mouse models, lower accumulations ranging from 15% to 30% ID g 1 were observed [38] (Figure 3a). Interestingly, tumor to blood selectivity was strongly dependent on the model and the dose used [39]. The authors hypothesized that this may be due to processing of the antibody by the tumor. The G250 antibody has also extensively been characterized by nuclear medicine in human patients and tested for therapeutic approaches both in its naked form and as a carrier for the targeted delivery of radionuclides into CAIX-expressing lesions [40]. Scientists at Bayer attached the potent cytotoxic drug monomethyl auristatin E (MMAE) to the surface of a different anti-CAIX antibody (i.e., synthesized an antibody–drug conjugate) to deliver the drug into CAIX-expressing xenograft tumors [41]. The conjugate showed potent activity against strongly CAIX-positive tumors but showed little or no activity in tumors with low antigen expression. Collectively, these data validate CAIX as an excellent antigen for targeted drug delivery applications. Antibodies may, however, not always be the ideal ligands for targeted drug delivery applications. Their large size can hinder homogenous penetration into solid tumors [42]. Long circulation half-lives together with labile linkers [43] can lead to the premature release of cytotoxic drugs attached to the antibody outside the tumor. Several attempts have thus been made to replace antibodies with small molecule ligands for targeted drug delivery applications [34]. Small molecules may penetrate tumors much more easily than large macromolecules and are typically cleared from circulation much more rapidly than antibodies. In the context of CAIX, Supuran and co-workers demonstrated that ligands of CAIX in conjugate with nearinfrared dyes could strongly accumulate in CAIX-expressing HT-29 tumors but did not exploit this interaction for drug delivery [44]. Our own laboratory has recently described conjugates consisting of CAIX ligands connected to a different near-infrared dye that strongly accumulated in CAIX-positive SK-RC-52 renal cell carcinoma xenografts (up to 5% ID g 1 24 h after injection) in mice [45,46] (Figure 3b). Theoretical considerations and experimental evidence suggest that very high affinities for the tumor antigen (typically Kd < 10 nM) are required for small molecules to become good delivery ligands. In our hands, a derivative Current Opinion in Chemical Biology 2015, 26:48–54

of the literature-known pan-CA ligand acetazolamide (AAZ, Kd = 13–20 nM) in conjugate with a near-infrared dye turned out to have sufficient affinity for CAIX to strongly accumulate inside antigen positive SK-RC-52 tumors. Our data, however, also suggest that ligands with a higher binding affinity for CAIX may perform even better for targeted delivery applications [46]. Indeed, by attaching two AAZ moieties to a peptide scaffold a bivalent binder with a higher functional affinity for CAIX in vitro and stronger tumor accumulation at late time points in vivo could be obtained [46] (Figure 3b). Conjugates of tumor-homing CAIX ligands with the cytotoxic maytansinoid DM1 through a disulfide linker [45,46] were prepared and injected into SK-RC-52 xenograft tumor-bearing mice. The CAIX ligand was envisioned to transport the drug into CAIX-expressing solid tumors where the disulfide linker was cleaved to release the drug. A monovalent conjugate using a derivative of AAZ as the targeting ligand showed a potent antitumor effect in CAIX-expressing SK-RC-52 renal cell carcinoma xenografts, which was strictly dependent on the CAIX ligand. A bivalent AAZ-based disulfide-linked DM1 conjugate (Figure 3c) could also cure a subset of mice bearing CAIX-expressing SK-RC-52 renal cell carcinoma xenografts. This suggests that small-molecule ligand-based delivery of potent cytotoxic drugs into CAIX-expressing tumors may be an encouraging new anticancer strategy.

Conclusion CAIX is a promising target for the treatment of cancer. It plays important roles in tumor physiology, is highly upregulated in many solid neoplastic lesions but mostly absent from healthy tissues. Functional inhibition of CAIX using small molecules in animal models has proven to be a viable anticancer strategy [19]. Additionally, CAIX has extensively been validated as an antigen for targeted drug delivery applications using antibodies in animals and humans [37–40]. More recently, the use of small molecule ligands of CAIX for the targeted delivery of potent cytotoxic drugs has been described [45,46]. We and others believe that small molecules may be particularly attractive vehicles for targeted drug delivery application [34]. Their small size may aid tumor penetration [42] and reduced circulation half-life in the blood stream may circumvent problems associated with the stability of the linker [43] between the drug and delivery vehicle associated with antibody–drug conjugates. In order to advance the development of small molecule based anticancer strategies directed against CAIX we believe that several challenges still need to be surmounted. For one, ligands with higher affinity and improved selectivity for CAIX over other isoforms may still be required. Innovative library technologies such as DNA-encoded chemical libraries [47,48] reviewed in www.sciencedirect.com

Targeting CAIX with small organic ligands Wichert and Krall 53

other contributions in this issue may greatly facilitate the identification of improved inhibitors. In a very recent report, Neri, Scheuermann and co-workers, for example, used a DNA-encoded self-assembling chemical library (ESAC) approach to identify auxiliary ligands that could enhance the affinity of AAZ towards CAIX when connected to AAZ by a short spacer. The resulting heterobidentate binder exhibited subnanomolar affinity towards CAIX in vitro and a substantially enhanced targeting performance in a CAIX-expressing tumor model in vivo [49]. In a second dimension, diagnostic tools such as nuclear imaging agents will be required to identify patient populations who are most likely to benefit from a treatment aimed at CAIX. In any case, promising strategies identified in an academic setting will have to be moved into the clinic. Only there we will ultimately learn about the advantages and drawbacks of new therapeutic approaches and truly advance the field of cancer therapy.

Funding M.W. and N.K. are members of the group of Prof. Dario Neri, ETH Zurich. Research in his group on CAIX is funded by ETH Zurich, the Swiss National Science Foundation (SNSF), the Swiss Commission for Technology and Innovation (CTI), Philochem AG, Krebsliga Schweiz/Krebsforschung Schweiz (KFS-2839-08-2011) and Oncosuisse.

Conflict of interest statement M.W. and N.K. receive shares of revenue from ETH Zurich from a licensing deal with Philochem AG covering small molecule-based technologies for the targeted delivery of cytotoxic drugs into CAIX-expressing solid tumors. N.K. is a consultant of Philochem AG.

Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at http://dx.doi.org/10. 1016/j.cbpa.2015.02.005.

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37. Chrastina A, Zavada J, Parkkila S, Kaluz S, Kaluzova M, Rajcani J, Pastorek J, Pastorekova S: Biodistribution and pharmacokinetics of 125I-labeled monoclonal antibody M75 specific for carbonic anhydrase IX, an intrinsic marker of hypoxia, in nude mice xenografted with human colorectal carcinoma. Int J Cancer 2003, 105:873-881. 38. van Schaijk FG, Oosterwijk E, Molkenboer-Kuenen JD, Soede AC, McBride BJ, Goldenberg DM, Oyen WJ, Corstens FH,  Boerman OC: Pretargeting with bispecific anti-renal cell carcinoma  anti-DTPA(In) antibody in 3 RCC models. J Nucl Med 2005, 46:495-501. The G250 antibody against CAIX strongly accumulates in CAIX-positive renal cell carcinoma xenografts in mice thus validating CAIX as an antigen for targeted drug delivery approaches. 39. Kranenborg MH, Boerman OC, de Weijert MC, OosterwijkWakka JC, Corstens FH, Oosterwijk E: The effect of antibody protein dose of anti-renal cell carcinoma monoclonal antibodies in nude mice with renal cell carcinoma xenografts. Cancer 1997, 80:2390-2397. 40. Stillebroer AB, Boerman OC, Desar IM, Boers-Sonderen MJ, van Herpen CM, Langenhuijsen JF, Smith-Jones PM, Oosterwijk E, Oyen WJ, Mulders PF: Phase 1 radioimmunotherapy study with lutetium 177-labeled anti-carbonic anhydrase IX monoclonal antibody girentuximab in patients with advanced renal cell carcinoma. Eur Urol 2013, 64:478-485. 41. Petrul HM, Schatz CA, Kopitz CC, Adnane L, McCabe TJ, Trail P, Ha S, Chang YS, Voznesensky A, Ranges G et al.: Therapeutic  mechanism and efficacy of the antibody-drug conjugate BAY 79-4620 targeting human carbonic anhydrase 9. Mol Cancer Ther 2012, 11:340-349. An antibody against CAIX conjugated to monomethyl auristatin E (MMAE) is highly efficacious against tumor xenografts with strong CAIX expression but not against weakly CAIX-expressing lesions. The paper thus demonstrates that targeted drug delivery into CAIX-expressing tumors may be an interesting anticancer strategy. 42. Dennis MS, Jin H, Dugger D, Yang R, McFarland L, Ogasawara A, Williams S, Cole MJ, Ross S, Schwall R: Imaging tumors with an albumin-binding Fab, a novel tumor-targeting agent. Cancer Res 2007, 67:254-261. 43. Shen BQ, Xu K, Liu L, Raab H, Bhakta S, Kenrick M, ParsonsReponte KL, Tien J, Yu SF, Mai E et al.: Conjugation site modulates the in vivo stability and therapeutic activity of antibody–drug conjugates. Nat Biotechnol 2012, 30:184-189. 44. Groves K, Bao B, Zhang J, Handy E, Kennedy P, Cuneo G, Supuran CT, Yared W, Peterson JD, Rajopadhye M: Synthesis and evaluation of near-infrared fluorescent sulfonamide derivatives for imaging of hypoxia-induced carbonic anhydrase IX expression in tumors. Bioorg Med Chem Lett 2012, 22:653-657. 45. Krall N, Pretto F, Decurtins W, Bernardes GJ, Supuran CT, Neri D:  A small-molecule drug conjugate for the treatment of carbonic anhydrase IX expressing tumors. Angew Chem Int Ed Engl 2014, 53:4231-4235. High affinity ligands for CAIX can be used to deliver potent cytotoxic drugs into CAIX-expressing solid tumors and substantially reduce tumor growth. Unlike antibodies, the small molecules quickly and deeply penetrate into target leasions and get excreted rapidly from circulation. 46. Krall N, Pretto F, Neri D: A bivalent small molecule-drug conjugate directed against carbonic anhydrase IX can elicit complete tumour regression in mice. Chem Sci 2014, 5:3640-3644. 47. Kleiner RE, Dumelin CE, Liu DR: Small-molecule discovery from DNA-encoded chemical libraries. Chem Soc Rev 2011, 40:5707-5717. 48. Mannocci L, Leimbacher M, Wichert M, Scheuermann J, Neri D: 20 years of DNA-encoded chemical libraries. Chem Commun (Camb) 2011, 47:12747-12753. 49. Wichert M, Krall N, Decurtins W, Franzini RM, Pretto F, Schneider P, Neri D, Scheuermann J: Dual-display of small  molecules enables the discovery of ligand pairs and facilitates affinity maturation. Nat Chem 2015, 7:241-249. A DNA-encoded self assembling chemical library (ESAC) approach is used to identify auxiliary ligands that can enhance the binding affinity of acetazolamide (AAZ) towards CAIX.

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