University of Florida Thesis Or Dissertation Formatting

STRUCTURE-GUIDED DESIGN OF CARBONIC ANHYDRASE IX SELECTIVE INHIBITORS AS A BREAST CANCER TREATMENT

CARRIE L. LOMELINO

A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY

UNIVERSITY OF FLORIDA

First and foremost, I would like to thank my mentor, Robert McKenna, for his expertise and guidance throughout the last four years. Not only has he prepared me for a career as an independent scientist in the field of structural biology, but he has also made my entire graduate school experience enjoyable and a time I will never forget. He has been patient with me at the times I am most stubborn and supportive when I am stressed and indecisive. I know I would not have survived graduate school without his constant sarcasm and humor. It would be remiss if I did not also thank Mavis Agbandje-

McKenna as she has essentially been my unofficial co-mentor. She is a wonderful role model for a woman starting her career in science and I have always appreciated her advice. Most importantly, she has consistently reminded me to be confident in myself and my work. I would also like to acknowledge the members of my committee, Linda

Bloom, Susan Frost, Nicole Horenstein, Robert Huigens, and David Ostrov. I greatly appreciate the advice you have all given over the last few years to improve my research.

I would like to recognize all the members of the McKenna and Agbandje-

McKenna labs for making the lab feel more like a family than a workplace. A special shout out to Brian Mahon for mentoring me when I joined the lab; Mam Mboge for helping me achieve my TL1 aims and being a fun travel buddy; Bri Murray for always listening to me vent and always making me feel self-assured; and Jacob Andring for keeping me sane and working through so many manuscripts and projects with me (team science!). Also, thank you to my IDP friends Andrea, Casey, Christina, Kevin, and

Maggie who have been there from the beginning- we survived!

4 Finally, there are not enough words to express the gratitude I have for the constant support from my family. I know that I would not be where I am today without them. Thank you to my parents, Dale and Lori, for encouraging me to chase my dreams. They have each set examples of what it means to succeed- every day my father demonstrates the importance of diligence and dedication while my mother proves that you can always achieve your goals with determination and persistence. Thank you for raising me to be responsible, hard working, independent, and honest. Thank you to my sister, Jenny, for always reminding me to be myself and have fun; my Aunt, Cheri

Cohen, for providing a home away from home; and my best friend, Brandi Cawthorn, for always being there no matter how far apart. One last special thank you to my grandfather, Carl Whitaker, for always believing in me and for telling me I couldn’t do something knowing full well I was capable and that it would push me to prove that I could- I hope my dissertation would make you proud.

5 TABLE OF CONTENTS

page

ACKNOWLEDGMENTS ...... 4

LIST OF TABLES ...... 8

LIST OF FIGURES ...... 9

LIST OF ABBREVIATIONS ...... 11

ABSTRACT ...... 13

CHAPTER

1 INTRODUCTION ...... 15

CA IX Expression, Regulation, and Function ...... 16 CA IX Structure ...... 18 CA Mechanism ...... 19 Inhibition of CA ...... 21 Structure-Guided Drug Design ...... 22

2 METHODS ...... 33

Protein Expression and Purification ...... 33 Stopped Flow CO2 Hydrase Assay ...... 34 Crystallization ...... 34 X-ray Crystallography ...... 35 MTT Assay ...... 35 Migration and Invasion ...... 36

3 SULFONAMIDE-BASED INHIBITION OF CA IX ...... 43

Benzene Sulfonamide Inhibitors ...... 44 Nitrogenous Base Inhibitors ...... 46 Multi-tail Inhibitors ...... 50 Summary ...... 52

4 CARBOXYLIC ACID-BASED INHIBITION OF CA IX...... 66

Heteroaryl-pyrazole Carboxylic Acids ...... 68 Nicotinic and Ferulic Acid ...... 69 Summary ...... 70

5 SWEETENER-BASED INHIBITION OF CA IX ...... 78

Acesulfame Potassium ...... 78

6 Sucralose ...... 81 Summary ...... 83

6 FRAGMENT-BASED INHIBITION OF CA IX ...... 92

Sulfamate ...... 92 Cyclic Sulfonamide ...... 95 Summary ...... 98

7 CONCLUSION ...... 107

APPENDIX

A MECHANISM OF WATER NETWORK REPLENISHMENT ...... 115

Overview ...... 115 Methods ...... 120 Protein Expression and Purification ...... 120 Crystallization ...... 120 CO2 Entrapment ...... 120 Data Collection ...... 121 Structure Determination and Refinement ...... 121 Results ...... 122 CO2 Binding Sites ...... 122 H64 and Water Network ...... 123 Entrance Conduit Waters ...... 124 Restoration of Water Network ...... 125 Summary ...... 126

B XFELs ...... 133

Overview ...... 133 Methods ...... 135 Macromolecule Production ...... 135 Microbatch Mixing ...... 135 Batch Crystallization and Seeding ...... 136 Data Collection and Processing ...... 137 Results ...... 139 Summary ...... 141

LIST OF REFERENCES ...... 145

BIOGRAPHICAL SKETCH ...... 157

7 LIST OF TABLES

Table page

1-1 CA II and CA IX active site residues...... 32

3-1 Inhibition profile of benzene sulfonamide inhibitors SLC-0111, B-E, and FC12-538E against CA II and CA IX...... 56

3-2 Inhibition profiles of pyrimidine (Y1-Y7) and purine (U1-U7) inhibitors against target CA IX and off-target CA II...... 60

4-1 Inhibition profiles of heteroaryl-pyrazole inhibitors against target CA IX and off-target CA II...... 73

5-1 Inhibition profiles of sweeteners against CA II and CA IX...... 86

6-1 Inhibition profiles of sulfamate inhibitors against CA II and CA IX...... 100

7-1 Isoform unique active site residues of CA II and CA IX...... 111

LIST OF FIGURES

Figure page

1-1 Kaplan Meier survival plots for CA IX mRNA expression in breast cancer...... 25

1-2 HIF1α mediated regulation of CA IX expression in hypoxic tumors...... 26

1-3 Depiction of CA IX mediated pH regulation of a tumor cell...... 27

1-4 Structural representation of CA IX domains...... 28

1-5 Structure of representative αCA (CA II)...... 29

1-6 CA ping-pong catalytic mechanism...... 30

1-7 Structural representation of catalytically active human CA isoforms...... 31

2-1 Representative SDS gel used to confirm the purity of CA IX-mimic following affinity chromatography...... 39

2-2 Crystals of CA II and CA IX-mimic...... 40

2-3 Representative X-ray diffraction pattern from a crystal of CA II in complex with an inhibitor...... 41

2-4 Migration and invasion assay protocol...... 42

3-1 Structures of benzene sulfonamide CA inhibitors...... 55

3-2 Binding of FC12-538 in the active site of CA II...... 57

3-3 Overlay of FC12-538E with benzene sulfonamide inhibitors...... 58

3-4 Structures of pyrimidine and purine CA inhibitors...... 59

3-5 Binding of Y2 in the active site of CA II...... 61

3-6 Binding of U6 in the active sites of CA II and CA IX-mimic...... 62

3-7 Structures of multi-tail CA inhibitors...... 63

3-8 Binding of multi-tail inhibitors in the active sites of CA II and CA IX-mimic...... 64

4-1 Structures of heteroaryl-pyrazole carboxylic acid inhibitors...... 72

4-2 Binding of 1D in the active site of CA II...... 74

4-3 Overlay of carboxylic acid CA inhibitors...... 75

9 4-4 Binding of nicotinic acid and ferulic acid in the active site of CA II...... 76

4-5 In silico model of ferulic acid directly binding to zinc...... 77

5-1 Structures of sweetener CA inhibitors...... 85

5-2 Binding of acesulfame potassium in the active site of CA II and CA IX-mimic.. .. 87

5-3 Secondary binding sites of acesulfame potassium...... 88

5-4 Competitive binding of acesulfame potassium and CO2 in CA II...... 89

5-5 Binding of sucralose in the active site of CA IX-mimic...... 90

5-6 Depiction of CO2 pathway to the active site of CA IX-mimic...... 91

6-1 Structures of sulfamate CA inhibitors...... 99

6-2 Binding of sulfamate inhibitors in the active sites of CA II and CA IX-mimic.. ... 101

6-3 Structures of cyclic sulfonamide CA inhibitors...... 103

6-4 Binding of ABM in the active sites of CA II and CA IX-mimic...... 104

6-5 MTT assays for increasing concentrations of CAI...... 105

6-6 Migration and invasion assays of lead compound saccharin and ABM...... 106

7-1 Zones of human α CAs...... 112

7-2 Binding of GV1-89 in the active site of CA II...... 113

7-3 Binding of AN11-741 in the active sites of CA II and CA IX-mimic...... 114

A-1 CA II active site with the ordered water network stabilized by hydrophilic residues and CO2 binding site in the hydrophobic region...... 128

A-2 Electron density of CO2 and active site water molecules upon CO2 release.. .. 129

A-3 Electron density of F226 in the secondary binding site upon CO2 release...... 130

A-4 Extended water network in a surface representation of CA II...... 131

A-5 Proposed water replenishment mechanism following CA II catalysis...... 132

B-1 CA II crystals...... 142

B-2 Diffraction...... 143

B-3 Proposed pump-probe XFEL experiments and 3NPAA binding...... 144

10 LIST OF ABBREVIATIONS

3NPAA 3-nitrophenyl acetate

AceK Acesulfame potassium

AE Anion exchanger

ATP Adenosine triphosphate

BMM Basement membrane matrix

CA Carbonic anhydrase

CAI Carbonic anhydrase inhibitor

CD Catalytic domain

CHESS Cornell High Energy Synchrotron Source

CXI Coherent X-ray imaging

DW Deep water

EC Entrance conduit

ER Estrogen receptor

GI Gastrointestinal

GLUT Glucose transporter

HER2 Human epidermal growth factor 2

HIF Hypoxia inducible factor

HRE Hypoxia response element

IC Intracellular Domain

LCP Lipid cubic phase

MBM Microbatch mixing

MCT Monocarboxylate transporter

PAGE Polyacrylamide gel electrophoresis

PAL Pohang Accelerator Laboratory

PEG Polyethylene glycol

PG Proteoglycan domain

PM Polycarbonate membrane

PR Progesterone receptor

PYP Photoactive yellow protein

RT Room temperature

SDS Sodium dodecyl sulfate

SFX Serial femtosecond crystallography

SSRL Stranford synchrotron radiation lightsource

TM Transmembrane domain

TNBC Triple negative breast cancer

TR Time resolved

VDW Van der Waals

VHL Von Hippel Lindau

WEC Entrance conduit waters

XFEL X-ray free electron

ZBG Zinc-binding group

ZBW Zinc-bound water

12 Abstract of Dissertation Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy

STRUCTURE-GUIDED DESIGN OF CARBONIC ANHYDRASE IX SELECTIVE INHIBITORS AS A BREAST CANCER TREATMENT

Carrie L. Lomelino

December 2018

Chair: Robert McKenna Major: Medical Sciences, Biochemistry and Molecular Biology

Carbonic anhydrase IX (CA IX) is a zinc metalloenzyme that catalyzes the hydration of CO2. Overexpression of CA IX has been observed in several cancer types, including breast cancer, and its activity is hypothesized to play an important role in the regulation of pH in hypoxic tumors. In addition, the inhibition of CA IX activity using small molecules has been shown to decrease tumor volume and metastasis in mouse models. Therefore, CA IX is recognized as a biomarker and emerging therapeutic target. Though several CA inhibitors are currently available, these compounds do not exhibit isoform selectivity and bind nonspecifically to the 15 CA isoforms expressed in humans. The following studies describe the structural characterization of three classes of CA inhibitors, including sulfonamides, carboxylic acids, and sweentener-based compounds, in order to identify compound properties and interactions that enhance selectivity for CA IX over off-target CA II. These structural analyses guided the design and synthesis of a new CA IX inhibitor that was subsequently shown to exhibit different modes of binding between CA II and CA IX-mimic. Furthermore, increasing concentrations of this inhibitor were shown to decrease viability and invasion of a breast cancer cell line. Overall, this work explores the derivatization of new pharmacophores

and lead compounds to be used in the design of CA IX selective inhibitors and the knowledge gained from these studies will be used to propose the next directions in CA

IX inhibitor drug design.

CHAPTER 1 INTRODUCTION

Last year in the US, ~1.7 million cancer cases were diagnosed and ~600,000 cancer-related deaths were recorded, 90% of which are attributed to metastatic disease.1 For women, breast cancer represents the most frequent type of newly developed cancer. Although detection techniques and targeted therapies have significantly improved in the last few decades, metastatic breast cancer remains the second leading cause of cancer-related deaths.2

Breast cancer is divided into four molecular subtypes that are defined by gene expression profiles for the estrogen, progesterone, and human epidermal growth factor receptors (ER, PR, HER2, respectively) and proliferation factor Ki67: luminal A (ER and/or PR +, HER2 -, low Ki67), luminal B (ER and/or PR +, HER2 +, high Ki67), HER2- enriched (ER and/or PR -, HER2 +), and basal like (ER and/or PR -, HER2 -).3 The BC subtype not only determines the most appropriate treatment strategy, but also correlates to prognosis, disease relapse, and therapy response rates.

The basal like subtype contains triple negative breast cancers (TNBC), which represents the most aggressive form of breast cancer, accounting for 15-20% of new cases and 25% of deaths.4 In comparison to receptor positive subtypes, patients with

TNBC experience the shortest overall survival, exhibiting high probabilities for both recurrence and metastasis. Targeted therapies have been developed to treat receptor

Adapted from Lomelino CL, Andring JT, McKenna R. (2018). Crystallography and its Impact on Carbonic Anhydrase Research. Int J Med Chem, Article ID 9419521; Singh S, Lomelino CL, Mboge MY, Frost SC, McKenna R. (2018). Cancer Drug Development of Carbonic Anhydrase Inhibitors Beyond the Active Site. Molecules, 23(5), 1045-1067; Lomelino CL, Andring JT, McKenna R. (2018). Structural Insight into the Catalytic Mechanism of Carbonic Anhydrase. In Carbonic Anhydrases: Biochemistry, Mechanism of Action and Therapeutic Applications; Penttinen J; Nova Publishers, 71-110, ISBN 978-1-53613-262-5.

positive subtypes: ER (tamoxifen, fulvestrant, aromatase inhibitors) and HER2

(trastuzumab, pertuzumab, lapatinib). However, TNBC by definition does not express these receptors and treatment options are therefore limited to systemic chemotherapies or radiation.5 Furthermore, metastatic tumors are often diagnosed too late for treatment and many aggressive cancers exhibit multi-drug resistance or insensitivity to common chemotherapeutic agents. Therefore, the identification of new prognostic markers and molecular targets has the potential to greatly improve the survival rates for TNBC patients.

Carbonic anhydrase IX (CA IX) is an isoform from a family of zinc metalloenzymes that catalyze the reversible interconversion of carbon dioxide and water to produce bicarbonate and a proton. As such, CAs play an important role in many physiological processes, such as respiration, pH regulation, ion transport, biosynthetic reactions, and bone resorption. In healthy tissue, CA IX expression is limited to the GI tract. However, overexpression of CA IX has been observed in several aggressive cancer cell lines and tumor biopsies, including breast cancer.6,7

Furthermore, high CA IX mRNA expression in patients with TNBC correlates with decreased in overall, relapse free, and metastasis free survival analyzed using Kaplan

Meier plots (Figure 1-1A, B, C). Therefore, CA IX is recognized as a biomarker and therapeutic target for TNBC.

CA IX Expression, Regulation, and Function

CA IX expression is observed to increase in hypoxic conditions characterized by low oxygen concentrations. The rapid growth and proliferation of tumor cells often results in hypoxic regions with low oxygen diffusion as the cells outgrow the available blood supplies.8 Tumor cells subsequently undergo a metabolic switch from oxidative

phosphorylation to anaerobic glycolysis, termed the Warburg effect. This phenomenon results in an increased production of lactic acid that is exported from the cell with a proton, acidifying the surrounding tumor microenvironment to pH ~6.5.9–11 Extracellular acidosis threatens cell viability by disrupting important biological activities in the cell, including ATP production, cell migration, proliferation, protein synthesis, and apoptosis.12 Tumor cells adapt to hypoxic conditions and an acidic microenvironment by inducing the expression of stress response genes regulated by the transcription factor hypoxia inducible factor 1 (HIF-1), promoting angiogenesis and cell proliferation.13 In fact, HIF-1 levels have been observed to increase in aggressive tumors.

One of the genes regulated by HIF-1 is CA IX. HIF-1 is composed of two subunits, HIF-1α and HIF-1β, and functions as a heterodimer. Under normoxic conditions, HIF-1α is hydroxylated by prolyl hydroxylase and is consequently recognized by the von Hippel Lindau (VHL) protein for ubiquitin-mediated degradation in the proteasome. In hypoxic conditions, however, HIF-1α is not hydroxylated and the subunit accumulates. HIF-1α is subsequently transported into the nucleus where it dimerizes with HIF-1β to form an active transcription factor. HIF1 regulates the expression of stress response genes, such as CA IX, by binding to a hypoxia response element

(HRE) upstream of the transcription start site (Figure 1-2).14 HIF-1 also regulates the expression of other genes that contain HRE sites and induces the transcription of proteins important for pH regulation, cell proliferation, cell adhesion, angiogenesis, and vascular remodeling.

CA IX activity is hypothesized to be critical for the regulation of pH in cancer cells that must thrive in an acidic tumor microenvironment.6,15,16 The overexpression of CA IX

enables neoplastic cells to survive these harsh conditions by catalyzing the production of bicarbonate. Because CA IX has an extracellular catalytic domain, bicarbonate produced during catalysis can buffer the surrounding milieu or be transported into the cell to maintain near physiological intracellular pH (Figure 1-3).17 CA IX has also been shown to be involved in cell proliferation and cell-cell interactions.18

Therefore, CA IX is recognized as a biomarker of hypoxia and an attractive therapeutic target due to its role in pH regulation and tumorigenesis. The inhibition of

CA IX catalytic activity has been shown to decrease the growth and proliferation of tumor cells.12,19 Furthermore, previous studies in breast cancer mouse models have shown that both the knock down of CA IX via shRNA or small molecule inhibition of catalytic activity decreases tumor volume, decreases metastasis, and prolongs overall survival.19

CA IX Structure

CA IX is a homodimeric metalloenzyme that is stabilized by a disulfide bond between the catalytic domains of two monomers (C199, C299). N-linked and O-linked glycosylation sites have been observed at residues N309 and T78, respectively. CA IX is composed of four domains: a proteoglycan domain (PG), catalytic domain (CD), transmembrane (TM) domain, and intracellular domain (IC) (Figure 1-4).20 The PG domain is unique to CA IX and is hypothesized to facilitate catalytic activity in an acidic microenvironment due to its composition of Asp and Glu residues.18,21 The PG domain is also predicted to participate in cell adhesion since both deletion of the PG domain and binding of an antibody decrease adhesion.18 CA IX activity has also been shown to be dependent on post-translational modifications of the IC tail, with deletion and targeted mutagenesis experiments preventing acidification of the extracellular

environment, possibly relating to interactions with transporters.22 For example, CA IX has been shown to interact and/or be in close proximity with cell membrane transporters such as anion exchanger (AE) to regulate the pH of tumors cells.23

Although the first structural studies were performed on CA II, the findings apply to the entire class of α CAs. The overall shape of the CA catalytic domain is an ellipsoid.

The active site is conically shaped and ~15 Å deep with a zinc situated at the base. The zinc is tetrahedrally coordinated by three His residues (H94, H96, and H119) and a water molecule. Seven right-handed α-helices are present on the surface of the enzyme, surrounding a 10 stranded β-sheet core. The CA active site can be divided into two halves consisting of hydrophobic (I91, V121, F131, V135, L141, V143, L198, P202,

L204, V207, and W209) and hydrophilic (N62, H64, N67, Q92, T199, and T200) residues (Figure 1-5A). The hydrophobic region contains the binding site for substrate

CO2 (V121, V143, L198, and W209) whereas the hydrophilic residues stabilize an ordered water network essential for proton transfer. Electron density was observed for this network of five ordered water molecules (ZBW, W1, W2, W3a, W3b), which are stabilized via hydrogen bonding with surrounding residues Y7, N62, N67, T199, and

T200 (Figure 1-5B).24,25

CA Mechanism

Many CA II structures have since been solved and recently sub-angström resolution was achieved, providing further insight into the CA catalytic mechanism. α

CAs exhibit a classic two-step, ping-pong mechanism. The first step is a nucleophilic attack of CO2 by the zinc bound hydroxyl, generating zinc-bound bicarbonate. The second step is the regeneration of the catalytic zinc-bound hydroxide via proton

transfer, which is facilitated by the ordered water network and a proton shuttle residue

(Figure 1-6).

In high resolution crystal structures of CA II, H64 was observed to exhibit two conformations, termed the “in” and “out” conformations. Due to its proximity to the catalytic zinc (~7.5 Å) and pKa value (7.1), H64 was proposed as the proton shuttle residue.26,27 As such, H64 accepts a proton from zinc-bound water (ZBW) when occupying the in conformation to regenerate the enzyme for catalysis. H64 then flips or rotates to occupy the out conformation and transfer the proton to bulk solvent.

Occupancy of the out conformation was observed to increase in acidic conditions when

H64 is protonated, likely due to repulsion of the charged side chain by the positively charged zinc. Conversely, the deprotonation of H64 at high pH resulted in a higher occupancy of the in conformation.28 This orientation and protonation state primes the imidazole ring for the acceptance of a proton, which is subsequently transferred to bulk solvent.29

In the hydration direction, the reaction starts with an open active site exposing a zinc-bound hydroxide primed for catalysis and an empty hydrophobic binding pocket.

The zinc-bound hydroxide interacts with the hydroxyl group of T199, which is further anchored by the carboxylate group of E106 via hydrogen bonding. These interactions enhance the nucleophilic nature of the zinc-bound hydroxide and position CO2 in a

30 favorable orientation for nucleophilic attack. CO2 diffuses into the active site, binding in the hydrophobic pocket approximately 2.8 Å from the zinc-bound hydroxyl.31,32 In response to CO2 binding, H64 undergoes a conformational change from the “out” to the

“in” position in preparation for proton transfer.33,34 Following the nucleophilic attack of

- CO2, the generated zinc-bound HCO3 molecule is subsequently displaced by a water molecule in the active site. This nucleophilic attack is nearly instantaneous and therefore does not contribute to the overall rate of the reaction.

The second, rate limiting step is the regeneration of the zinc-bound hydroxyl via proton transfer from the ZBW to bulk solvent coordinated by H64 and the ordered water

- network. While HCO3 is released, H64 completes its conformational change to fully occupy the “in” conformation.35 The ordered water network in the active site acts as a proton wire, allowing intramolecular proton transfer over a distance of 8-10 Å out of the active site. Proton transfer is the rate limiting step of the CA catalytic mechanism and can be divided into a two-part process: shuttling of the proton through the five waters to

H64 followed by transfer of the proton from H64 to bulk solvent.36,37

Inhibition of CA

Multiple α CA isoforms are recognized as therapeutic targets in diseases and a significant portion of CA research has focused on the design of CA inhibitors (CAIs).

Approximately twenty systemic CAIs are currently available for the treatment of glaucoma, altitude sickness, and epilepsy.38,39 However, there are fifteen CA isoforms expressed in humans. These CA isozymes differ by both cellular localization and catalytic efficiency. CA I, CA II, CA III, CA VII, CA VIII, CA X, CA XI, and CA XIII are cytosolic; CA IV, CA IX, CA XII, and CA XIV are membrane-bound; CA Va and CA Vb are mitochondrial; and CA VI is secreted (Figure 1-7A). Furthermore, CA I-CA XIV share high sequence and structural homology, particularly within the active site (Figure

1-7B). Therefore, systemic CAIs do not exhibit sufficient isoform selectivity and bind to multiple CA isoforms, often causing undesired side effects. Nonspecific binding can also result in sequestration of the inhibitor by ubiquitous isoforms, decreasing the

bioavailability and efficacy of the inhibitor and requiring higher doses for treatment.

Hence, the design of CA IX selective inhibitors is necessary for the use of CAIs as a breast cancer therapy.

Structure-Guided Drug Design

Structure-guided drug design is a technique that uses high resolution crystal structures of a molecular target to rationalize the design of high affinity, small molecule inhibitors. This process often begins with high throughput screening of different classes of inhibitors to identify lead compounds that inhibit CA activity. The most promising compounds, which exhibit binding affinities in the nano to micromolar range, are then studied using X-ray crystallography to identify interactions between the compound and target molecule that promote selective binding.

As previously described, structural biology has provided a thorough characterization of the α CA active site. This knowledge can subsequently be used to analyze the binding of CAIs and guide the design of isoform specific inhibitors. For example, small inhibitors that contain aromatic functional groups have been shown to orient in the hydrophobic region of the active site whereas compounds with longer tails or charged groups preferentially bind in the hydrophilic region.40 The hydrophobic half of the active site was further divided into two pockets separated by residue 131 (Pocket 1:

L198, F131, V135, and L204; Pocket 2: I91, V121, and F131). The superposition of nearly 30 CAIs identified that the majority of compound tails that bind in the hydrophobic region orient below F131 into Pocket 2. Crystallographic studies showed that the incorporation of flexible linkers promotes tail binding in Pocket 1 and increases affinity for CA.41 In 2013, a structural comparison of the binding of all non-redundant inhibitors in complex with CA II was performed. Of the 145 compounds, only 14 were observed to

orient toward a region between the hydrophobic and hydrophilic areas of the active site.

This region was termed the “selective pocket” and contains residues 67, 69, 91, and

131, which differ between the human CA isoforms.42

As mentioned above, the design of isoform selective inhibitors is complicated by similaries in sequence and active site structure. In fact, the catalytic domain of CA IX shares 34% sequence identity with cytosolic CA II. From a structural point of view, CA II and CA IX also exhibit significant tertiary structure similarity, with a Cα rmsd value of 0.6

Å. The mapping and comparison of residues between CA II and CA IX has identified several isoform unique residues in the active site, including those in the selective pocket

(Table 1-1). CAIs are therefore designed to extend into the selective pocket with the incorporation of functional groups that will promote interactions with these isoform unique residues.

As CA IX is a membrane-bound isoform with an extracellular catalytic domain,

CAI selectivity can be enhanced by designing membrane impermeable compounds to prevent the off-target binding of cytosolic CAs. For example, positively or negatively charged hydrophilic moieties can be added to promote impermeability. However, these properties make it unlikely that such compounds would enter the bloodstream or be developed into a drug to be taken orally. Therefore, inhibitors can be designed as prodrugs with hydrophobic moieties that mask the desired, inhibitory substituents until the compound is present in the reductive conditions of a hypoxic environment where it will be hydrolyzed and become an active inhibitor.43

It is important to recognize that the affinity of a CAI is dependent on the free energy of binding (ΔG = ΔH - TΔS) with both enthalpic and entropic contributions. A

ligand loses rotational freedom upon binding, decreasing ΔS. This entropic penalty can be counteracted by enthalpic gains upon the formation of interactions between the ligand and target molecule in addition to increases in entropy as water molecules are displaced from the active site.44 Both terms of the free energy calculation must therefore be considered during the selection of chemical moieties in the drug design process. For example, the addition of hydrophobic functional groups has been shown to induce entropic penalties that are not balanced by the increase in enthalpy, decreasing the binding affinity for the target.45 One must also consider how compound derivatization will impact drug delivery and pharmacological properties such as absorption, distribution, metabolism, and excretion. Compounds that are to be delivered orally should follow Lipinski’s rule of 5: fewer than 5 Hydrogen bond donors and 10 acceptors, molecular weight < 500 g/mol, and the log P < 5.46 However, these are not steadfast rules and many FDA approved drugs do not fulfill each of the five rules.47

Figure 1-1. Kaplan Meier survival plots for CA IX mRNA expression in breast cancer. Patients with high CA IX expression are shown in red and low CA IX expression in black. A) overall survival, B) relapse free survival in TNBC patients, C) metastasis free survival in TNBC patients.

Figure 1-2. HIF1α mediated regulation of CA IX expression in hypoxic tumors. The color gradient represents normoxic cells with normal oxygen levels (red) to hypoxic cells with low oxygen diffusion (blue).

Figure 1-3. Depiction of CA IX mediated pH regulation of a tumor cell. Glucose and lactate are represented as sticks (yellow).

Figure 1-4. Structural representation of CA IX domains. PG domain (pink), catalytic domain (cyan), transmembrane domain (yellow), and intracellular domain (green).

Figure 1-5. Structure of representative αCA (CA II). A) surface representation with hydrophic and hydrophilic residues colored orange and purple, respectively. B) proton transfer water network. Hydrogen bonds are shown as black dashes.

Figure 1-6. CA ping-pong catalytic mechanism. Open active site (top left) before CO2 enters the active site. Nucleophilic attack is represented by arrows (top right), - - resulting in zinc-bound HCO3 (bottom right). HCO3 is displaced by a water molecule and proton transfer is shown as a transparent arrow (bottom left). The relative transparency of H64 correlates to occupancy of in and out - conformations. CO2 and HCO3 are represented as green sticks.

Figure 1-7. Structural representation of catalytically active human CA isoforms. A) cellular distribution of CAs. CA VI is secreted; CA IX, CA XII, and CA XIV are membrane-bound; CA IV is GPI-anchored; CA I, CA II, CA III, CA VII, and CA XIII are cytosolic; and CA Va/CA Vb are mitochondrial. B) overlay of CAs, highlighting conservation of the active site. Structure colors correspond to the isoform.

Table 1-1. CA II and CA IX active site residues. Residue CA II CA IX 60 Leu Arg 62 Asn Asn 64 His His 65 Ala Ser 67 Asn Gln 69 Glu Thr 91 Ile Leu 92 Gln Gln 94 His His 96 His His 119 His His 121 Val Val 131 Phe Val 135 Val Leu 141 Leu Leu 143 Val Val 198 Leu Leu 199 Thr Thr 200 Thr Thr 202 Pro Pro 204 Leu Ala 207 Val Val 209 Trp Trp CA II numbering

CHAPTER 2 METHODS

Due to the challenges in expressing, purifying, and crystallizing wild type CA IX, a CA IX-mimic was utilized for structural studies. The CA IX-mimic was produced by mutating seven active site residues of CA II to mimic the active site of CA IX (A65S,

N67Q, E69T, I91L, F131V, K170E, L204A).48

Protein Expression and Purification

CA II and CA IX-mimic were expressed in BL21(DE3) competent cells. Cells were grown in 1 L luria broth (LB, containing 100 mg Ampicillin) at 37°C until an OD600

~0.6. Protein expression was induced with 1 mL Isopropyl β-D-1-thiogalactopyranoside

(IPTG, 100 mg/mL) in the presence of 1 mM ZnSO4. The cells were incubated at 37°C for an additional 3-4 hrs and then harvested via centrifugation. The cell pellets were resuspended in buffer (0.2M sodium sulfate, 0.1M Tris-HCl, pH 9.0) and then lysed via a microfluidizer or homogenization followed by an overnight incubation at 4°C on stir plate. The lysate was centrifuged and the supernatant filtered prior to purification.

CA II and CA IX-mimic were purified by affinity chromatography on a p-

(aminomethyl)benzenesulfonamide agarose column. After running the lysate through the column two times, nonspecific proteins were removed with two separate wash steps

(0.2M sodium sulfate, 0.1M Tris-HCl, pH 9.0 followed by 0.2M sodium sulfate, 0.1M

Tris-HCl, pH 7.0). The protein was eluted with a buffer containing 0.4 M sodium azide,

50mM Tris-HCl, pH 7.8. Azide was subsequently removed from the purified protein via buffer exchange into 50 mM Tris, pH 7.8 using Amicon Ultra-15 centrifugal filter devices with a 10,000 MWCO. The final purity was verified via SDS-PAGE and protein concentration calculated from A280 measurements (Figure 2-1).

Stopped Flow CO2 Hydrase Assay

CA activity was measured using an Applied Photophysics stopped-flow instrument with a phenol red indicator (0.2 mM).49 The CAI of interest was complexed with CA II or CA IX and rapidly mixed with a carbon dioxide solution (1.7 to 17 mM) that contains the pH indicator. This experiment is repeated for at least four concentrations of inhibitor. The hydrase activity of CA produces bicarbonate and a proton, which decreases the pH of the solution and changes the color of the pH indicator. Changes in absorbance are then measured at 557 nm over a time period of 10–100 sec in order to calculate kinetic parameters and inhibition constants.

Protein solutions were prepared in a buffer of 20 mM Hepes (pH 7.5) and 20 mM

Na2SO4 (maintains ionic strength). Stock solutions of the CAI of interest were prepared in deionized water to a concentration of 0.1 mM and diluted in buffer. Inhibitor and enzyme solutions were preincubated for 15 min at RT to allow complex formation. IC50 values were obtained by fitting the dose response curves using the non-linear least- squares methods in PRISM 3 (mean values from three independent measurements).

The Cheng–Prusoff equation was then used to calculate the inhibition constants (Ki).

Crystallization

Purified CA II or CA IX-mimic was diluted with storage buffer (50 mM Tris, pH

7.8) to a final concentration of 10 mg/mL. Crystal drops were set in a 1:1 ratio of protein to precipitant solution (1.6 M NaCitrate, 50 mM Tris, pH 7.8) for a total volume of 5 µL.

Crystals were grown at room temperature using the hanging drop vapor diffusion method and crystal growth was observed within 3-5 days (Figure 2-2).

Complexes of CA II or CA IX-mimic with an inhibitor were produced via co- crystallization or drug soaks. For co-crystallization, the inhibitor is diluted in the

precipitant solution and then the crystal drop set up in a 1:1 ratio as previously described; this method ensures the inhibitor is present in the protein solution prior to crystal formation. For crystal soaks, the inhibitor is diluted in the equilibrated well solution and 1 µL is added to the 5 µL crystal drop. Crystals were soaked at RT overnight. Both co-crystals and soaked crystals were transferred into a cryoprotectant solution (20% glycerol) prior to flash-cooling in liquid nitrogen for shipment to the synchrotron.

X-ray Crystallography

X-ray diffraction data was collected on the F1 beamline at the Cornell High

Energy Synchrotron Source (CHESS) or 14-1 beamline at the Stanford Synchrotron

Radiation Lightsource (SSRL). CHESS has a Pilatus 6M detector whereas SSRL has a

Rayonix MX325 CCD detector. Data sets were collected at CHESS (SSRL) with a crystal to detector distance of 225-270 mm (120-180 mm), 1° (0.15°) oscillation angle, and exposure time of 3-5 (1-2) sec for a total of 180-360 (1200) images (Figure 2-3).

Diffraction data was indexed and integrated in HKL2000 or XDS and scaled to the P21 space group. Phases were determined via molecular replacement using the structure of

CA II (PDB code: 3KS3) as a search model. Modifications to the models were performed in Coot50 whereas refinements and ligand restraint files were generated in

Phenix51. All figures were produced using PyMol (The PyMOL Molecular Graphics

System, Version 2.0 Schrödinger, LLC).

MTT Assay

The MTT assay is a colorimetric experiment that measures the enzyme mediated reduction of a tetrazolian dye (MTT) into a colored product (formazan), which can be quantified based on absorbance readings. The MTT assay directly measures metabolic

activity but is used as an indicator of cell viability. Increasing concentrations of CAIs are tested for their ability to decrease MTT reduction, suggesting a decrease in cell viability.

Media was aspirated from the plate of UFH-001 breast cancer cells and the cells incubated with 2 mL of trypsin for 5 min at 37°C. The cells were then resuspended, centrifuged, trypsin aspirated, and 6 mL media added to stop the reaction. The number of cells were quantified using a hemacytometer. The volume of cell suspension necessary for 2,500 cells/well was calculated to produce a stock of 25 mL. A volume of

200 μL was then added to each well and the plate incubated at 37°C overnight.

Stock concentrations of CAIs (1 M saccharin; 100 mM ABM, GAL, SBC) were diluted 100-fold in media. Then, 200 μL of drug-free media was added to all rows of the

96-well plate with the exception of row B. Next, 210 μL of media containing the CAI of interest was added to row B. Serial dilutions were performed by taking 10 μL from row B and adding to the drug-free media in row C; this was repeated for the remaining rows of the plate with 10 μL from row H being discarded so that total volume remains consistent in each row. The plate was then incubated at 37°C for 48 hrs.

A 5 mg/mL stock of MTT was made in PBS, pH 7.4. A volume of 20 μL MTT stock was then added to each well of the plate. The plate was then incubated at 37°C for 4 hrs. Following incubation, MTT solvent is added to each well to dissolve the formazan product. The absorbance of each well is then measured at 590 nm.

Migration and Invasion

The impact of CAIs on the migration and invasion of a breast cancer tumor cell line was measured using a colorimetric assay. A kit (Cell Biolab CytoSelect) was used to measure the migration of a cancer cell line through an insert coated in a polycarbonate membrane (PM) toward a chemoattractant. Because invasive cells are

able to degrade basement membrane, the PM is also coated in an even layer of basement membrane matrix (BMM) in the invasion assay. Cells that pass through the

PM or PM/BMM can then be stained and quantified (Figure 2-4).

For the invasion assay, the PM/BMM insert was rehydrated in 300μL serum free media, ensuring the insert is fully covered. Media was aspirated from the plate of UFH-

001 breast cancer cells and the cells incubated with 2 mL dissociation buffer for 5 min at

37°C. The cells were dissociated and resuspended by pipetting until cells could no longer be seen adhered to plate and then transferred to falcon tube. In order to stop the reaction, 2.5 mL of serum free media was added. The cells were harvested by centrifugation for 5 min at speed 5. The remaining media was aspirated and the cells resuspended in 2 mL of serum free media. A cell count was determined using a Coulter

Counter ZM. The volume of resuspension containing 50,000 cells was calculated so that the number of cells added to each well remains constant. This calculated volume of resuspended cells is added to each well in addition to the volume of drug required for each tested inhibitor concentration. Serum free media was then added to each well for a total volume of 300 μL per insert. 500 μL DMEM + 10% FBS was added to bottom of well to serve as a chemoattractant. Each plate was swirled to ensure coverage of the insert and avoid bubbles. The plates were then incubated at 37°C for 24 hrs (migration) or 48 hrs (invasion). Following incubation, the insert is removed from the plate, disposing of excess media, and emerged in a new well containing 500 μL of dye. The inserts were incubated for 10 min at RT and then rinsed in deionized water. A Q-tip was used to gently remove excess dye from inside the insert. The inserts are then imaged

and the number of cells that migrated (or invaded) through the PM (+BMM) were quantified.

Figure 2-1. Representative SDS gel used to confirm the purity of CA IX-mimic following affinity chromatography. The samples are labeled with the final, concentrated sample run as a 5-fold dilution.

Figure 2-2. Crystals of A) CA II and B) CA IX-mimic.

Figure 2-3. Representative X-ray diffraction pattern from a crystal of CA II in complex with an inhibitor.

Figure 2-4. Migration and invasion assay protocol.

CHAPTER 3 SULFONAMIDE-BASED INHIBITION OF CA IX

CA inhibitor research was initiated in the 1940s with the observation that sulfanilamide (p-aminobenzene-sulfonamide) inhibited CA activity. This inhibitory effect was shown to relate directly to the sulfonamide group (SO2NH2) with a loss of inhibition upon its removal.52 Sulfonamides and their bioisosteres have been studied since and remain the primary scaffold in CAI design, referred to as the classical CAIs.

Sulfonamide-based inhibitors bind to the active site zinc in a tetrahedral geometry, displacing the zinc-bound water/hydroxide. The sulfonamide ZBG also forms hydrogen bonds with T199 and E106, two active site residues conserved amongst the α

CAs and referred to as “gate keepers”. Sulfonamides exhibit three possible protonation states in solution. Initial solution state NMR studies hypothesized that sulfonamide- based compounds bind in the deprotonated state and interact with zinc through the sulfonamide nitrogen.53 Joint refinement of X-ray and neutron crystallography data was used to determine the protonation state of sulfonamide-based inhibitors, confirming that a representative compound, acetazolamide, binds in the anionic form with the negatively charged sulfonamido coordinating to the zinc.

Early studies revealed that heterocyclic sulfonamides exhibited >1,000-fold increase in affinity in comparison to benzene derivatives with a general increase in activity as the acidity of the compound increased.54 Such experiments led to the development of a drug design strategy termed the “ring approach” in which a ring system is added to a sulfonamide ZBG to increase affinity for the target CA.55 Such

Adapted from Lomelino CL, Andring JT, McKenna R. (2018). Crystallography and its Impact on Carbonic Anhydrase Research. Int J Med Chem, Article ID 9419521.

work led to the development of first generation CA inhibitors that are still available in the clinic, including acetazolamide (Diamox), methazolamide (Neptazane), ethoxzolamide, and dichlorphenamide (Keveyis). However, many of these compounds exhibit poor water solubility. Therefore, polar functional groups were then added to the aromatic/heterocyclic sulfonamide scaffold to modulate the physico-chemical properties and solubility of the compound. This approach resulted in the development of second generation CAIs such as dorzolamide (Trusopt) and brinzolamide (Azopt). Several additional sulfonamide-based CAIs are clinically available, including topiramate, celecoxib (Celebrex), sulpiride (Dogmatil), sulthiame, valdecoxib, zonisamide, irosustat

(COUMATE), and esterone sulfamate (EMATE).

SLC-0111 (4-(4-fluorophenylureido)-benzenesulfonamide) was the first CA IX inhibitor to reach phase I clinical trials as an antitumor/antimetastatic agent after showing significant effects in animal models.56 The initial pharmacokinetic and efficacy data led to the recent progression of SLC-0111 to phase II trials for the treatment of CA

IX expressing tumors. However, the 20-fold selectivity of SLC-0111 for CA IX over CA II is not sufficient for the prevention of off-target binding and drug sequestration.

Therefore, three subclasses of sulfonamide-based CAIs have been studied to identify properties that further improve isoform selectivity.

Benzene Sulfonamide Inhibitors

SLC-0111 was identified from a group of 4-substituted ureido benzenesulfonamide inhibitors (compounds B-E, Figure 3-1). These inhibitors were shown to exhibit a wide range in affinity for CA II and CA IX-mimic, ranging between 0.5 and 1,000 nM (Table 3-1). This diverse inhibition profile was attributed to the flexibility of the ureido linker, allowing the tails of the compounds to rotate and adopt the most

energetically favorable conformation. As such, the compound tails were observed to interact with different pockets in the CA active site. For example, inhibitors containing 2- isopropyl-phenyl and 3-nitrophenyl tails (C and D, respectively) oriented toward the hydrophilic region of the active site, which correlated to a loss of selectivity for CA IX. As described above, was determined to exhibit the greatest selectivity for tumor-associated

CA IX.41

Based on the selectivity of SLC-0111 for CA IX, a new derivative (FC12-538E) was synthesized that replaces the ureido linker between the benzenesulfonamide ZBG and aromatic tail with a thioureido linker (Figure 3-1). Few structural characterizations have been performed for compounds incorporating a thioureido linker, therefore a kinetic and X-ray crystallographic study of FC12-538E was performed. FC12-538E was determined to exhibit 25-fold selectivity for CA IX over CA II (Table 3-1).

The X-ray crystal structure of CA II in complex with FC12-538E was determined to 2.0 Å resolution. Electron density was observed in the active site with inhibitor tail oriented toward the hydrophobic region (Figure 3-2A). As is expected for sulfonamide inhibitors, FC12-538E binds directly to the zinc via the deprotonated nitrogen of the sulfonamide ZBG, forming a hydrogen bond with the backbone amide of T199 (2.9 Å)

(Figure 3-2B). Binding of the inhibitor not only displaces the ZBW that is essential for nucleophilic attack, but also displaces the majority of the water molecules in the proton transfer water network. Interactions were also observed between the thioureido sulfur of the linker and amide of Q92 (3.3 Å). The linker is further stabilized by a hydrogen bond

Adapted from Lomelino CL, Mahon BP, McKenna R, Carta F, Supuran CT. (2016). Kinetic and X-ray crystallographic investigations on carbonic anhydrase isoforms I, II, IX and XII of a thioureido analog of SLC-0111. Bioorg. Med. Chem, 24(5), 976-981.

between an NH moiety of FC12-538E and a water molecule within the active site that bridges to the backbone carbonyl of P201 (3.0 Å and 2.6 Å, respectively). The thioureido sulfonamide primarily interacted with the hydrophobic side of the CA II active site, with the 3-iodophenyl tail participating in van der Waals interactions with F131 and

P202. Several interactions with hydrophobic residues within the active site further stabilize the inhibitor (V135, L198, and L204) (Figure 3-2B).

The crystal structure of CA II in complex with FC12-538E was then compared to the previously determined structures of the 4-substituted ureido benzenesulfonamide series to rationalize the improved inhibition profile. FC12-538E and SLC-0111 are nearly superimposable with a slight variance in the C=S and C=O moieties of the linker

(Figure 3-3A). The bulkier C=S group in FC12-538E is shifted toward the hydrophilic region of the active site, allowing the formation of a hydrogen bond with Q92 whereas the C=O of SLC-0111 is too far for this interaction (3.6 Å). The orientation of FC12-538E is least similar to compounds C and D, which showed the highest affinities for CA II. The superimposition of FC12-538E with compounds C and D showed that only the benzenesulfonamide ZBG are superimposable whereas the linker and tail components exhibit significantly different orientations when bound to the enzyme (Figure 3-3B). The steric hindrance of F131 in CA II is predicted to limit the interactions of this series of compounds with the hydrophobic region of the active site, reducing their affinity for the off-target CA II.

Nitrogenous Base Inhibitors

Molecular hybridization is a technique that covalently combines two or more drug pharmacophores into a single molecule and has been shown to be an effective tool in the design of antitumor agents. In this study, pyrimidine and purine moieties are

incorporated into the tails of benzenesulfonamide scaffolds for the design of novel compounds that both inhibit CA IX activity and exploit the antitumor effects of uracil and adenine derivatives.

A “privileged scaffold” in drug design describes a compound that serves as a ligand for multiple biological targets.57 As such, pyrimidine and purine scaffolds are considered privileged due to their natural abundance and important roles in metabolic and cellular processes.58 Because large quantities of nucleotides are required to complete DNA replication in highly proliferative tumor cells, compounds that interfere with DNA synthesis have proven to be effective chemotherapeutics. In fact, pyrimidine and purine antimetabolites were among the first FDA approved anticancer drugs and account for 20% of drugs currently used to treat cancer.59 Furthermore, pyrimidine and purine scaffolds are synthetically accessible, confer drug-like properties to the compounds in which they are incorporated, and enhance solubility in water.60 Thus, nitrogenous bases such as uracil and adenine represent components of a number of useful drugs. Moreover, many pyrimidine-like scaffolds have been developed that exhibit potent cytotoxic activity against different human cell lines by interfering with DNA synthesis in tumors.61

Hence, two series of CAIs have been synthesized that combine a benzenesulfonamide ZBG with an uracil or adenine scaffold tail through an ether, amide, or triazole linker (Figure 3-4). The amide and triazole linkers were pursued due to their stability in vivo and previously reported anticancer activity.62,63 The uracil and

Adapted from Nocentini A, Bua S, Lomelino CL, Mckenna R, Bartolucci G, Tenci B, Di Cesare Mannelli L, Ghelardini C, Gratteri P, Supuran CT. (2017). Discovery of New Sulfonamide Carbonic Anhydrase IX Inhibitors Incorporating Nitrogenous Bases. ACS Med. Chem. Lett., 8(12), 1314-1319.

adenine moieties were incorporated at various positions on the benzenesulfonamide

ZBG and the length of the linker varied in order to test diverse positions of the compound tails within the pockets of the CA active site. Uracil and adenine were appended through the N1 and N9 moieties, respectively, in order to maintain the pharmacophore and the connection by which such nitrogenous bases are incorporated in nucleotides and nucleic acids.64

The inhibiton profiles of the pyrimidine and purine series were shown to be more dependent on the length and positioning of the linker rather than the identity of the linker or nitrogenous base (Table 3-2). For example, para-substitution of the benzenesulfonamide scaffold showed higher affinity for both CA II and CA IX (1−495 nM) in comparison to compounds with a meta-substitution (390−4360 nM). For compounds containing an amide linker at the para position, a direct or two carbon spacer between the amide and the benzene improved CA inhibitory properties (10−50 nM) over those with a single carbon spacer (45−495 nM).

Several of the reported compounds (Y3, Y5, U2, U3, U5, and U7) exhibited preferential binding for CA IX over the off-target CA II. Compounds Y7 and U2, which incorporate a triazole linker, were determined to be the most efficacious CA IX inhibitor from each series (5 and 2 nM, respectively) whereas Y5 and U7 exhibited the highest selectivities for CA IX over CA II (9.9 and 2.7-fold, respectively). For inhibitors containing a para-substituted amide linker, the addition of a single carbon spacer between the amide moiety and benzene ring decreased the affinity for CA IX in both the pyrimidine (from 25 to 405 nM) and purine series (from 20 to 45 nM). Conversely, the increase of the spacer to two carbons improved CA IX inhibiton in the pyrimidine (from

405 to 50 nM) and purine series (from 45 to 10 nM). The meta-substituted derivatives exhibited the weakest affinity for CA IX, regardless of the nitrogenous base incorporated as the tail (430−3120 nM).

X-ray crystallography was utilized to demonstrate the binding of a representative compound from the pyrimidine and purine series, Y2 and U6, respectively. The crystal structure of CA II in complex with Y2 was determined to a resolution of 1.4 Å and the structures of CA II and CA IX-mimic in complex with Y2 to resolutions of 1.6 Å (Figures

3-5A and 3-6A, B). As previously observed, the benzenesulfonamide ZBG displaced the

ZBW and bound directly to the zinc. The orientation of this pharmacophore was conserved in both CA II and CA IX-mimic with a hydrogen bond between the sulfonamide oxygen and backbone amide of T199 (3.0-3.1 Å).

The tail component of Y2 was observed to exhibit dual conformations due to the freedom of rotation about the amide linker, resulting in one conformation oriented in the hydrophobic region and a second toward the hydrophilic region. The tail oriented toward the hydrophobic pocket exhibited lower occupancy as indicated by weaker electron density (Figure 3-5A). In this position, the carbonyl of the Y2 linker forms a hydrogen bond with a water molecule that bridges to the side chain of T199 (2.2 and 2.9 Å, respectively). In the higher occupancy conformation, the linker carbonyl forms a hydrogen bond with the side chain of Q92 (2.8 Å) and the uracil moiety interacts with

N67 (3.2 Å). This increase in hydrogen bonding is expected to further stabilize the compound tail, resulting in the observed higher occupancy. Both conformations are further stabilized by interactions with active site residues F131, V135, L198, P202, L204 and F131, I91, V121 (Figure 3-5B).

The binding of U6 is shown in both CA II and CA IX-mimic and the compound was determined to display significantly different orientations between the two isofomrms

(Figure 3-6C). In complex with CA II, the free amine of the adenine moiety forms a weak hydrogen bond with the backbone carbonyl of G132 (3.5 Å). The linker and tail components are further supported by VDW interactions with residues Q92, F131, V135,

L198, and P202 (Figure 3-5D). In CA IX-mimic, the linker carbonyl and N of the adenine ring form hydrogen bonds with an active site solvent molecule (2.6 and 3.1 Å, respectively), which anchors to Q92 (3.0 Å). Again, the binding of U6 in the CA IX active site is further stabilized by VDW interactions with residues Q67, V131, V135, and L198

(Figure 3-5E). The decrease in steric hindrance of residue V131 (in relation to F131 of

CA II) is predicted to allow the rotation of the tail about the linker to form more energetically favorable interactions within the hydrophilic region of the CA active site.

Multi-tail Inhibitors

The tail method is a strategy often used in CA drug design in which an aromatic

ZBG is chemically linked to a derivatized tail component in order to promote interactions and modulate chemical properties. In previous work, a series of two tail CAIs were synthesized based on the hypothesis that a sulfonamide-based compound with both hydrophobic and hydrophilic tail oriented in opposing directions would be able to interact with both halves of the CA active site, inhibiting both steps of the CA catalytic mechanism. These compounds were shown to inhibit CA activity, but with lower potency in comparison to the single tail derivatives.65 Therefore, the tail method was similarly used to design a new set of CA inhibitors that contain three tails linked to a benzene sulfonamide ZBG through an amide linker (Figure 3-7). The incorporation of a third tail is expected to further improve the CA affinity and isoform selectivity by increasing the

number of interactions with isoform unique residues that line the entrance of the CA active site. The inclusion of two bulky, hydrophobic tails has the potential to block the pathway of CO2 movement along the hydrophobic region of the active site whereas the third hydrophilic tail can orient in the hydrophilic region to disrupt the proton transfer water network. Furthermore, the incorporation of the hydrophilic tail improves solubility, which is important for delivery.

Crystal structures of CA II and CA IX-mimic in complex with multi-tail CAIs AB2-

103, AB2-134, and AB2-166 were determined to resolutions of 1.4, 1.4, 1.5, 1.3, and 1.4

Å, respectively (Figures 3-8A, B, C, E, F). As previously observed, the conserved benzene sulfonamide ZBG binds directly to zinc and displaces the ZBW with a hydrogen bond between the oxygen of the sulfonamide and the backbone amide of

T199 (2.9-3.0 Å). Furthermore, the orientation of the ZBG is shared between isoforms.

Because the ZBG and amide linker components are shared between the three compounds, observed differences in binding orientations are dictated by the inhibitor tails. It is important to note, however, that weak electron density was observed for some of the inhibitor tails, indicating flexibility.

In complex with CA II, the carbonyl oxygen of compound AB2-103 forms a hydrogen bond with an active site solvent that bridges to Q92 (2.8 and 3.0 Å, respsectively). The two hydrophobic tails are stabilized by VDW interactions with residues I91, V121, F131, V135, L198, P202, and L204 whereas the hydrophilic CN tail extends from the active site into bulk solvent (Figure 3-8H).

Similar to AB2-103, the carbonyl oxygen of compound AB2-134 forms a hydrogen bond with an active site water molecule, which anchors to Q92 (2.7 and 2.9 Å,

respsectively) in CA II. The carboxyl tail is oriented toward the hydrophilic region and also binds to a water molecule that bridges to E69 (2.9 and 2.7 Å, respsectively) (Figure

3-8I). Similar interactions were observed in CA IX-mimic with the carbonyl and carboxyl tail of compound 134 forming hydrogen bonds with solvent that is anchored to Q92 (2.8,

2.8, and 3.0 Å, respectively) (Figure 3-8J). In both isoforms, the tail is stabilized by VDW interactions with residues 67, 91, 121, 131, 135, 198, and 202. The superposition of

AB2-134 bound in CA II and CA IX-mimic shows that the amide linker is shifted toward the hydrophobic region in CA IX, likely due to decrease in steric hindrance of residue

131 (Figure 3-8D). This shift causes the two cyclic tails to rotate in toward the active site to prevent clashes with residues on the surface of the enzyme.

In CA II, the carbonyl oxygen of AB2-166 forms a hydrogen bond with an active site solvent molecule that bridges to Q92 (3.0 and 3.0 Å, respsectively). The carboxyl tail also binds to a water that anchors to N67 (3.1 and 3.2 Å, respsectively) (Figure 3-

8K). Interestingly, the linker carbonyl reorients in CA IX-mimic, preventing the formation of any hydrogen bonds (Figure 3-8L). As hypothesized for AB2-134, the decrease in steric hindrance of residue V131 is predicted to allow the rotation of the phenyl tails about the linker (Figure 3-8G).

Summary

Overall, sulfonamide-based inhibitors remain the primary class of CAIs represented both in the clinic and drug design research. However, many of these compounds do not exhibit sufficient selectivity for the development of a CA IX inhibitor as an efficacious breast cancer treatment. The above research has explored three subclasses of sulfonamide-based CAIs (benezene sulfonamides, nitrogenous bases,

and multi-tail inhibitors), using various drug design approaches to improve isoform selectivity.

For the benzene sulfonamides, the flexibility of the ureido or thioureido linker was shown to promote the most energetically favorable binding orientation for this series of bezene sulfonamide CAIs. The orientation of compound tails in the hydrophobic region of the active site was shown to be more selective for CA IX. Incorporation of a thioureido linker over ureido in FC12-538E was shown to improve selectivity for CA IX in relation to the CAI in clinical trials, SLC-0111, due to the increase in hydrogen bonding of the bulker linker.

Molecular hybridization was utilized to design nitrogenous base compounds that inhibit both CA IX activity and DNA synthesis. Inhibitors containing a triazole linker were shown to be the most potent CA IX inhbitors. Among both pyrimidine and purine inhibitors, the para-substituttion of the benzene sulfonamide ZBG with a 0 or 2 carbon spacer between the ZBG and amide were shown to be more inhibitory than the meta derivatives.

Lastly, the tail method was used to synthesize multi-tail CAIs incorporating two hydrophobic tails and one hydrophilic tail oriented in opposing directions. Multiple tails increases the number of interactions with isoform unique residues in the CA active site to improve selectivity.

Throughout the three subclasses of sulfonamide-based CAIs, the length and freedom of rotation about the linker was shown to greatly impact the binding orientation of the inhibitor in the CA active site. Furthermore, residue 131 was shown to induce

steric hindrance in CA II (Phe), which is decreased in CA IX (Val) to allow rotation of inhibitor tails.

Figure 3-1. Structures of benzene sulfonamide CA inhibitors.

Table 3-1. Inhibition profile of benzene sulfonamide inhibitors SLC-0111, B-E, and FC12-538E against CA II and CA IX. Ki (nM) Compound CA II CA IX Selectivity Ratio SLC-0111 960 45 20 B 50 5 10 C 5 0.5 10 D 15 1 15 E 230 7.5 30 FC12-538E 1025 40 25

Figure 3-2. Binding of FC12-538 in the active site of CA II. A) Electron density of FC12- 538E in a surface representation of CA II. The 2Fo−Fc electron density map is contoured to σ = 0.8. B) CA II active site residue interactions with FC12- 538E. Residues are labeled and hydrogen bonds shown as black dashes.

Figure 3-3. Overlay of FC12-538E with benzene sulfonamide inhibitors A) SLC-0111 (orange) and B) inhibitors B-E.

Figure 3-4. Structures of pyrimidine and purine CA inhibitors.

Table 3-2. Inhibition profiles of pyrimidine (Y1-Y7) and purine (U1-U7) inhibitors against target CA IX and off-target CA II. Ki (nM) Compound CA II CA IX Selectivity Ratio Y1 705 3120 0.2 Y2 20 25 0.8 Y3 495 405 1.2 Y4 45 50 0.9 Y5 4360 440 9.9 Y6 390 460 0.8 Y7 1 5 0.2 U1 655 1310 0.5 U2 5 2 2.5 U3 890 430 2.1 U4 10 20 0.5 U5 60 45 1.3 U6 10 10 1 U7 80 30 2.7

Figure 3-5. Binding of Y2 in the active site of CA II. A) Electron density of Y2 in a surface representation of CA II. The 2Fo−Fc electron density map is contoured to σ = 0.8. B) CA II active site residue interactions with Y2. Residues are labeled and hydrogen bonds shown as black dashes.

Figure 3-6. Binding of U6 in the active sites of CA II and CA IX-mimic. Electron density of FC12-538E in surface representations of A) CA II and B) CA IX-mimic. The 2Fo−Fc electron density map is contoured to σ = 0.8. C) Overlay of U6 binding orientations in CA II and CA IX-mimic. Interactions of U6 in the active sites of D) CA II and E) CA IX-mimic. Residues are labeled and hydrogen bonds shown as black dashes.

Figure 3-7. Structures of multi-tail CA inhibitors.

Figure 3-8. Binding of multi-tail inhibitors in the active sites of CA II and CA IX-mimic. Electron density of AB2-103 in surface representations of A) CA II; AB2-134 in B) CA II and C) CA IX-mimic; AB2-166 in E) CA II and F) CA IX-mimic. The 2Fo−Fc electron density map is contoured to σ = 0.8. Overlay of D) AB2-134 and G) AB2-166 binding orientations in CA II and CA IX-mimic. Interactions of AB2-103 in the active sites of H) CA II; AB2-134 in I) CA II and J) CA IX- mimic; AB2-166 in K) CA II and L) CA IX-mimic. Residues are labeled and hydrogen bonds shown as black dashes.

Figure 3-8. Continued

CHAPTER 4 CARBOXYLIC ACID-BASED INHIBITION OF CA IX

As sulfonamide-based CAIs have been studied for several decades, studies are recently being directed toward the discovery of original pharmacophores and chemotypes to explore the molecular diversity of novel CAIs. The design of non- classical CA inhibitors deselects from interactions with the active site zinc and has the potential to improve isoform selectivity by interacting with isoform unique residues in and around the entrance of the CA active site.66,67 Several classes of compounds have recently been identified as non-classical CAIs, including phenols, polyamines, coumarins, and fullerenes.68–75 While it is possible for non-classical inhibitors to maintain the traditional tetrahedral geometry by binding directly to the catalytic zinc, several classes participate in different binding mechanisms. For example, non-classical

CAIs have been identified that inhibit CA catalytic activity by anchoring to the ZBW, binding to the enzyme outside the active site, or occluding the entry of substrate.

Carboxylic acids represent a class of compounds that inhibit metalloenzymes through various mechanisms of action, such as coordinating to the metal ion in a mono- or bidentate manner.76 Carboxylic acid-based compounds have recently been identified as one such promising class of CAIs. Previous structural studies have shown that this class of compounds exhibit multiple binding sites and therefore mechanisms of inhibition. Similar to sulfonamide-based compounds, some inhibitors have been shown to bind directly to the active site zinc, displacing the ZBW that is essential for catalytic activity.77–79 Alternatively, a second binding mode inhibits enzymatic activity by

Adapted from Lomelino CL, Supuran CT, McKenna R. (2016). Non-Classical Inhibition of Carbonic Anhydrase. Int. J. Mol. Sci., 17(7), 1150-1163.

anchoring to the ZBW, primarily in the anionic state.77,80 Lastly, carboxylic acid inhibitors have been observed to bind outside the active site in a pocket on the surface of the enzyme; binding in this pocket is hypothesized to restrict movement of the proton shuttle residue H64 to the "out" conformation, preventing regeneration of the nucleophilic hydroxide for catalysis.75

The scaffold of carboxylic acid-based inhibitors can vary in both size and chemical properties in order to promote interactions with residues in the hydrophobic/hydrophilic regions of the active site or isoform-unique residues of the selective pocket. Furthermore, the orientation of functional groups in relation to the carboxylic acid ZBG has been shown to be an important determinant in binding affinity due to steric hindrance, further promoting selectivity based upon the size of amino acids lining the active site cavity.81 Compounds that incorporate a tail that extends from the

CA active site have been shown to increase the binding affinity of carboxylic acid-based inhibitors over 100-fold, highlighting the importance of interactions with isoform unique residues. For example, structures of CA ΙΙ in complex with butenoic acid inhibitors (PDB:

5FNM and 5FLS) exhibit Ki values between 700–900 μM whereas a more compact benzoic acid derivative (PDB: 4E3F) has a Ki of only ~5 mM. As these three compounds demonstrate the same binding mode by anchoring to the ZBW and forming hydrogen bonds with conserved active site residue T199, the increase in specificity must be dictated by the additional VDW interactions with hydrophobic residues formed by the butenoic acid derivatives.77,80 Inhibitors containing a cyclic imide scaffold have been proven to selectively inhibit CA IX over off-target, cytosolic CA ΙΙ. Furthermore, the carboxylic acid derivatives were shown to exhibit a better selectivity ratio for CA IX over

CA II in comparison to the sulfonamide-based derivates containing the same cyclic imide scaffold.82

Heteroaryl-pyrazole Carboxylic Acids

This study focuses on the structural optimization of a previously identified lead compound, 3-(1-ethyl-1H -indol-3-yl)-1H -pyrazole-5-carboxylic acid (1A), to generate and evaluate a new series of indolylpyrazole-5-carboxylic acids/esters with an improved inhibition profile toward CA IX (Figure 4-1, Table 4-1). This new series of CAIs was designed by varying the substituents at the nitrogen atoms on the indole and pyrazole rings (1A-1D) in addition to esterification of the carboxylic acid moiety (2A−2D) (Figure

4-1). N-alkylation of the indole was introduced to test the impact of changes in lipophilicity whereas N-methylation of the pyrazole prevented possible hydrogen bonding. Because the carboxylic acid is hypothesized to serve as the ZBG and is therefore essential for inhibition, esterification of this group was performed to measure the effects on the inhibition profile. Several derivatives showed selectivity for CA IX over

CA II, with Ki values ranging from 0.5 to 4.5 μM (Table 4-1). Overall, the inhibition profile was observed to be independent of the substituents on both the indole and the pyrazole rings with no general trend correlating to lipophilicity. Furthermore, no significant differences were measured between the carboxylic acid and ester derivatives. The most potent CA IX inhibitors include 1D and 2A whereas the most selective over CA II is 1A.

The indolylpyrazole-5-carboxylate backbone represents an original pharmacophore for CA inhibition. Therefore, the interactions of a representative

Adapted from Cadoni R, Pala N, Lomelino CL, Mahon BP, McKenna R, Dallocchio R, Dessì A, Carcelli M, Rogolino D, Sanna V, Rassu M, Iaccarino C, Vullo D, Supuran C, Sechi M. (2017). Exploring Heteroaryl- pyrazole Carboxylic Acids as Human Carbonic Anhydrase XII inhibitors. ACS Med. Chem. Lett., 8(9), 941-946.

compound (1D) in complex with CA II were studied by X-ray crystallography and determined to 1.2 Å resolution (Figure 4-2A). Interestingly, 1D binding is unique from classical sulfonamide-based CAIs in that it does not bind directly to the active site zinc.

Instead, 1D anchors to the ZBW through the carboxylic acid moiety (2.6 Å). As such, the compound resides buried in the CA II active site and disrupts the ordered water network by displacing W1. 1D binding is primarily stabilized by interactions with residues adjacent to the catalytic zinc. The carboxyl oxygen of 1D forms hydrogen bonds with the backbone amides of both T199 and T200 (2.9 and 3.1 Å, respectively). In addition, the hydroxyl of the 1D ZBG interacts with an active site solvent molecule (3.2 Å) that bridges to the side chain of Q92 (3.1 Å). The indole tail of 1D is further stabilized by

VDW interactions with the hydrophobic pocket of the CA II active site, including residues

F131, V135, L141, L198 and P202 (Figure 4-2B).

Nicotinic and Ferulic Acid

Here we present the X-ray crystal structures of CA II in complex with two carboxylic acid-based inhibitors, nicotinic acid (NA) and ferulic acid (FA), and discuss properties of carboxylic acid-based CAIs that determine the mode of binding. As previously described, carboxylic acid-based inhibitors have been observed to bind multiple sites in and around the CA active site. Therefore, crystal structures of CA in complex with carboxylic acids deposited in the PDB were examined to rationalize inhibitor properties that contribute to the preferred mode of binding. But-2-enoic acids and compounds containing a carboxylic acid attached to a 5- or 6-member ring were observed to bind "indirectly" by anchoring to the ZBW at a distance of ~2.7 Å (Figure 4-

3A). Such compounds would be unable to bind directly to the zinc due to steric hindrance of active site residues such as L198, T200, and V121. Alternatively, the

inclusion of a linker between the carboxylic acid and inhibitor tail allows for rotation about the linker, preventing clashes and enabling direct binding to zinc (Figure 4-3B).

Based on these observed patterns, NA was predicted to inhibit CA II activity by anchoring to ZBW whereas FA was expected to bind directly to the zinc, displacing

ZBW.

The crystal structures of CA II in complex with NA and FA were determined to resolutions of 1.7 and 1.5 Å, respectively. Unambiguous electron density in the initial

Fo-Fc omit map was observed in the active site of each complex (Figure 4-4A, B).

Interestingly, both NA and FA were observed to anchor to the ZBW with the carboxylic acid of both compounds similarly oriented in comparison to previously determined anchoring carboxylic acid-based inhibitors. The carboxyl oxygen of both NA and FA was shown to form hydrogen bonds with the amide of T199 and hydroxyl of T200.

Additionally, the two inhibitors were further stabilized by van der Waals interactions with active site residues Q92, V121, F131, L198, and P202 (Figure 4-4C, D).

In order to understand the unexpected binding mode of FA, a model of FA interacting directly with zinc was generated using the structure of a similar acrylic acid- based compound (PDB accession code: 5EHW) as a template. This model shows that direct binding would result in obstruction of the methoxy group by active site residue

P201 or F131, depending on the orientation of the ring (Figure 4-5). Therefore, derivatization of the tail in linker-containing carboxylic acid-based compounds must also be taken into consideration during drug design.

Summary

Carboxylic acid-based compounds represent a promising class of non-classical

CAIs that exhibit multiple mechanisms of inhibition. First, the derivatization of an indole

and pyrazole scaffold was shown to have little effect on isoform selectivity. Then, several deposited structures were analyzed to understand the properties of carboxylic acid-based compounds that promote direct or "indirect" binding to the active site zinc.

The information gained from these studies provides guidance in the design of isoform specific CAIs. For example, the derivatization of aromatic compounds or tails of linker- containing inhibitors will promote anchoring the ZBW due to steric hindrance, increasing interactions with isoform unique residues that are more frequent extending radially outward from the active site zinc.

Figure 4-1. Structures of heteroaryl-pyrazole carboxylic acid inhibitors.

Table 4-1. Inhibition profiles of heteroaryl-pyrazole inhibitors against target CA IX and off-target CA II. Ki (μM) Compound CA II CA IX Selectivity Ratio 1A 1820 7.8 1000 1B 4.7 4.5 1 1C 0.7 19 0.04 1D 0.8 2.9 0.3 2A 6.7 0.5 13 2B 0.5 7.9 0.06 2C 0.8 7.4 0.1 2D 0.4 3 0.1

Figure 4-2. Binding of 1D in the active site of CA II. A) Electron density of 1D in a surface representation of CA II. The 2Fo−Fc electron density map is contoured to σ = 0.8. B) Interactions of 1D in the active site of CA II. Residues are labeled and hydrogen bonds shown as black dashes. .

Figure 4-3. Overlay of carboxylic acid CA inhibitors. A) Compounds that bind "indirectly" by anchoring to ZBW: 4-hydroxybenzoic acid (orange, PDB code: 4E3G), 2-hydroxybenzoic acid (blue, PDB code: 5M78), 2-sulfanylbenzoic acid (light purple, PDB code: 4E4A), 2,5-dihydroxybenzoic acid (dark purple, PDB code: 4E3D), 2,6-dihydroxybenzoic acid (dark pink, PDB code: 4E3F), 3- phenoxybenzoic acid (light pink, PDB code: 5FLT), (E)-3-(4-chlorophenyl)but- 2-enoic acid (gold, PDB code: 5FLS), (E)-3-(4-methoxyphenyl)but-2-enoic acid (green, PDB code: 5EH8), 3-(1-ethyl-1Hindol-3-yl)-1-methyl-1H-pyrazole- 5-carboxylic acid (yellow, PDB code: 6B4D). B) Compounds that bind directly to zinc and displace ZBW: (1,1-dioxido-3-oxo-1,2-benzothiazol-2(3H)-yl)acetic acid (raspberry, PDB code: 5CLU), 2-(4-ethoxypheyl)ethanoic acid (cyan, PDB code: 5FNJ), 2-(4-phenylmethoxyphenyl)ethanoic acid (olive, PDB code: 5FLQ), (E)-3-(2,4-dichlorophenyl)prop-2-enoic acid (teal, PDB code: 5EHW), (E)-3-[3-[[3-(2-hyddroxy-2-oxoethyl)phenyl]methoxy]phenyl]prop-2-enoic acid (purple, PDB code: 5EHV), cholic acid (light orange, PDB code: 4N16).

Figure 4-4. Binding of nicotinic acid and ferulic acid in the active site of CA II. Electron density of A) NA and B) FA in surface representations of CA II. The 2Fo−Fc electron density map is contoured to σ = 0.8. Interactions of C) NA and D) FA in the active site of CA II. Residues are labeled and hydrogen bonds shown as black dashes.

Figure 4-5. In silico model of ferulic acid directly binding to zinc. Note this would result in steric clashes with CA II active site residues F131 and P201 (colored red).

CHAPTER 5 SWEETENER-BASED INHIBITION OF CA IX

CA inhibitors with an alternative ZBG are being studied to deselect from the zinc so that binding affinity is dictated by interactions with active site residues, which are more likely to differ between CA isoforms. Saccharin, the artificial sweetener in “Sweet’

N Low”, was analyzed as a potential CA inhibitor due to the incorporation of a cyclic sulfonamide. Saccharin was shown to bind directly to the zinc and displace ZBW, exhibiting 60-fold selectivity for CA IX over CA II.83,84 Further interactions with active site residues H64, V131, and L198 were expected to contribute to CA IX selectivity. The selective inhibition of saccharin led to the examination of another sweetener, sucrose, as a potential CA inhibitor. Sucrose was determined to bind at the entrance of the active site, but not inhibit catalytic activity. The study of artificial sweeteners as CA inhibitors is continued in the following studies with the report of structure-activity relationships for acesulfame potassium and sucralose.

Acesulfame Potassium

Similar to saccharin, acesulfame K (aceK) incorporates a cyclic sulfonamide and exhibits 10-fold selectivity for CA IX over CA II (Figure 5-1, Table 5-1). The crystal structures of CA II and CA IX-mimic in complex with aceK were determined to 1.5 Å resolution in order to rationalize this difference in affinity (Figure 5-2A, B). Interestingly, aceK was observed to exhibit different binding modes between the two isoforms (Figure

5-2C). In CA IX-mimic, aceK binds directly to the zinc via the deprotonated nitrogen and

Adapted from Murray AB, Lomelino CL, Supuran CT, McKenna R. (2018). “Seriously Sweet”: Acesulfame K Exhibits Selective Inhibition Using Alternative Binding Modes in Carbonic Anhydrase Isoforms. J. Med. Chem., 57(7), 1096-1107.

displaces ZBW. As observed in other compounds containing a sulfonamide ZBG, the oxygen of the cyclic sulfonamide forms a hydrogen bond with the backbone nitrogen of

T199 (3.0 Å). The compound is further stabilized by a hydrogen bond between the carbonyl oxygen of aceK and the hydroxyl of T200 (3.4 Å) (Figure 5-2D). In contrast, aceK anchors to the ZBW via the deprotonated nitrogen in CA II (2.6 Å). This orientation shifts the sulfonamide oxygen away from T199, disrupting the hydrogen bond. However, the carbonyl oxygen of aceK forms hydrogen bonds with the hydroxyl group and backbone nitrogen of T200 (2.6 and 2.9 Å, respectively) (Figure 5-2E).

Two additional aceK molecules were observed to bind CA IX-mimic in the cleft

(site II) and on the surface (site III). These molecules exhibited higher B factors (site II:

36.9, site III: 29.5 Å2) in comparison to the active site aceK (site I: 14.6 Å2). The site II aceK is stabilized through a hydrogen bond with the backbone nitrogen of Y7 (2.8 Å) whereas the site III aceK binds within a hydrophobic pocket and is stabilized by VDW interactions. (Figure 5-3B)

The binding and interactions of sites II and III aceKs were conserved in CA II in addition to the binding of three more aceK molecules. One aceK molecule was observed at the entrance of the active site (site IV) and two additional molecules on the surface (sites V and VI). Again, the B factors of these sites (site II: 39.7, site III: 26.9, site IV: 41.5, site V: 39.9, site VI: 25.7 Å2) are higher than that of aceK in the active site

(site I: 19.8 Å2). The sulfonyl group of site IV aceK is bound ~7 Å from that of site I and is stabilized by hydrogen bonds with water molecules that further bridge to active site residues T200 and P201. In addition, the methyl group of site IV aceK interacts with

F131, which may stabilize this binding site and justify its observation in the crystal

structure. As residue 131 is replaced by a Val in CA IX-mimic, this interaction is lost and the site IV aceK molecule is not stabilized for observation in the complexed structure.

The sulfonyl and aromatic oxygens of site V aceK form hydrogen bonds with the side chain of K225 (2.6 and 3.5 Å, respectively). The sulfonyl and carbonyl oxygens of site VI aceK form hydrogen bonds with the backbone and side chain of K113 (2.9 and 2.6 Å, respectively). The observation of these transient aceK molecules was utilized to propose two possible pathways of aceK movement to and from the CA active site:

VIIIIVI or IIIVIIIVI. Sites III, V, and VI on the protein surface are expected to act as local minima when trafficking to the active site with site IV the penultimate position before binding within the active site (Figure 5-3A).

Active site water molecules have been proven to play an important role in the free energy of ligand binding (ΔG = ΔH - TΔS). In comparison to bulk solvent, water molecules in an enzyme active site are often entropically unfavorable due to interactions with the protein surface.85 Therefore, ligand binding and subsequent displacement of water molecules creates a more favorable environment. In both CA II and CA IX-mimic, site I aceK displaces DW, W1, and W2 of the proton wire. However, aceK also displaces the ZBW in CA IX-mimic, implying a more favorable binding energy to correlate to the observed increase in affinity.

The crystal structures of CA II and CA IX-mimic in complex with aceK were then

32 superimposed with the known CO2 binding site (PDB accession code: 3D92). In CA

IX-mimic, the sulfonyl oxygens of site I aceK occupy both oxygen positions of CO2.

Conversely, the sulfonyl oxygens of site I aceK in CA II only occupy one oxygen position of CO2 (Figure 5-4A). This observation implies that aceK binding in CA IX-mimic would

inhibit catalytic activity more effectively than in CA II. A competition assay was performed to determine if CO2 is capable of exhibiting partial occupancy and binding in the presence of aceK in CA II. Therefore, CA II crystals were incubated under high pressure CO2 prior to cryo-cooling and the crystal structures determined using X-ray crystallography (Figure 5-4B, C). Under 54 atm, CO2 binding shifted the site I and site IV aceK molecules 2-3 Å toward bulk solvent (Figure 5-4D). As pressure decreased to 34 atm, three orientations of aceK were observed with the site I aceK exhibiting partial occupancy with CO2 (Figure 5-4E). These structures suggest that aceK binding may not prevent the binding of CO2 in CA II, further rationalizing the observed differences in inhibitory potential between the two isoforms.

Sucralose

Here, the X-ray crystal structure of CA IX-mimic in complex with sucralose is presented at ~1.5 Å resolution and compared to the binding of aforementioned sweeteners (saccharin, sucrose, and aceK) in order to identify interactions that promote preferential binding. Unambiguous electron density in the initial Fo− Fc omit map was observed at the entrance of the active site (Figure 5-5A). Sucralose binding is primarily stabilized through hydrogen bonds with residues on the hydrophilic side of the active site. More specifically, a hydrogen bond was observed between the C3 hydroxyl of the fructofuranose moiety and Q92 (3.2 Å). Additionally, several hydrogen bonds are observed between hydroxyl groups of sucralose and solvent molecules in the active site, which bridge to active site residues Q67 and T200. Sucralose is further stabilized

Adapted from Lomelino CL, Murray AB, Supuran C, McKenna R. (2018). Sweet Binders: Carbonic Anhydrase IX in Complex with Sucralose. ACS Med. Chem. Lett., 9(7), 657-661.

by VDW interactions with residues on the hydrophobic side of the active site, including

L91, V121, V131, and L141 (Figure 5-5B). The CO2 binding site was not impeded by sucralose binding and the proton wire remained conserved. In the presence of sucralose, there is still a small opening of ∼ 6 Å on the hydrophilic side of the active site entrance, but it is energetically unfavorable for CO2 to enter through this opening due to its hydrophobic nature (Figure 5-6A). Therefore, inhibition of CA activity is most likely a result of the obstruction of the hydrophobic side of the active site, inhibiting the entry of

CO2.

To further annotate the binding of sucralose, it was compared to the structures of

CA IX-mimic in complex with sucrose, saccharin, and aceK.84,86 The sweeteners containing a cyclic sulfonamide bind further in the active site and exhibit a smaller buried surface area in comparison to the dissacharides. Furthermore, the cyclic sulfonamides displace more active site water molecules, including two waters of the proton wire (ZBW and W1), and form fewer hydrogen bonds than the disaccharides.

Sucralose binding was further compared to sucrose due to their common chemical structures.86 Sucralose is produced by substituting chlorine atoms for hydroxyl groups at the C4 and C1, C6 positions of the glucopyranose and fructofuranose rings in sucrose, respectively.87 Both compounds bind in a similar position outside the active site and interact primarily through hydrogen bonding, not displacing the solvent molecules of the proton wire. Additionally, both sucralose and sucrose maintain VDW interactions with residues in the hydrophobic cleft. Interestingly, sucralose inhibits CA IX activity with a micromolar binding affinity whereas sucrose is able to bind but does not inhibit activity

(Table 5-1). This difference in inhibitory potential is most likely attributed to the

aforementioned chlorine atom substitutions. In comparison to sucrose binding, the fructofuranose ring of sucralose is rotated nearly 180° and shifted ~4-5 Å toward the hydrophobic pocket. This conformational shift is likely caused by the increase in bond length of C−Cl (1.8 Å) in comparison to C−OH (1.4-1.5 Å), which induces the rotation of the ring to prevent steric clashes with active site residues. This reorientation also allows the formation of interactions between hydrophobic chloride ions of the fructofuranose ring and residues of the hydrophobic pocket.88,89 These interactions are not observed in sucrose binding, leaving an opening along the hydrophobic pocket through which CO2 could enter to initiate catalysis (Figure 5-6B).

Summary

Sweetener-based compounds have been studied due to their inhibition of CA activity and selectivity for target CA IX. The cyclic sulfonamides saccharin and ace K were shown to bind in the CA IX active site and interact directly with the zinc, inhibiting activity by displacement of the ZBW. In contrast, disaccharides sucralose and sucrose binding were observed at the entrance of the active site. The insights gained from the structural comparisons of saccharin, ace K, sucrose, and sucralose binding can be utilized to rationalize structure-guided drug design of CA IX specific inhibitors. The fructofuranose ring of a disaccharide was confirmed to orient toward the active site and interact with residues in the selective pocket, determining the specificity of binding. A disaccharide can therefore be utilized as a lead compound and the stereochemistry or composition of the six membered ring sugar altered to optimize hydrogen bonding with isoform specific residues. Combining such compounds with sweeteners that bind directly to the catalytic zinc demonstrates the potential to develop a single compound that inhibits CA activity through two simultaneous mechanisms.

Futhermore, sweeteneres are attractive lead molecules for drug development as these compounds have already been approved for safe human consumption (Title 21

US Code of Federal Regulations (CFR) Sec. 172.800 (aceK), 172.831 (sucralose),

180.37 (saccharin), and 184.1854 (sucrose)). Additional benefits of sweetener-based compounds include favorable properties for delivery and digestion. Sweeteners are typically water-soluble but can also be modified to ensure impermeability of the cell membrane to select for the extracellular catalytic domain of CA IX, avoiding off -target inhibition of cytosolic CAs. In addition, artificial sweetners such as sucralose are not naturally digested and have therefore been optimized for stability in the range of conditions and temperatures experienced during digestion. Overall, sweetener-based compounds provide a promising class of lead compounds for the design of CA IX specific inhibitors.

Figure 5-1. Structures of sweetener CA inhibitors.

Table 5-1. Inhibition profiles of sweeteners against CA II and CA IX. Ki (nM) Compound CA II CA IX Selectivity Ratio Saccharin 6,000 100 60 Acesulfame K >20,000 2,400 8 Sucrose >20,000 >20,000 1 Sucralose 300 2,200 0.1

Figure 5-2. Binding of acesulfame potassium in the active site of CA II and CA IX- mimic. Electron density of aceK in surface representations of A) CA II and B) CA IX-mimic. The 2Fo−Fc electron density map is contoured to σ = 0.8. C) Overlay of aceK binding orientations in CA II and CA IX-mimic. Interactions of aceK in the active sites of D) CA II and E) CA IX-mimic. Residues are labeled and hydrogen bonds shown as black dashes.

Figure 5-3. Secondary binding sites of acesulfame potassium. Six molecules are bound in A) CA II whereas only three were observed in B) CA IX-mimic.

Figure 5-4. Competitive binding of acesulfame potassium and CO2 in CA II. A) model superimposing aceK binding with CO2 binding site (PDB: 3D92). Electron density of aceK molecules under B) 54 atm and C) 34 atm pressurized CO2. The 2Fo−Fc electron density map is contoured to σ = 0.8. Superimposition of D) 54 atm and E) 34 atm structures with AceK binding sites in the absence of CO2 (pink).

Figure 5-5. Binding of sucralose in the active site of CA IX-mimic. A) Electron density of sucralose in surface representation of CA IX-mimic. The 2Fo−Fc electron density map is contoured to σ = 0.8. B) Interactions of sucralose in the active site of CA IX-mimic. Residues are labeled and hydrogen bonds shown as black dashes.

Figure 5-6. Depiction of CO2 pathway to the active site of CA IX-mimic. Sphere representation of A) sucralose (pink) and B) sucrose (yellow) with the size of each sphere representative of the van der Waals radii of that atom. Proposed CO2 pathways are indicated by gray arrows.

CHAPTER 6 FRAGMENT-BASED INHIBITION OF CA IX

Fragmentation studies have shown that the most potent inhibitors result from the combination of small molecules. The identification of an inhibitor binding site at the entrance of the CA active site has led to the use of a fragment-based approach. Using this technique, small molecules that inhibit CA activity by binding to different areas in and around the active site are chemically linked to synthesize a single compound.90,91

Such inhibitors are expected to be more efficacious by simultaneously displacing the

ZBW and obstructing substrate entry into the active site. Therefore, the combination of a

ZBG linked to a carbohydrate tail has the potential to produce CA IX selective inhibitors.

Modifications of the chemical linker would provide the spacial requirements to optimize interactions with residues in the selective pocket of the active site, improving selectivity for CA IX and preventing off-target inhibition of CA II.

Sulfamate

Glycoconjugates have been proposed as a promising class of CA IX inhibitors due to their poor membrane diffusion properties, targeting the extracellular catalytic domain of CA IX.92 Therefore, inhibitors were designed that combine a sulfamate ZBG with a glucose carbohydrate tail. Three compounds were synthesized using the following linkers: n-pentyl chain (623), pyrrolidine ring (625), and piperidine ring (626)

(Figure 6-1). Acetylation of the glucose tail allows these compounds to exhibit properties of ester prodrugs. A prodrug is a compound that exhibits little to no activity until it is

Adapted from Mahon BP, Lomelino CL, Ladwig J, Rankin GM, Driscoll JM, Salguero AL, Pinard MA, Vullo D, Supuran CT, Poulsen SA, McKenna R. (2015). Mapping Selective Inhibition of the Cancer-Related Carbonic Anhydrase IX Using Structure-Activity Relationships of Glucosyl-Based Sulfamates. J. Med. Chem., 58(16), 6630-6638.

transformed to an active molecule through chemical or enzymatic cleavage.93 The acetyl groups mask the hydrophilic hydroxyls of glucose to improve oral bioavailability.94

The ester bonds are then hydrolyzed in the bloodstream to produce the active compound. This hydrolysis reaction exposes the hydrophilic hydroxyl groups, preventing passive diffusion through the cell membrane, which further improves selectivity for the extracellular catalytic domain of CA IX.43

These compounds were shown to be potent inhibitors of CA IX with nanomolar binding affinities (Table 6-1). X-ray crystallography was used to study the binding of these three compounds and rationalize the differences in the selectivity ratios for CA IX over CA II (Figure 6-2A, B, D, E, G, H). In both CA II and CA IX-mimic, the deprotonated nitrogen of the sulfamate group binds directly to zinc, displacing ZBW. A sulfonyl oxygen also forms a hydrogen bond with the backbone amide of T199 (2.7-3.0 Å).

Because the tail group is conserved between the three compounds, the differences in the selectivity ratio can be directly attributed to the linker.

Compound 623 is the least selective for CA IX with a ratio of only ~5-fold over CA

II (Table 6-1). Therefore, it is not surprising that 623 binds in a similar orientation between the two isoforms (Figure 6-2C). In both isoforms, 623 binding displaces ZBW,

DW, and DW′. In CA II, the NH of the sulfonamide linker forms a hydrogen bond with an active site solvent molecule that bridges to the backbone of P201. The acetyl group at position 6 of the glucose ring also bonds with W5 (2.8 Å) (Figure 6-2J). In CA IX-mimic, the sulfamate oxygen interacts with a water molecule that is stabilized by the side chain of T199. The acetyl group at position 6 of the glucose ring also forms a hydrogen bond with the backbone of P201 (3.5 Å) (Figure 6-2K). Interactions with residues 5, 20, 92,

121, 131, 135, 141, 198, 202 further stabilize 623 binding. The length of the linear pentyl linker causes the carbohydrate tail to extend out of the active site, limiting interactions with isoform unique residues.

Compound 625 shows the highest selectivity (~400-fold) for CA IX over CA II and also shows the greatest difference in binding orientation between the isoforms (Table 6-

1, (Figure 6-2F). In CA IX-mimic, the compound extends from the active site along the hydrophobic region whereas 625 bends to interact with residues in the hydrophilic half of the active site. 625 binding displaces all water molecules of the proton wire except

W2 and W3a in CA IX-mimic whereas W2 is also displaced in CA II. In CA IX-mimic, the five-membered pyrrolidine linker group forms a hydrogen bond with the side chain of

H64 (3.2 Å). This interaction further inhibits CA catalytic activity by preventing proton transfer. In addition, a hydrogen bond is formed between the OH at position 2 of the glucose moiety with Q92 (2.8 Å). VDW interactions are observed with hydrophobic residues L91, V121, V131, V135, L141, and L198 (Figure 6-2L). The difference in orientation of compound 625 in CA II results in the loss of hydrogen bonding with H64 and Q92. However, the acetyl group at position 6 of the glucose ring interacts with the amide of N62 (3.0 Å). Similar to binding in CA IX-mimic, 625 forms VDW interactions with residues I91, V121, F131, L141, L198, and P202 (Figure 6-2M). Residue 131 (Phe in CA II and Val in CA IX) is predicted to significantly impact the affinity of compound

625. The decrease in steric hindrance of V131 allows 625 to reorient and bind in a more favorable conformation.

Compound 626 shows ~130-fold selectivity for CA IX over CA II with the most significant observable difference in the orientation of the glucose tail (Table 6-1, Figure

6-2I). In both isoforms, there is a displacement of DW, DW′, ZBW, and W1 upon 626 binding. In CA II, the sulfonyl linker of 626 is stabilized by hydrogen bonds with N67 and

Q92 (3.3 and 2.6 Å, respectively). In addition, 626 exhibits VDW interactions with V121,

F131, V135, L198, P202, and L204 (Figure 6-2N). In CA IX-mimic, the sulfonyl linker of

626 forms a hydrogen bond with the amide of Q92 (3.1 Å). The acetyl group at position

2 of the glucose ring also forms a weak bond with H64 (3.5 Å). VDW interactions also occur between the glucose ring and active site residues L91, V121, V131, V135, L141 and P202 (Figure 6-2P). Similar to compound 625, the decrease of steric hindrance of

V131 in comparison to F131 allows reorientation of the 626 tail to bind in a more favorable conformation in CA IX-mimic, resulting in the formation of the hydrogen bond with H64.

Cleavage of the acetyl groups on the glucose tail was observed throughout the crystal structures. However, the extent and positions of such cleavage was not conserved between the isoforms. CA exhibits weak esterase activity and it is possible that this activity is more prevalent in CA II. For example, compound 623 is deacetylated at positions 3 and 4 CA II whereas the acetyl groups are conserved in CA IX-mimic. The only acetyl group that remains in compound 625 is at position 6 (CA II). Lastly, 626 remains acetylated at position 6 in CA II and position 2 in CA IX-mimic. It is unknown if the arrangement of an acetyl group on the glucose ring determines the propensity for cleavage and how this may translate to the measured Ki values of the acetylated compounds.

Cyclic Sulfonamide

Based on the CA IX selectivity exhibited by artificial sweeteners, a saccharin- based compound (SBC) was designed that combines a cyclic sulfonamide ZBG,

saccharin, with a carbohydrate tail, glucose, through a triazole linker. This compound was shown to exhibit >1000-fold selectivity for CA IX over CA II, which is significantly higher than the SLC-0111 compound currently in clinical trials.95 Following these studies, two new derivatives were designed that replace the glucose tail of SBC with galactose. The linker component of these new compounds incorporates a one (GAL) or two carbon (ABM) spacer between the triazole and galactose tail (Figure 6-3). The freedom of rotation about the two carbon linker of ABM is predicted to promote the formation of interactions between the galactose tail and isoform unique residues surrounding the CA IX active site, further improving selectivity for CA IX.

X-ray crystallography was utilized to study the binding of ABM in complex with

CA II and CA IX-mimic and the structures were both determined to 1.4 Å resolution, respectively (Figure 6-4A, B). As observed in the crystal structures of CA in complex with aceK, ABM exhibits a different mechanism of binding based upon the isoform

(Figure 6-4C). The saccharin ZBG binds directly to zinc via the deprotonated nitrogen in

CA IX-mimic, displacing ZBW. ABM displaces DW, DW′, and W1 in CA IX-mimic. As observed for sulfonamide-based CA inhibitors, a sulfonyl oxygen of saccharin forms a hydrogen bond with the backbone amide of T199 (3.0 Å). The linker is stabilized by a solvent molecule that bridges to Q92 (2.5 and 3.1 Å, respectively) (Figure 6-4D).

Alternatively, ABM anchors to the ZBW in CA II. ABM binding also displaces the remaining waters of the proton wire except W3a. This change in orientation disrupts the hydrogen bonding of the sulfonyl oxygen, however the carbonyl of the saccharin ZBG is within hydrogen bonding distance of the T199 backbone amide and T200 side chain

(2.9 and 2.7 Å, respectively). The triazole linker is stabilized through an interaction with

Q92 (2.6 Å) (Figure 6-4E). An overlay of ABM in the two isoforms shows that the tail is oriented in the hydrophilic region of the active site in mimic whereas hydrophobic in CA

II, which is expected to be more favorable in the mimic due to the polar nature of galactose (Figure 6-4C). Were ABM to bind directly to the zinc in CA II, the rigidity of the saccharin ZBG directly attached to the triazole linker would cause a steric clash with

F131. Although the affinity has yet to be measured, ABM is expected to have higher affinity for CA IX due to the direct zinc binding and more favorable orientation of the galactose tail.

Increasing concentrations of ABM, GAL, and lead compound saccharin were then tested for their effect on cell viability using an MTT assay. These measurements were performed in the UFH-001 breast cancer cell line. The efficacy of ABM and GAL were also compared to the previously synthesized compound, SBC, which contains the glucose tail. Though not statistically significant, a general trend was observed for all four

CAIs in the UFH-001 cell line- cell growth was observed to slightly increase until an inhibitor concentration of 10 mM (saccharin) or 10 uM (ABM, GAL, SBC), after which cell viability decreased in a dose dependent manner (Figure 6-5A, B).

Saccharin and ABM were then tested for their effect on migration and invasion of the breast cancer tumor cell line. As the greatest effect on cell viability was observed above 10 mM saccharin (maximum tolerated dose is 270 mM), concentrations of 10,

100, and 200 mM saccharin were tested. Similarly, migration and invasion were measured at 100 uM and 1 mM ABM because ABM was shown to decrease cell viability above 10 uM. Saccharin was shown to significantly decreases both migration and

invasion in a dose dependent manner (Figure 6-6A, B). In contrast, ABM had little effect on migration but was shown to significantly decreases invasion (Figure 6-6C, D).

Summary

A fragment approach was used to rationally design CA inhibitors that simultaneously inhibit catalytic activity through two mechanisms using a single compound- displacing ZBW and obstructing substrate entry into the active site.

Furthermore, the length of the linker can be modified so that the tail binds at the entrance of the active site where the majority of isoform unique residues reside, promoting interactions and improving selectivity. Two sets of compounds were designed incorporating either a sulfamate or cyclic sulfonamide ZBG chemically linked to a carbohydrate tail. The length and identity of the linker was shown to greatly impact binding affinity. Furthermore, steric hindrance of residue 131 dictates orientation of the compound tail, allowing for more favorable interactions in CA IX. Both sets of compounds conserved the ZBG and tail components, indicating that observed differences in binding affinities and orientations were a direct result of the linker modifications. However, these compounds were synthesized using a click chemistry scheme and the carbonhydrate tail can easily be changed to a different sugar in order to develop a full series of CAIs. As previous studies have shown that sweetners that bind the entrance of the active site exhibit significantly different affinities for CA IX even when binding in a similar location, it is important to test inhibitors that also vary the tail component of the compound.

Figure 6-1. Structures of sulfamate CA inhibitors.

Table 6-1. Inhibition profiles of sulfamate inhibitors against CA II and CA IX. Ki (nM) Compound CA II CA IX Selectivity Ratio 623 10 2 5 625 725 2 363 626 265 2 133

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Figure 6-2. Binding of sulfamate inhibitors in the active sites of CA II and CA IX-mimic. Electron density of 623 in surface representations of A) CA II and B) CA IX- mimic; 625 in D) CA II and E) CA IX-mimic; 626 in G) CA II and H) CA IX- mimic. The 2Fo−Fc electron density map is contoured to σ = 0.8. Overlay of C) 623, F) 625, and I) 626 binding orientations in CA II and CA IX-mimic. Interactions of 623 in the active sites of J) CA II and K) CA IX-mimic; 625 in L) CA II and M) CA IX-mimic; 626 in N) CA II and P) CA IX-mimic. Residues are labeled and hydrogen bonds shown as black dashes.

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Figure 6-2. Continued

102

Figure 6-3. Structures of cyclic sulfonamide CA inhibitors.

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Figure 6-4. Binding of ABM in the active sites of CA II and CA IX-mimic. Electron density of ABM in surface representations of A) CA II and B) CA IX-mimic. The 2Fo−Fc electron density map is contoured to σ = 0.8. C) Overlay of ABM binding orientations in CA II and CA IX-mimic. Interactions of ABM in the active sites of D) CA II and E) CA IX-mimic. Residues are labeled and hydrogen bonds shown as black dashes.

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Figure 6-5. MTT assays for increasing concentrations of CAI.

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Figure 6-6. Migration and invasion assays of lead compound saccharin and ABM.

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CHAPTER 7 CONCLUSION

CA IX is overexpressed in hypoxic tumors and recognized as a biomarker and therapeutic target in breast cancer. High expression levels of CA IX have been shown to correlate to shorter overall, progression free, and metastasis free survival. Inhibition of

CA IX activity has been shown to result in decreased tumor volume and improved survival in mouse models, likely due to the essential role of CA IX in pH regulation. CAIs currently available in the clinic lack selectivity for CA IX, resulting in off-target binding and sequestration of the inhibitor. This work has explored several classes of CAIs, including sulfonamides, carboxylic acids, and artificial sweeteners, and discussed inhibitor properties that improve CA IX selectivity in order to guide the design of a new

CA IX inhibitor.

Sulfonamide-based compounds are considered the classical CA inhibitors and represent the most common class of studied compounds with one inhibitor, SLC-0111, currently in clinical trials. However, these compounds do not exhibit sufficient selectivity for CA IX over off-target CA II. Three subclasses of sulfonamide CAIs were structurally characterized. Overall, the length and flexibility of the linker component in addition to steric hindrance of active site residues were shown to greatly impact binding. A new derivative of SLC-0111 was shown to improve CA IX selectivity by modifying the linker and increasing hydrogen bonding. Another technique used to improve the efficacy of CA

IX inhibitors was molecular hybridization, which targets multiple biological processes with a single compound. Inhibitors were designed that combined a sulfonamide ZBG with pyrimidine and purine tails to simultaneously inhibit CA IX activity and DNA

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synthesis. Lastly, multi-tail inhibitors were shown to increase the number of interactions with isoform unique residues.

Research is now moving toward nonclassical compounds that deselect from zinc binding and improve isoform selectivity by promoting interactions with isoform unique residues. Carboxylic acid-based compounds were shown to exhibit multiple mechanisms of inhibition and substituents shown to dictate whether a compound binds directly to zinc or anchors to zinc-bound water.

Aritificial sweeteners such as the cyclic sulfonamide aceK was shown to demonstrate different binding modes between isoforms. Alternatively, disaccharides such as sucralose bind the entrance of the CA active site. Therefore, a fragment approach was employed to design new CA IX inhibitors that simultaneously inhibit CA activity by displacing ZBG and occluding substrate entry. Furthermore, one such inhibitor was shown to decrease both cell viability and invasion in a breast cancer cell line. Due to the click chemistry synthetic scheme, the carbohydrate tail can be easily changed to expand the series and test a number of inhibitors.

Although structure-guided drug design has led to several classes of high affinity

CAIs, the majority of these compounds still do not exhibit sufficient isoform selectivity to prevent off-target binding and subsequent seqeuestration of the inhibitor. Therefore, interactions between an inhibitor tail and the four residues of the selective pocket do not adequately differentiate between the fifteen human α CAs. Structural biologists are beginning to move beyond the active site and map residues on the surface of the enzyme (Table 7-1). Residues in and around the CA active site can be divided into three zones, 5-10 Å, 10-15 Å, and 15-20 Å extending radially from the catalytic zinc

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(Figure 7-1). With the exception of residue 67, the majority of residues in the 5-10 Å zone are conserved between the isozymes. This observation is reinforced by the lack of selectivity observed for compact CAIs that bind deep within the active site. The 10-15 Å zone contains the selective pocket and has therefore been the targeted region for drug design over the past several years. However, the number of isoform unique residues increases even further in the 15-20 Å zone. In addition to the functionalization of a compound tail, the extension of the linker and/or tail components of CAIs is therefore predicted to further improve selectivity by promoting interactions with the unique residues beyond the active site. For example, the crystal structure of CA II in complex with a benzene sulfonamide inhibitor, GV1-89, demonstrates that the tail of this compound orients between the phobic and philic regions of the active site, extending toward the 15-20 Å zone (Figure 7-2A). An extension of the inhibitor tail and addition of hydrophobic substituents could promote interactions with P72 and L123 due to the decrease in steric hindrance of V131 in CA IX (Figure 7-2B). Following this hypothesis, the crystal structures of CA II and CA IX-mimc were solved in complex with a benzene sulfonamide CAI, AN11-741, which contains a longer linker and hydrophic tail (Figure 7-

3A, B). A superposition of the inhibitor in the two isoforms shows that that tail binds in significantly different orientations, extending between the hydrophobic and hydrophilic regions in CA II and bending into a hydrophobic pocket in CA IX-mimic (Figure 7-3C).

VDW interactions with residues in the hydrophobic region are shown to be important for stabilizing the tail in the CA IX-mimic active site whereas the longer, my hydrophilic side chains of 69 and 72 in CA II allow the formation of hydrogen bonds (Figure 7-3D, E).

These structures also highlight how the flexibitliy of the linker component is important

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and can allow the inhibitor to reorient and bind in the most energetically favorable conformation.

However, as inhibitors are designed to form interactions with isoform unqiue residues beyond the active site, the CA IX-mimic will no longer be useful for structural characterization as the majority of mutations, with the exception of residue 170, are located in zones 1 and 2. Recently, a CA IX surface variant was designed that mutates six residues on the surface of the CA IX catalytic domain in order to remove the disulfide that stabilizes dimerization (C174S), improve solubility (L183S and M350S), and mimic residues on the surface of CA II (A213K, A258K, and F259Y). These mutations correspond to residues 41, 50, 217, 80, 127, and 128 in the CA II sequence, respectively.96

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Table 7-1. Isoform unique active site residues of CA II and CA IX. Residue CA II CA IX 5-10 Å 65 Ala Ser 67 Asn Gln 10-15 Å 60 Leu Arg 69 Glu Thr 91 Ile Leu 131 Phe Val 135 Val Leu 204 Leu Ala 15-20 Å 19 Asp Val 20 Phe Ser 22 Ile Ala 72 Asp Pro 123 Trp Leu 130 Asp Arg 132 Gly Asp 136 Gln Gly 170 Lys Glu 173 Ser Glu

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Figure 7-1. Zones of human α CAs. The three colored zones represent active site residues in human CAs located 5-10 (pink), 10-15 (blue), and 15-20 (yellow) Å from the catalytic zinc.

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Figure 7-2. Binding of GV1-89 in the active site of CA II. A) Electron density of GV1-89 in surface representations of CA II (zones colored as in Figure 7-1). The 2Fo−Fc electron density map is contoured to σ = 0.8. Interactions of GV1-89 in the active site of CA II. Residues are labeled and hydrogen bonds shown as black dashes.

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Figure 7-3. Binding of AN11-741 in the active sites of CA II and CA IX-mimic. Electron density of AN11-741 in surface representations of A) CA II and B) CA IX- mimic. The 2Fo−Fc electron density map is contoured to σ = 0.8. Overlay of AN11-741 binding orientations in CA II and CA IX-mimic. Interactions of AN11-741 in the active sites of D) CA II and E) CA IX-mimic. Residues are labeled and hydrogen bonds shown as black dashes.

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APPENDIX A MECHANISM OF WATER NETWORK REPLENISHMENT

CA II is a zinc metalloenzyme that catalyzes the reversible hydration of CO2.

Although the proton-transfer process has been extensively studied, its underlying mechanism is not fully understood. Here, crystallographic structures of CA II cryocooled under CO2 pressures of 7.0 and 2.5 atm reveal new intermediate solvent states that provide crystallographic “snapshots” during the restoration of the water network in the active site. Specifically, a new intermediate water (WI′) is observed stabilized by five water molecules at the entrance to the active site (the entrance conduit, EC). Based on these structures, a water network replenishment mechanism is proposed following the nucleophilic attack of CO2. This mechanism explains how the ZBW and W1 are replenished to reconnect the proton wire which is broken upon CO2 binding. This study provides the first “physical” glimpse of a water reservoir flowing into the CA II active site during catalytic activity.

Overview

The reversible interconversion of CO2 and water to bicarbonate and a proton occurs at a rate limited by diffusion in the presence of the zinc metalloenzyme CA

17,97–99 II. In the hydration direction, the nucleophilic attack of CO2 by the zinc-bound hydroxide generates bicarbonate, which is subsequently displaced by a water molecule.27 The next step of catalysis is the transfer of a proton from the ZBW to bulk solvent, regenerating the catalytic zinc-bound hydroxide.

Adapted from Kim JK, Lomelino CL, Avvaru BS, Mahon BP, McKenna R, Park SY, Kim CU. (2018). Active site solvent replenishment observed during human carbonic anhydrase II catalysis. IUCrJ., 5(1), 93-102; Lomelino CL, Andring JT, McKenna R. (2018). Structural Insight into the Catalytic Mechanism of Carbonic Anhydrase. In Carbonic Anhydrases: Biochemistry, Mechanism of Action and Therapeutic Applications; Penttinen J; Nova Publishers, 71-110, ISBN 978-1-53613-262-5.

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The first crystal structure of CA II was determined by Liljas and coworkers in

1972 and was further refined in 1988.24,25 These studies laid the foundation for understanding the mechanism of CA activity. In CA II, the active-site zinc is located within a 15 Å deep cleft and is tetrahedrally coordinated by three His residues (H94,

H96 and H119) and a hydroxide ion/water molecule. Furthermore, the active site can be divided into two distinct sides, formed by hydrophilic residues (Y7, AN62, H64, N67,

T199, and T200) and hydrophobic residues (V121, V143, L198, V207 and W209). The

100 hydrophobic side sequesters and positions CO2 for nucleophilic attack. The hydrophilic side stabilizes an ordered water network that spans the distance between the catalytic zinc and H64 proton shuttle residue predicted to facilitate proton transfer to bulk solvent (Figure A-1).25

The binding site of CO2 was structurally determined by cryocooling CA II crystals under 15 atm pressurized CO2 gas to induce complex formation. Such high CO2 concentrations create an acidic pH that ensures a water molecule is bound to the catalytic zinc, preventing catalysis during data collection. As predicted in previous MD simulations and mutagenesis experiments, CO2 was observed to bind within the hydrophobic region of the CA II active site, displacing DW. A weak hydrogen bond with the amide nitrogen of T199 optimizes the orientation of CO2 for nucleophilic attack by the zinc-bound hydroxide. CO2 binding is conserved in the absence of zinc, indicating that binding and orientation of CO2 relies solely on interactions with residues in the hydrophobic pocket. A secondary CO2 molecule was observed to bind in a hydrophobic region outside the active site, displacing the phenyl ring of F226.32,101

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The proton wire is made up of five water molecules (ZBW, W1, W2, W3a, W3b) that are stabilized through a hydrogen bonding network, interacting with residues located in the hydrophilic region of the active site (Y7, N62, N67, T199, and T200). The localization of these water molecules remains conserved over pH range 5.1–10.0. This hydrogen-bonded network is believed to act as a proton wire that reduces the work required to transfer a proton from zinc to bulk solvent.37,102–107

Hydrogen bonding interactions within the proton wire were first hypothesized based on the high-resolution crystal structure of wild type CA II.34 DW is positioned the

- deepest into the active site pocket and interacts with ZnH2O/OH (2.5 Å) in the absence of substrate. The hydrogen bond between the zinc-bound hydroxide and hydroxyl of

T199 (2.7 Å) orients the lone pair of electrons on the oxygen for nucleophilic attack of

108 - CO2. ZnH2O/OH also interacts with W1 (2.6 Å), which is further stabilized by T200

(2.8 Å). W1 coordinates to W2 (2.7 Å), which then branches off in opposite directions to

W3a and W3b (2.7 Å). W3a is stabilized by Y7 (2.8 Å) whereas W3b anchors to N62

(3.0 Å) and N67 (2.7 Å).28 Waters W2 and W3a also stabilize the in conformation of H64 through weak hydrogen bonds (3.3 Å).37 W2 was the only water molecule observed to lie in the plane of the H64 imidazole ring and was therefore predicted to be responsible for the final transfer of the proton to ND1 of H64.102 However, there was debate over the role of W2 since the distance between the water molecule and the H64 imidazole ring

(in conformation) was too far for the formation of a hydrogen bond (3.8 Å).

Although complete electron density was observed for the placement of proton wire water molecules, X-ray crystal structures could not confirm the hypothesized hydrogen bonding patterns. Therefore, neutron crystallography was utilized to

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determine the orientation of water molecules, which was determined to be pH dependent. At pH 10.0, W1 serves as a hydrogen bond donor to both DW and T200, resulting in a broken wire. The deuterium atoms on W2, W3a, and W3b are oriented toward the imidazole ring of H64 occupying the in conformation but do not form hydrogen bonds as previously predicted.29 However, the orientation of W1 changes near physiological pH (pH 7.8). W1 now acts as a hydrogen bond acceptor of T200 and forms a hydrogen bond with W2. Consequently, W2 and W3a reorient in a concerted fashion to complete the proton wire. W2 now behaves as a hydrogen bond donor to

- H64, indicating that the most likely path for proton transfer is ZnH2O/OH -W1-W2-H64.

Reorientation of active site water molecules was hypothesized to be caused by changes in protonation states of residues in the hydrophilic half of the active site, such as H64 and Y7.109

CA II crystal structures collected over a range of pH values have shown that H64 exhibits two conformations termed the “in” and “out” positions, which orient toward and away from the active site, respectively. Neutron studies demonstrated that H64 is uncharged when occupying the in position and the deuterium atom of ND1 faces away from the active site. This orientation and protonation state prime the imidazole ring for the acceptance of a proton, which is subsequently transferred to bulk solvent.29 The pH- induced movement of H64 highlights the flexibility necessary to facilitate proton transfer between the zinc-bound water and bulk solvent.

As previously mentioned, the capture of CO2 in the active site of CA II was

32,110 achieved by cryocooling CA II crystals under a 15 atm CO2 pressure. More recently, attempts have been made to track the intermediate changes during gradual

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CO2 release by incubating 15 atm CO2-pressurized CA II crystals at RT for various time intervals (50 s, 3 min, 10 min, 25 min and 1 h) prior to flash cooling, resulting in

111 decreases in the internal CO2 pressure. The structures revealed that two waters (DW and DW′) replace the vacated space as CO2 leaves the active site. In addition, a new, ordered water molecule, termed intermediate water (WI), was observed in the crystal

32 structure of CA II under 15 atm CO2. WI is too far from W2 to form a hydrogen bond

(4.8 Å), so the wire is broken upon CO2 binding. In the presence of CO2, H64 primarily occupies the out conformation and a water molecule was observed between W2 and

H64, termed W2’. Electron density for W2’ diminished as CO2 released and the occupancy for the in conformation of H64 increased. The wire is restored as the partial pressure of CO2 decreases and WI shifts to the W1 position. It is therefore plausible to

- 111 suggest a completed proton wire consisting of ZnH2O/OH -W1-W2-W2’-H64.

In this study, we present structures of CA II from crystals cryocooled under CO2 pressures of 7.0 atm and 2.5 atm, referred to as ‘7.0 atm CA II’ and ‘2.5 atm CA II’, respectively. These two structures are compared with previous structures of CA II crystals cryocooled under 15 atm CO2 pressure and then incubated at room temperature for 0 s (PDB: 5DSI; ‘15 atm CA II’), 50 s (PDB: 5DSJ; ‘15 atm CA II-50s’) and 1 h (PDB: 5DSN; ‘15 atm CA II-1h’). The structural comparison reveals that 7.0 atm

CA II and 2.5 atm CA II are previously unknown intermediate states between 15 atm CA

II and 15 atm CA II-50 s. Together, these studies provide a view of how CA II utilizes a water reservoir to fill the void in the active site as CO2 is released.

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Methods

Protein Expression and Purification

CA II was expressed in Escherichia coli BL21(DE3) transformed with a plasmid encoding the CA II gene. Purification was carried out using affinity chromatography as previously described.49 Briefly, cells were lysed and the lysate loaded onto agarose resin coupled with p-(aminomethyl)benzenesulfonamide. The protein was eluted with

400 mM sodium azide in 100 mM Tris followed by extensive buffer exchange into 10 mM Tris, pH 8.0.

Crystallization

Crystals of CA II were obtained using hanging-drop vapor diffusion. A 10 mL drop consisting of equal volumes of protein solution (5 mL) and well solution (5mL) was equilibrated against 1mL well solution (1.3 M sodium citrate, 100 mM Tris, pH 7.8) at

RT. Crystal growth was observed after 3 days.

CO2 Entrapment

32,111 CO2 entrapment was carried out as described in previous reports. The CA II crystals were first soaked in a cryosolution consisting of the reservoir solution supplemented with 20% (v/v) glycerol. The crystals were then coated with mineral oil to prevent dehydration and loaded into the base of high-pressure tubes. Once in the pressure tubes, the crystals were pressurized with CO2 gas to two different pressures

(7.0 and 2.5 atm) at room temperature. After 10 min, the crystals were cryocooled in liquid-nitrogen without releasing the CO2 gas. Once the CO2-bound crystals had been fully cryocooled, the crystal-pressurizing CO2 gas was released and the crystal samples were stored in liquid-nitrogen until data collection.

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Data Collection

Diffraction data was collected on CHESS beamline F1 (wavelength of 0.9180 Å).

Data were collected using the oscillation method in intervals of 1° on an ADSC

Quantum 270 CCD detector with a crystal-to-detector distance of 100 mm. For the 7.0 atm CA II data set (0.9 Å resolution), an initial data set consisting of 180 images was collected with 1 sec exposures to cover diffraction resolution up to 1.1 Å. The detector was then offset to cover diffraction resolution up to 0.88 Å, and a second data set consisting of 360 images was collected with 10 sec exposures. For the 2.5 atm CA II data set (1.0 Å resolution), a single data set consisting of 360 images was collected with

10 sec exposure for each image. Indexing, integration, merging and scaling were performed using HKL2000.112

Structure Determination and Refinement

The structures of CA II at CO2 pressures of 7.0 and 2.5 atm were determined using the CCP4 program suite.113 Prior to refinement, a random 5% of the data were flagged for Rfree analysis. The previously determined 1.1 Å resolution crystal structure

(PDB: 3D92) was used as the initial phasing model. Maximum-likelihood (MLH) refinement was carried out using REFMAC5 and the water molecules were automatically modeled using ARP/wARP during the MLH cycles.113,114 The refined structures were manually checked using the molecular graphics program

Coot.50Reiterations of MLH refinement were carried out with anisotropic B factors and riding H atoms. The partial occupancies of W1 in 7.0 and 2.5 atm CA II were estimated such that the electron density in the Fo-Fc map disappears. All structural figures were rendered in PyMOL.

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Results

CO2 Binding Sites

The overall structures are similar with all-protein-atom rmsd values between 15 atm CA II and the other four structures (7.0 atm CA II, 2.5 atm CAII,15 atm CAII-50s, and 15 atm CA II-1h) of 0.14, 0.12, 0.10 and 0.13 Å, respectively. As expected, differences in the electron densities for the CO2 binding sites were observed. The 15 and 7.0 atm CA II structures show unambiguous electron density in the initial Fo-Fc omit map for the active site CO2 (Figure A-2A, B). At 2.5 atm, electron density for the CO2 site is represented by sparsely connected lobes, suggesting that the CO2 site is partially occupied by both CO2 and two water molecules (DW and DW′) (Figure A-2C). In 15 atm

CA II-50s, the electron density for the CO2 binding site is further shifted towards Zn and

ZBW, correlating with the known positions of DW and DW′ (Figure A-2D). This argues that 2.5 atm CA II has a higher internal CO2 pressure than 15 atm CA II-50s. Finally, in

15 atm CA II-1h, the electron density becomes two distinct lobes, indicating that the

CO2 site is completely replaced by DW and DW′ (Figure A-2E).

A secondary binding site has been previously observed in a hydrophobic pocket created by V223 and F226.32 Comparison of the 15 atm CA II and 15 atm CA II-1h structures indicates that the F226 side chain must rotate to accommodate CO2 (Figure

A-3A, E). In the 7.0 atm CA II structure, decreased occupancy of CO2 results in dual conformations of the F226 side chain (Figure A-3B). In the cases of 2.5 atm CA II and

15 atm CA II-50s, the secondary CO2 was not present and the F226 side chain occupies the position observed in the 15 atm CA II-1h (Figure A-3C, D). These observations imply that 7.0 atm CA II has a higher internal CO2 pressure than 15 atm

CA II-50s.

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H64 and Water Network

As described above, structural examinations of the CO2 binding sites suggest that both 7.0 atm CA II and 2.5 atm CA II have a higher internal CO2 pressure than 15 atm CA II-50 s. Furthermore, 7.0 atm CA II intuitively has a higher internal CO2 pressure than 2.5 atm CA II, hence leading to the conclusion that the internal CO2 pressure decreases in the sequence 15 atm CA II, 7.0 atm CA II, 2.5 atm CA II, 15 atm CA II-50s,

15 atm CA II-1h. Such an interpretation ascertains that 7.0 atm CA II and 2.5 atm CA II are intermediate states that fill the gaps between the 15 atm CA II and the earliest time

111 point of CO2 release (15 atm CA II-50s) observed in the previous study. On this foundation, H64 and the water network near the active site were analyzed in order of decreasing internal CO2 pressure.

The side chain of H64 lies predominantly in the ‘out’ position in 15 atm CA II whereas it occupies the in position 15 atm CA II-1h (Figure A-2A, E). At intermediate pressures, H64 occupies dual conformations (Figure A-2B, C, D). In concert with H64 moving from the ‘out’ to the ‘in’ position, the density for W2′ is observed to gradually dissipate.

In previous studies, WI was observed in the presence of CO2 whereas only

111 density for W1 was seen upon release of CO2. In this study, we observed the dynamic replacement of WI with W1 as the internal CO2 pressure decreases. Dual occupancy of W1 and WI were seen at 7.0 and 2.5 atm and the ~2.0 Å distance between them suggests that these waters exhibit partial occupancies. A reduction in electron density of WI is observed as CO2 pressure decreases from 15, 7.0, to 2.5 atm

CA II, with complete disappearance in 15 atm CA II-50s and 15 atm CA II-1h. In contrast, W1 electron density starts to emerge in the 7.0 atm CA II, is more pronounced

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in 2.5 atm CA II, and is fully occupied in 15 atm CA II-50s and 15 atm CA II-1h. The concerted changes in electron density suggests that WI moves to the W1 position upon

CO2 release (Figure A-2).

Entrance Conduit Waters

This study reveals newly observed features in the water network at the entrance connecting the CA II active site to bulk solvent, termed the entrance conduit (EC). The

EC consists of hydrophobic residues L198, V135, L204, P202 and F131 and hydrophilic residues H64, Q92 and T200 with H64 positioned perpendicularly to the EC. Along with the previously reported intermediate water WI, another well ordered intermediate water

WI′ was observed in the 7.0 and 2.5 atm CA II structures (Figure A-2B, C). Five water molecules (named entrance-conduit waters or WECs) were observed along the surface of the EC throughout the five structures (Figure A-4). However, alternate positions of

WEC1 (WEC1′ and WEC1′′), WEC2 (WEC2′) and WEC5 (WEC1′) were observed as internal pressure of CO2 decreased. Hydrogen bonds exist between the five WEC waters (WEC1-WEC2, WEC2-WEC3, WEC3-WEC4, and WEC4-WEC5), which are further stabilized by residues lining the EC. For instance, the side-chain amide of Q92 binds to WEC2, and the main-chain carbonyl of P201 and the side-chain hydroxyl of

T200 bind to WEC5, which are conserved throughout all of the internal CO2 pressure structures. Furthermore, WEC2, WEC3, and WEC5 form hydrogen bonds with WI′ whereas WEC2 stabilizes WI and W1.

The dynamic motions of the WECs imply a direct interplay with the proton- transfer water network in the active site. Specifically, interactions between WEC1,

WEC2, and W3b were observed. The positions of W3a and W3b were previously thought to be invariant in order to stabilize W2 in the proton-transfer wire In this study,

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however, an alternate position of W3b (W3b′) was observed along with alternate positions of WEC1 (WEC1′) and WEC2 (WEC2′) in the 15 and 7.0 atm CA II structures

(Figure A-2A, B). The distances between these water molecules are so close (1.3-1.7 Å) that they organize into a continuous tube of electron density. W3b′ and WEC1′, and

WEC2′ disappear as CO2 pressure decreases, with W3b, WEC1, and WEC2 recovering to a singly occupied position upon release of CO2. These results suggest that the waters in the proton wire (W1, W2, W2′, W3a, W3b, W3b′), the intermediate waters (WI and WI′), and the EC water network (WEC1, WEC2, WEC3, WEC4, WEC5) act interdependently with concerted motions.

Restoration of Water Network

As previously described, the observation of WI, and not W1, in the fully occupied

CO2 state suggests a disruption of the proton wire upon substrate binding. Therefore, the replenishment of ZBW following bicarbonate production must coincide with a reconnection of the proton wire in order to complete proton transfer. The stabilization of

WI and WI′ by EC water molecules and rearrangement during CO2 release has led the proposal of the following mechanism.

WI is predicted to fill the positions of both W1 (2.0 Å) and DW (2.4 Å). This interpretation is supported by the observation that the electron density of W1 emerges as that of WI dissipates and the electron density of WI is fused to the electron density of

DW. Subsequently, W1 can move to ZBW (2.6 Å) and DW can shift to either ZBW or

DW′ (2.4 and 2.2 Å, respectively). The distance between WI and ZBW (2.6 Å) suggests

WI can also directly flow into the ZBW position. WI replenishes multiple water positions

(W1, DW, DW′, ZBW), so bulk solvent serves as a water reservoir to rapidly supply new water molecules the WI position, a process that seems to be facilitated by WI′. WI′ is

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separated from WI by 2.2 Å, is located closer to the bulk solvent, and is transiently stabilized by the dynamic motions of WECs, which take place in concert with the changes of solvent in the active site. As the W1/DW/DW′/ZBW positions are filled, the emergence of W1 and stabilization of WECs results in sterically induced destabilization of WI and WI′, respecitvely. Therefore, WI and WI′ are no longer ordered and the active site water network is fully restored for proton transfer from ZBW to His64 (uncharged and in the ‘in’ position) via W1/W2/W2′ (Figure A-5).

Summary

Structural comparisons between CA II in complex with CO2 and during its release revealed intermediate snapshots during the water-network rearrangement in the active site as the waters fill the void following CO2 liberation. Based on our observations, insight into the water-network restoration prior to proton transfer is proposed. While previous studies of the catalytic activity of CA II have mainly focused on the CO2 binding site (Zn/ZBW/DW) and the proton-transfer water network (W1/W2/W3a/W3b), our results indicate that the intermediate (WI, WI′, W2′ and W3b′) and the entrance-conduit waters (WEC1, WEC2, WEC3, WEC4, WEC5) are critically involved in catalysis by CA

II. The substrate CO2 enters via the hydrophobic half of the active site, while the product

- HCO3 , being a charged molecule, exits by perturbing the ordered waters that fill the hydrophilic half of the active site.107,115 Thus, the ordered waters within the active site and its vicinity are likely to exist in a state of intermittent rearrangement during the forward and reverse reactions of catalysis. Taken collectively, our results provide snapshots of low-energy stages of water rearrangement during catalysis.

Future mutation studies to perturb the protein regions that stabilize these waters would provide more evidence of their roles in the reaction. Moreover, our results

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suggest that the catalytic activity of CA II can be more thoroughly understood with the

‘extended’ catalytic waters (DW/DW′/ZBW/W1/WI/WI′/W2/W2′/W3a/W3b/W3b′/WEC1-

WEC5) (Figure A-4). Molecular dynamics simulations on this extended water network may reveal further insights into the bioenergetic mechanisms utilized by CA II to replenish ordered water networks from the surrounding disordered bulk solvent.116,117

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Figure A-1. CA II active site with the ordered water network stabilized by hydrophilic residues (purple) and CO2 binding site in the hydrophobic region (orange).

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Figure A-2. Electron density of CO2 and active site water molecules upon CO2 release. A)15 atm, B) 7 atm, C) 2.5 atm, D) 15 atm-50 sec E) 15 atm-1 hr.

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Figure A-3. Electron density of F226 in the secondary binding site upon CO2 release. A)15 atm, B) 7 atm, C) 2.5 atm, D) 15 atm-50 sec E) 15 atm-1 hr.

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Figure A-4. Extended water network in a surface representation of CA II. Water molecules of the proton wire are colored red, entrance conduit waters pink, and bulk solvent light blue.

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Figure A-5. Proposed water replenishment mechanism following CA II catalysis. Water molecules of the proton wire are colored red, entrance conduit waters pink, and intermediate waters dark red.

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APPENDIX B XFELs

Recent advances in X-ray free-electron laser (XFEL) sources now permit the study of protein dynamics with femtosecond X-ray pulses that allow the visualization of intermediate states in enzyme catalysis. However, the production of usable crystalline slurries for serial crystallographic experiments has been a limiting factor in experimentation. In this study, we propose two methods for the growth of CA II microcrystals suitable for the collection of XFEL diffraction data. First, microbatch mixing

(MBM) takes advantage of secondary nucleation induced by mixing via the application of steady agitation during the crystallization process, resulting in large quantities of well- diffracting microcrystals. Second, a combination of seeding and batch crystallization increases the number of nucleation points. This work presents methods that can be applied to a range of macromolecules and introduces a simple protocol for the production of microcrystals for serial crystallographic experiments. Preliminary results collected from these microcrystal slurries provides the necessary framework for time resolved experiments to study CA catalysis at XFEL beamlines.

Overview

Over the last decade, the development of X-ray free-electron lasers (XFELs) has presented new opportunities for experiments in the field of structural biology. With a peak spectral brightness many orders of magnitude higher than a third generation

Adapted from Mahon BP, Kurian JJ, Lomelino CL, Smith IR, Socorro L, Bennett A, Hendon AM, Chipman P, Savin DA, Agbandje-McKenna M, McKenna R. (2016). Microbatch Mixing: ‘Shaken not Stirred’, a Method for Macromolecular Microcrystal Production for Serial Crystallography. Cryst. Growth Des., 16(11), 6214-6221; Lomelino CL, Kim JK, Lee C, Im S, Andring JT, Mahon BP, Chung M, Kim CU, McKenna R. (2018). Carbonic Anhydrase II Microcrystals Suitable for XFEL Studies. Acta Crystallogr. Sect. F Struct. Biol. Commun., 74(6), 327-330.

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synchrotron source and X-ray pulses on the femtosecond timescale, XFEL studies can produce useful diffraction from nanocrystals at RT before the crystal becomes subject to radiation damage.118 As the time resolution of synchrotron sources is limited to ~100 ps, a femtosecond X-ray pulse provides a means to measure biological reactions that typically occur within picoseconds. Furthermore, the collection of diffraction data at RT is important for the visualization of reaction intermediates, as cryogenic temperatures have been shown to restrict the occupancy of alternate conformational states.119 Thus, serial femtosecond crystallography (SFX) provides the ability to generate ‘molecular movies’ of enzyme catalytic mechanisms.120

The emergence of SFX and XFELs in conjunction with photoactivation strategies allows the study of protein dynamics and kinetics in real time, which has been demonstrated in studies of photosystems I and II, myoglobin, photoactive yellow protein

(PYP), and bacteriorhodopsin.121–125 In previous Laue pump-probe time-resolved experiments, large-scale crystals were needed and achieved only 10-12%reaction initiation whereas ~40% photoconversion was achieved upon direct exposure of PYP microcrystals to the X-ray beam.126 The diffraction-before-destruction aspect of SFX and the tendency for low hit rates during data collection therefore demands large volumes of microcrystals for the collection of a complete data set.118 Furthermore, the microcrystals must be homogenous in size not only to aid delivery but also to ensure uniform photoactivation.

CA II, a zinc metalloenzyme that catalyzes the reversible hydration of CO2, is one of the fastest known enzymes, with a kcat of 106 s-1.127 This reaction follows a classic two-step, ping-pong enzymatic mechanism. In the hydration direction, a zinc-bound

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- hydroxide performs a nucleophilic attack on CO2, resulting in a zinc-bound HCO3 product that is subsequently displaced by a water molecule. The zinc-bound solvent is regenerated through a proton-transfer mechanism facilitated by a His residue at the entrance to the active site.

Although this reaction has been extensively studied and X-ray crystal structures of CA II in complex with substrate and product have been elucidated, there is still little structural information regarding the dynamics of the catalytic mechanism.32,101,111,128

Hence, the use of pump–probe, time-resolved SFX (TR-SFX) would be the preferred methodology to visualize the pathways of substrate/product entry/exit from the active site. This can be achieved using photoactivatable compounds such as 3- nitrophenylacetic acid (3NPAA), which releases CO2 upon photolysis (ƛ = 350 nm).

Therefore, CA II is an excellent candidate for the use of SFX-XFEL studies. Here, we demonstrate the production of CA II microcrystals suitable for the collection of XFEL diffraction data and present preliminary XFEL data and processing results.

Methods

Macromolecule Production

The expression and purification of CA II were performed as described previously.129,130 In brief, CA II was expressed in Escherichia coli BL21(DE3) competent cells via IPTG induction. The protein was then purified using affinity chromatography with p-(aminomethyl)-benzenesulfonamide resin. The purity was verified by SDS-PAGE and the concentration was determined via UV-Vis spectroscopy.

Microbatch Mixing

MBM utilizes the effect of applying agitation via mixing during the crystallization process to intentionally increase, by orders of magnitude, the secondary nucleation

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points for micro- and nanosized crystal formation. The general procedure consists of a batch crystallization setup that is followed by agitation by mixing. For our study, we utilized 24-well VDX plates with sealant for crystallization of all samples. Briefly, 300-

500 μL of precipitant solution was pipetted into each well. Then, the protein was added to the wells in ratios of 1:5, 1:10, and 1:15 (sample:precipitant) to determine effects of sample concentration on microcrystallization. Trays were then sealed with a coverslip and placed at RT on a tabletop shaker set to 72, 200, or 400 rpm and left overnight. For

CA II, the crystallization condition used was 1.6 M sodium citrate, 50 mM Tris, pH 7.8.

To determine if crystal size and quantity could be manipulated, several trays were set up containing crystallization buffer at a pH of either 7.8 or 11.0 and with various concentrations of sodium citrate from 1.6−2.0 M. It should be noted that crystallization conditions for CA II containing high sodium citrate concentrations (≥1.8 M) readily formed large salt crystals once exposed to air (during sample transfer). Therefore, CA II microcrystals were also produced using MBM in 0.2 M MgCl2, 0.1 M Tris, pH 8.5, and

30% (w/v) PEG 4000 to limit the formation of salt crystals. Concentrations of crystal density were estimated by diluting crystal slurries 1:1000, staining with coomassie, and counting using a hemocytometer or alternatively by visually counting unstained crystals in a volume of 0.5 μL and extrapolating to total well volume.

Batch Crystallization and Seeding

Crystals of CA II were grown at 298 K using the hanging drop vapor-diffusion method with a precipitant solution consisting of 1.6 M sodium citrate, 50 mM Tris, pH

7.8. Upon visual inspection, a single high-quality crystal (~200 x 50 x 50 mm) was transferred from the crystal drop into a new 10 mL drop of precipitant solution and crushed using a needle. The needle was then immersed into a secondary 10 mL droplet

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of precipitant solution to dilute the crystals and create a CA II seed stock. CA II microcrystals were grown at 298 K utilizing a combination of seeding and batch crystallization methods by adding CA II seed stock and purified protein directly to the precipitant solution. In a 24-well culture plate, 5 mL seed stock, 300 mL CA II (30 mg/mL) and 1200 mL precipitant solution were added to each well. Microcrystal growth was observed after 12 hrs. The microcrystal suspension was diluted in precipitant solution (1:4 ratio) and syringe-filtered through a metal filter, removing crystals of greater than 100 mm in length. After five successive filtrations, the microcrystal suspension was concentrated by centrifugation at ~840g for 5 min. The microcrystals were then mixed with monoolein in a 1:1 ratio in gas-tight syringes and transferred into a lipid cubic phase (LCP) injector for data collection.

Data Collection and Processing

CA II microcrystals were first characterized for proteinaceous content by collecting “powder diffraction” patterns of the microcrystal slurries. The CA II microcrystals were syringed through a filter containing a 0.2 μm pore size to remove crystals >10 μm in size. It should be noted that crystals with sizes >0.2 μm were observed even after filtering, as determined by the apparent Bragg peaks in the diffraction image and measurements from TEM images. This is most likely due to the rectangular shape of the observed crystalline samples allowing for passage through filter pores, or due to the viscosity of crystal slurry solution causing damage to the syringe filter and allowing larger crystals to pass. Nonetheless, from negative stain EM images, it is predicted that this method of filtering did not induce any damage to the crystals within slurry solutions. Prior to collecting a diffraction image, microcrystal slurries were pelleted by centrifugation at 15000 rpm for 30 min with a limited volume of

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buffer left to ensure the crystals would not dry out while reducing potential for background scattering from excess solvent. The CA II slurry was loaded into a quartz capillary and sealed with wax. Diffraction patterns of the CA II crystal slurries were collected at the CHESS F1 beamline using a wavelength of 0.98 Å. The images were collected at RT using a Dectris Pilatus 6M detector at a crystal-to-detector distance of

500 mm, where the theoretical maximum resolution is determined to be ∼3 Å with exposure times of 0.5, 1, and 5 s per image. For noise from background scatter to be reduced, several images (for each exposure time) were summed and smoothed background subtracted. The resolution of diffraction was obtained by measuring the distance from beam center to the outermost visible concentric ring of diffraction scattering.

Diffraction data was then collected at the Coherent X-ray Imaging (CXI) station at

Pohang Accelerator Laboratory XFEL (PAL-XFEL). The CXI station at PAL-XFEL is specifically designed for SFX and time-resolved SFX experiments, with Kirkpatrick-Baez mirrors for ~2 mm microfocusing, an LCP injector system for microcrystal delivery, an optical (pump) femtosecond laser and capabilities to collect data at a 60 Hz repetition rate. For our data collection, we utilized single-shot X-ray pulses at 10 Hz and a microfocus of 5 mm diameter. The X-ray pulse widths were 20 fs at 1.2 x 1011 photons per pulse (photon energy = 9.715 keV). The subsequent X-ray diffraction data were collected on a Rayonix detector with a readout rate of 10 Hz. The microcrystal suspension in monoolein allowed sample injection using an isocratic flow mode at a flow rate of 150 nL/min. The injector diameter was 100 mm and the sample-to-detector distance was 130 mm.

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Three methods of data pre-processing were tested with NanoPeakCell: methods

A, B and C using images containing more than ten detector pixels with I > 10 000, I >

5000 and I > 1000, respectively.131 CrystFEL was utilized for data indexing using the peakfinder 8 and MOSFLM functions.132

Results

Utilizing MBM, crystals from all samples were successfully grown. CA II microcrystal slurries formed overnight and, using negative stain EM, crystals within these slurries were of submicrometer size. On the basis of previous studies, overnight shaking was chosen as the optimum time to induce secondary nucleation. Further, it was shown that ~24 hr shaking followed by “static” incubation of the crystallization tray still resulted in a high level secondary nucleation and formation of microcrystalline products. Compared to a standard batch crystallization method, the crystal size and growth rate were greatly decreased using MBM (Figure B-1A, B). This is observed directly by comparing crystals of CA II that were grown using both methods. More specifically, the average crystal size of CA II grown using MBM were estimated (using negative stain EM images) at 1.5 μm3. This is compared to CA II grown in a standard batch crystallization setup, which yielded crystals with average sizes estimated at 0.008 mm3. In addition, MBM resulted in overnight crystal formation in CA II whereas a standard batch method saw crystals in 3-5 days. Salt and protein concentration had little effect on crystal size during MBM and only contributed to crystal density. Interestingly, both of these parameters had a more significant effect on crystal size when crystals of

CA II were grown under standard batch methods. This may suggest that successful crystallization conditions determined using a standard batch method may be transitioned with limited manipulation to produce microcrystals using the MBM method.

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The pH of the crystallization conditions appeared to have limited effect on secondary nucleation as microcrystals were grown in both pH 7.8 and 11 without significant difference in crystal size or quantity. In the case of CA II, it appeared that mixing speed had a significant influence on microcrystal formation using MBM. Specifically, it was shown that fast mixing speeds often resulted in a reduction in crystallization “hits”. The reason for this observation is likely related to protein stability, where at 200 rpm there is a significant amount of shear force that potentially causes CA II to lose conformational stability and thus reduces the propensity to crystallize

The batch crystallization method, in which purified CA II was directly mixed with precipitant solution, resulted in only a few CA II crystals (Figure B-1C). Hence, this method alone was inadequate for producing the large volumes of crystals necessary for

XFEL experiments and delivery via an LCP injector. Therefore, microcrystals were grown via seeding in conjunction with batch crystallization, promoting multiple crystal nucleation points. As this method resulted in a mixture of crystal sizes, syringe-driven filtration was used to exclude larger crystals, resulting in crystals ranging between 40 and 80 mm in size (Figure B-1D). The microcrystal suspension was combined with monoolein as a homogenous mixture that was effectively injected via an LCP injector without clogging

Powder diffraction images were collected on CA II MBM microcrystal slurries.

Diffraction of CA II microcrystal slurries indicated a pattern consistent with those previously observed for protein samples. In addition, samples diffracted to a high resolution of ~3 Å (Data not shown). Utilizing a more brilliant source or altering the crystal-to detector distance is predicted to improve the resolution for SFX. The CA II

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microcrystals produced from seeding and batch crystallization were then shown to diffract to a maximal resolution of 1.7 Å and belonged to space group P21, with unit-cell parameters a = 42.2, b = 41.2, c = 72.0 Å, β = 104.2°. A total of 380,000 images were collected in 4 hrs, of which 199,812 and 15,996 images were selected during pre- processing using methods A, B, and C, respectively (Figure B-2). This resulted in hit rates of 0.05, 0.21, and 4.21%, respectively.

Summary

Overall, these experiments demonstrate the successful growth of CA II microcrystals suitable for XFEL data collection. These techniques can potentially be used as a general method for microcrystal growth of a number of macromolecular targets that can be applied to a range of SFX experiments. Furthermore, our diffraction data confirm the feasibility of pump-probe TR-SFX experiments to study the catalytic mechanism of CA II in conjunction with photoactivatable compounds like 3NPAA, which releases CO2 upon laser exposure (Figure B-3A). In theory, CA II can be incubated with

3NPAA, CO2 released into the CA active site upon laser exposure (pump), and then X- ray diffraction data collected (probe) (Figure B-3A). Preliminary strucures have shown that 3NPAA binds within the active site of CA II, anchoring to ZBW through the nitro group (Figure B-3C). The determination of the CA II_3NPAA following exposure to a laser showed the loss of electron density for the carboxyl group, indicating the release of CO2 (Figure B-3D). The ability to reproducibly generate CA II microcrystal slurries and show the binding of the photolabile compound 3NPAA demonstrates that CA II represents an ideal candidate for pump-probe experiments at an XFEL beamline.

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Figure B-1. CA II crystals grown via A) hanging drop vaporization B) microbatch mixing C) batch crystallization D) batch crystallization in conjunction with seeding.

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Figure B-2. Diffraction. More than ten detector pixels with A) I > 10 000, B) I > 5000, and C) I > 1000, respectively.

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Figure B-3. Proposed pump-probe XFEL experiments and 3NPAA binding. A) mechanism of CO2 release. B) photoactivation of 3NPAA releases CO2 in CA active site. Binding of C) 3NPAA and D) 3NPAA following laser exposure in the active site of CA II. The 2Fo−Fc electron density map is contoured to σ = 0.8.

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156 BIOGRAPHICAL SKETCH

In 2014, Carrie graduated from the University of North Carolina at Chapel Hill with a B.S. in chemistry and a focus in biochemistry. During the summer of 2012, she completed an internship at RJ Lee Group under the supervision of Julianne Wolfe.

During her senior year, Carrie performed research in the lab of Dr. Leslie Parise, chair of the biochemistry and biophysics department at UNC. Her project focused on the design of an endogenously fluorescent protein that could be followed throughout a cell in order to identify key protein interactions that could be targeted by small molecule inhibition.

In 2014, Carrie joined the graduate program in biomedical sciences at the

University of Florida. She begain a focus in structural biology by joining the lab of Dr.

Robert McKenna, which specializes in X-ray crystallography. Her thesis project focused on the design of isoform selective inhibitors of Carbonic Anhydrase IX (CA IX) as a potential treatment of breast cancer. Carrie was awarded the TL1 training grant by the

UF Clinical and Translational Science Institute (CTSI), which is a predoctoral training program for students interested in a career in multidisciplinary, clinical and translational science. This program expanded her thesis project to include a clinical mentor and a more clinically focused aim. Carrie was awarded the UF medical guild advancement to candidacy award, recognized as the student with top honors in the qualifying exam competition, and awarded the 2018 Boyce Award for a presentation of her research accomplishments. Throughout the 4.5 years in Dr. McKenna’s lab, Carrie published six chapters and nineteen papers.

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