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The microtubule (MT) cytoskeleton provides the architecture that governs intracellular organization and the regulated motion of macromolecules through the crowded cytoplasm. The key to establishing a functioning cytoskeletal architecture is regulating when and where new MTs are nucleated. Within the spindle, the vast majority of MTs are generated through a pathway known as branching MT nucleation, which exponentially amplifies MT number in a polar manner. Whereas other MT nucleation pathways generally require a complex organelle such as the centrosome or Golgi apparatus to localize nucleation factors, the branching site is based solely on a simple, preformed MT, making it an ideal system to study MT nucleation. In this review, we address recent developments in characterizing branching factors, the branching reaction, and its regulation, as well as branching MT nucleation in systems beyond the spindle and within human disease.

To establish the microtubule cytoskeleton, the cell must tightly regulate when and where microtubules are nucleated. This regulation involves controlling the initial nucleation template, the γ-tubulin ring complex (γTuRC). Although γTuRC is present throughout the cytoplasm, its activity is restricted to specific sites including the centrosome and Golgi. The well-conserved γ-tubulin nucleation activator (γTuNA) domain has been reported to increase the number of microtubules (MTs) generated by γTuRCs. However, previously we and others observed that γTuNA had a minimal effect on the activity of antibody-purified Xenopus γTuRCs in vitro (Thawani et al., eLife, 2020; Liu et al., 2020). Here, we instead report, based on improved versions of γTuRC, γTuNA, and our TIRF assay, the first real-time observation that γTuNA directly increases γTuRC activity in vitro, which is thus a bona fide γTuRC activator. We further validate this effect in Xenopus egg extract. Via mutation analysis, we find that γTuNA is an obligate dimer. Moreover, efficient dimerization as well as γTuNA’s L70, F75, and L77 residues are required for binding to and activation of γTuRC. Finally, we find that γTuNA’s activating effect opposes inhibitory regulation by stathmin. In sum, our improved assays prove that direct γTuNA binding strongly activates γTuRCs, explaining previously observed effects of γTuNA expression in cells and illuminating how γTuRC-mediated microtubule nucleation is regulated.

In this work, the X-ray crystal structures of four different deuterium-labelled versions of a surface variant of human carbonic anhydrase IX are compared and discussed. The results show that the overall structure and active-site organization of each version are essentially the same, paving the way for future neutron protein crystallography studies., Human carbonic anhydrase IX (CA IX) expression is upregulated in hypoxic solid tumours, promoting cell survival and metastasis. This observation has made CA IX a target for the development of CA isoform-selective inhibitors. To enable structural studies of CA IX–inhibitor complexes using X-ray and neutron crystallography, a CA IX surface variant (CA IXSV; the catalytic domain with six surface amino-acid substitutions) has been developed that can be routinely crystallized. Here, the preparation of protiated (H/H), H/D-exchanged (H/D) and deuterated (D/D) CA IXSV for crystallographic studies and their structural comparison are described. Four CA IXSV X-ray crystal structures are compared: two H/H crystal forms, an H/D crystal form and a D/D crystal form. The overall active-site organization in each version is essentially the same, with only minor positional changes in active-site solvent, which may be owing to deuteration and/or resolution differences. Analysis of the crystal contacts and packing reveals different arrangements of CA IXSV compared with previous reports. To our knowledge, this is the first report comparing three different deuterium-labelled crystal structures of the same protein, marking an important step in validating the active-site structure of CA IXSV for neutron protein crystallography.

Up-regulation of carbonic anhydrase IX (CA IX) expression is an indicator of metastasis and associated with poor cancer patient prognosis. CA IX has emerged as a cancer drug target but development of isoform-specific inhibitors is challenging due to other highly conserved CA isoforms. In this study, a CA IX mimic construct was used (CA II with seven point mutations introduced, to mimic CA IX active site) while maintaining CA II solubility that make it amenable to crystallography. The structures of CA IX mimic unbound and in complex with saccharin (SAC) and a saccharin-glucose conjugate (SGC) were determined using joint X-ray and neutron protein crystallography. Previously, SAC and SGC have been shown to display CA isoform inhibitor selectivity in assays and X-ray crystal structures failed to reveal the basis of this selectivity. Joint X-ray and neutron crystallographic studies have shown active site residues, solvent, and H-bonding re-organization upon SAC and SGC binding. These observations highlighted the importance of residues 67 (Asn in CA II, Gln in CA IX) and 130 (Asp in CA II, Arg in CA IX) in selective CA inhibitor targeting.

Upregulation of carbonic anhydrase IX (CA IX) is associated with several aggressive forms of cancer and promotes metastasis. CA IX is normally constitutively expressed at low levels in selective tissues associated with the gastrointestinal tract, but is significantly upregulated upon hypoxia in cancer. CA IX is a multi-domain protein, consisting of a cytoplasmic region, a single-spanning transmembrane helix, an extracellular CA catalytic domain, and a proteoglycan-like (PG) domain. Considering the important role of CA IX in cancer progression and the presence of the unique PG domain, little information about the PG domain is known. Here, we report biophysical characterization studies to further our knowledge of CA IX. We report the 1.5 &Aring, resolution crystal structure of the wild-type catalytic domain of CA IX as well as small angle X-ray scattering and mass spectrometry of the entire extracellular region. We used matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry to characterize the spontaneous degradation of the CA IX PG domain and confirm that it is only the CA IX catalytic domain that forms crystals. Small angle X-ray scattering analysis of the intact protein indicates that the PG domain is not randomly distributed and adopts a compact distribution of shapes in solution. The observed dynamics of the extracellular domain of CA IX could have physiological relevance, including observed cleavage and shedding of the PG domain.

Multidrug resistance to current Food and Drug Administration-approved HIV-1 protease (PR) inhibitors drives the need to understand the fundamental mechanisms of how drug pressure-selected mutations, which are oftentimes natural polymorphisms, elicit their effect on enzyme function and resistance. Here, the impacts of the hinge-region natural polymorphism at residue 35, glutamate to aspartate (E35D), alone and in conjunction with residue 57, arginine to lysine (R57K), are characterized with the goal of understanding how altered salt bridge interactions between the hinge and flap regions are associated with changes in structure, motional dynamics, conformational sampling, kinetic parameters, and inhibitor affinity. The combined results reveal that the single E35D substitution leads to diminished salt bridge interactions between residues 35 and 57 and gives rise to the stabilization of open-like conformational states with overall increased backbone dynamics. In HIV-1 PR constructs where sites 35 and 57 are both mutated (e.g. E35D and R57K), x-ray structures reveal an altered network of interactions that replace the salt bridge thus stabilizing the structural integrity between the flap and hinge regions. Despite the altered conformational sampling and dynamics when the salt bridge is disrupted, enzyme kinetic parameters and inhibition constants are similar to those obtained for subtype B PR. Results demonstrate that these hinge-region natural polymorphisms, which may arise as drug pressure secondary mutations, alter protein dynamics and the conformational landscape, which are important thermodynamic parameters to consider for development of inhibitors that target for non-subtype B PR.

The α-class carbonic anhydrase (CA) from the thermophilic gammaproteobacterium Thiomicrospira crunogena XCL-2 (TcruCA) has been recently proposed as a candidate enzyme for carbon dioxide removal (CDR) in flue gas scrubbing systems. However during this process, the enzyme may be exposed to anionic impurities which can limit the use of CA for CDR. The inhibition of TcruCA by various anions, some of which are found in flue gas, is reported. The majority of the anions tested display weak (mM) inhibition but some, such as the metal poisons and anionic acids, exhibit stronger (μM) inhibition. Most relevant to the use of CA in CDR is the inhibition by SOx, NOx, and Hg2+, which can be removed through a pre-scrubbing treatment. In addition, the active site structural architecture of TcruCA is compared with other α-class CAs and differences in catalytic efficiency and anion inhibition are discussed. This study provides information regarding anionic inhibition of CAs as bio-catalytic CDR agents and the results should therefore be considered in the design of CA-mediated flue gas scrubbing systems.

Recent advances in X-ray free-electron laser (XFEL) sources have permitted the study of protein dynamics. Femtosecond X-ray pulses have allowed the visualization of intermediate states in enzyme catalysis. In this study, the growth of carbonic anhydrase II microcrystals (40–80 µm in length) suitable for the collection of XFEL diffraction data at the Pohang Accelerator Laboratory is demonstrated. The crystals diffracted to 1.7 Å resolution and were indexed in space group P21, with unit-cell parameters a = 42.2, b = 41.2, c = 72.0 Å, β = 104.2°. These preliminary results provide the necessary framework for time-resolved experiments to study carbonic anhydrase catalysis at XFEL beamlines.

Acetylation of surface lysine residues of proteins has been observed in Escherichia coli (E. coli), an organism that has been extensively utilized for recombinant protein expression. This post-translational modification is shown to be important in various processes such as metabolism, stress-response, transcription, and translation. As such, utilization of E. coli expression systems for protein production may yield non-native acetylation events of surface lysine residues. Here we present the crystal structures of wild-type and a variant of human carbonic anhydrase II (hCA II) that have been expressed in E. coli and exhibit surface lysine acetylation and we speculate on the effect this has on the conformational stability of each enzyme. Both structures were determined to 1.6 A resolution and show clear electron density for lysine acetylation. The lysine acetylation does not distort the structure and the surface lysine acetylation events most likely do not interfere with the biological interpretation. However, there is a reduction in conformational stability in the hCA II variant compared to wild type (∼ 4°C decrease). This may be due to other lysine acetylation events that have occurred but are not visible in the crystal structure due to intrinsic disorder. Therefore, surface lysine acetylation events may affect overall protein stability and crystallization, and should be considered when using E. coli expression systems.

Carbonic anhydrase IX (CA IX) is a key modulator of aggressive tumor behavior and a prognostic marker and target for several cancers. Saccharin (SAC) based compounds may provide an avenue to overcome CA isoform specificity, as they display both nanomolar affinity and preferential binding, for CA IX compared to CA II (>50-fold for SAC and >1000-fold when SAC is conjugated to a carbohydrate moiety). The X-ray crystal structures of SAC and a SAC-carbohydrate conjugate bound to a CA IX-mimic are presented and compared to CA II. The structures provide substantial new insight into the mechanism of SAC selective CA isoform inhibition.

Ureido-substituted benzenesulfonamides (USBs) show great promise as selective and potent inhibitors for human carbonic anhydrase hCA IX and XII, with one such compound (SLC-0111/U-F) currently in clinical trials (clinical trials.gov, NCT02215850). In this study, the crystal structures of both hCA II (off-target) and an hCA IX-mimic (target) in complex with selected USBs (U-CH3, U-F, and U-NO2), at resolutions of 1.9 A or better, are presented, and demonstrate differences in the binding modes within the two isoforms. The presence of residue Phe 131 in hCA II causes steric hindrance (U-CH3, 1765 nM; U-F, 960 nM; U-NO2, 15 nM) whereas in hCA IX (U-CH3, 7 nM; U-F, 45 nM; U-NO2, 1 nM) and hCA XII (U-CH3, 6 nM; U-F, 4 nM; U-NO2, 6 nM), 131 is a Val and Ala, respectively, allows for more favorable binding. Our results provide insight into the mechanism of USB selective inhibition and useful information for structural design and drug development, including synthesis of hybrid USB compounds with improved physiochemical properties.

We report the synthesis, biological evaluation, and structural study of a series of substituted heteroaryl-pyrazole carboxylic acid derivatives. These compounds have been developed as inhibitors of specific isoforms of carbonic anhydrase (CA), with potential as prototypes of a new class of chemotherapeutics. Both X-ray crystallography and computational modeling provide insights into the CA inhibition mechanism. Results indicate that this chemotype produces an indirect interference with the zinc ion, thus behaving differently from other related nonclassical inhibitors. Among the tested compounds, 2c with Ki = 0.21 μM toward hCA XII demonstrated significant antiproliferative activity against hypoxic tumor cell lines. Taken together, the results thus provide the basis of structural determinants for the development of novel anticancer agents.

Human carbonic anhydrase IX (hCA IX) expression in many cancers is associated with hypoxic tumors and poor patient outcome. Inhibitors of hCA IX have been used as anticancer agents with some entering Phase I clinical trials. hCA IX is transmembrane protein whose catalytic domain faces the extracellular tumor milieu, which is typically associated with an acidic microenvironment. Here, we show that the catalytic domain of hCA IX (hCA IX-c) exhibits the necessary biochemical and biophysical properties that allow for low pH stability and activity. Furthermore, the unfolding process of hCA IX-c appears to be reversible, and its catalytic efficiency is thought to be correlated directly with its stability between pH 3.0 and 8.0 but not above pH 8.0. To rationalize this, we determined the X-ray crystal structure of hCA IX-c to 1.6 Å resolution. Insights from this study suggest an understanding of hCA IX-c stability and activity in low-pH tumor microenvironments and may be applicable to determining pH-related effects on enzymes.

Correlation of the solvent effects through application of the extended Grunwald-Winstein equation to the solvolysis of isopropyl chlorothioformate results in a sensitivity value of 0.38 towards changes in solvent nucleophilicity (l) and a sensitivity value of 0.72 towards changes in solvent ionizing power (m). This tangible l value coupled with the negative entropies of activation observed indicates a favorable predisposition towards a modest rear-side nucleophilic solvation of a developing carbocation. Only in 100% ethanol was the bimolecular pathway dominant. These observations are very different from those obtained for the solvolysis of isopropyl chloroformate, where dual reaction channels were proposed, with the addition-elimination reaction favored in the more nucleophilic solvents and a unimolecular fragmentation-ionization mechanism favored in the highly ionizing solvents.

The pH of the tumor microenvironment drives the metastatic phenotype and chemotherapeutic resistance of tumors. Understanding the mechanisms underlying this pH-dependent phenomenon will lead to improved drug delivery and allow the identification of new therapeutic targets. This includes an understanding of the role pH plays in primary tumor cells, and the regulatory factors that permit cancer cells to thrive. Over the last decade, carbonic anhydrases (CAs) have been shown to be important mediators of tumor cell pH by modulating the bicarbonate and proton concentrations for cell survival and proliferation. This has prompted an effort to inhibit specific CA isoforms, as an anti-cancer therapeutic strategy. Of the 12 active CA isoforms, two, CA IX and XII, have been considered anti-cancer targets. However, other CA isoforms also show similar activity and tissue distribution in cancers and have not been considered as therapeutic targets for cancer treatment. In this review, we consider all the CA isoforms and their possible role in tumors and their potential as targets for cancer therapy.

Human carbonic anhydrase (CA; EC 4.2.1.1) isoform IX (CA IX) is an extracellular zinc metalloenzyme that catalyzes the reversible hydration of CO2 to HCO3 −, thereby playing a role in pH regulation. The majority of normal functioning cells exhibit low-level expression of CA IX. However, in cancer cells CA IX is upregulated as a consequence of a metabolic transition known as the Warburg effect. The upregulation of CA IX for cancer progression has drawn interest in it being a potential therapeutic target. CA IX is a transmembrane protein, and its purification, yield and crystallization have proven challenging to structure-based drug design, whereas the closely related cytosolic soluble isoform CA II can be expressed and crystallized with ease. Therefore, we have utilized structural alignments and site-directed mutagenesis to engineer a CA II that mimics the active site of CA IX. In this paper, the X-ray crystal structure of this CA IX mimic in complex with sucrose is presented and has been refined to a resolution of 1.5 Å, an R cryst of 18.0% and an R free of 21.2%. The binding of sucrose at the entrance to the active site of the CA IX mimic, and not CA II, in a non-inhibitory mechanism provides a novel carbohydrate moiety binding site that could be further exploited to design isoform-specific inhibitors of CA IX.

Acetylation of surface lysine residues of proteins has been observed in Escherichia coli (E. coli), an organism that has been extensively utilized for recombinant protein expression. This post-translational modification is shown to be important in various processes such as metabolism, stress-response, transcription, and translation. As such, utilization of E. coli expression systems for protein production may yield non-native acetylation events of surface lysine residues. Here we present the crystal structures of wild-type and a variant of human carbonic anhydrase II (hCA II) that have been expressed in E. coli and exhibit surface lysine acetylation and we speculate on the effect this has on the conformational stability of each enzyme. Both structures were determined to 1.6 Å resolution and show clear electron density for lysine acetylation. The lysine acetylation does not distort the structure and the surface lysine acetylation events most likely do not interfere with the biological interpretation. However, there is a reduction in conformational stability in the hCA II variant compared to wild type (∼ 4°C decrease). This may be due to other lysine acetylation events that have occurred but are not visible in the crystal structure due to intrinsic disorder. Therefore, surface lysine acetylation events may affect overall protein stability and crystallization, and should be considered when using E. coli expression systems.

Thiomicrospira crunogena XCL-2 expresses an α-carbonic anhydrase (TcruCA). Sequence alignments reveal that TcruCA displays a high sequence identity (>30%) relative to other α-CAs. This includes three conserved histidines that coordinate the active site zinc, a histidine proton shuttling residue, and opposing hydrophilic and hydrophobic sides that line the active site. The catalytic efficiency of TcruCA is considered moderate relative to other α-CAs ( k cat / K M = 1.1 × 10 7 M −1 s −1 ), being a factor of ten less efficient than the most active α-CAs. TcruCA is also inhibited by anions with Cl − , Br − , and I − , all showing K i values in the millimolar range (53–361 mM). Hydrogen sulfide (HS − ) revealed the highest affinity for TcruCA with a K i of 1.1 μM. It is predicted that inhibition of TcruCA by HS − (an anion commonly found in the environment where Thiomicrospira crunogena is located) is a way for Thiomicrospira crunogena to regulate its carbon-concentrating mechanism (CCM) and thus the organism’s metabolic functions. Results from this study provide preliminary insights into the role of TcruCA in the general metabolism of Thiomicrospira crunogena .

Biocatalytic CO2sequestration to reduce greenhouse-gas emissions from industrial processes is an active area of research. Carbonic anhydrases (CAs) are attractive enzymes for this process. However, the most active CAs display limited thermal and pH stability, making them less than ideal. As a result, there is an ongoing effort to engineer and/or find a thermostable CA to fulfill these needs. Here, the kinetic and thermal characterization is presented of an α-CA recently discovered in the mesophilic hydrothermal vent-isolate extremophileThiomicrospira crunogenaXCL-2 (TcruCA), which has a significantly higher thermostability compared with human CA II (melting temperature of 71.9°Cversus59.5°C, respectively) but with a tenfold decrease in the catalytic efficiency. The X-ray crystallographic structure of the dimeric TcruCA shows that it has a highly conserved yet compact structure compared with other α-CAs. In addition, TcruCA contains an intramolecular disulfide bond that stabilizes the enzyme. These features are thought to contribute significantly to the thermostability and pH stability of the enzyme and may be exploited to engineer α-CAs for applications in industrial CO2sequestration.

The alpha carbonic anhydrases (α-CAs) are a group of structurally related zinc metalloenzymes that catalyze the reversible hydration of CO2toHCO3-. Humans have 15 differentα-CAs with numerous physiological roles and expression patterns. Of these, 12 are catalytically active, and abnormal expression and activities are linked with various diseases, including glaucoma and cancer. Hence there is a need for CA isoform specific inhibitors to avoid off-target CA inhibition, but due to the high amino acid conservation of the active site and surrounding regions between each enzyme, this has proven difficult. However, residues towards the exit of the active site are variable and can be exploited to design isoform selective inhibitors. Here we discuss and characterize this region of “selective drug targetability” and how these observations can be utilized to develop isoform selective CA inhibitors.

Inhibition of human carbonic anhydrase IX (hCA IX) has shown to be therapeutically advantageous for treating many types of highly aggressive cancers. However, designing selective inhibitors for hCA IX has been difficult due to its high structural homology and sequence similarity with off-target hCAs. Recently, the use of glucosyl sulfamate inhibitors has shown promise as selective inhibitors for hCA IX. In this study, we present five X-ray crystal structures, determined to a resolution of 1.7 A or better, of both hCA II (a ubiquitous CA) and an engineered hCA IX-mimic in complex with selected glucosyl sulfamates and structurally rationalize mechanisms for hCA IX selectivity. Results from this study have allowed us, for the first time, to empirically “map” key interactions of the hCA IX active site in order to establish parameters needed to design novel hCA IX selective inhibitors.

HIV drug resistance continues to emerge; consequently, there is an urgent need to develop next generation antiretroviral therapeutics.1 Here we report on the structural and kinetic effects of an HIV protease drug resistant variant with the double mutations Gly48Thr and Leu89Met (PRG48T/L89M), without the stabilizing mutations Gln7Lys, Leu33Ile, and Leu63Ile. Kinetic analyses reveal that PRG48T/L89M and PRWT share nearly identical Michaelis–Menten parameters; however, PRG48T/L89M exhibits weaker binding for IDV (41-fold), SQV (18-fold), APV (15-fold), and NFV (9-fold) relative to PRWT. A 1.9 Å resolution crystal structure was solved for PRG48T/L89M bound with saquinavir (PRG48T/L89M-SQV) and compared to the crystal structure of PRWT bound with saquinavir (PRWT-SQV). PRG48T/L89M-SQV has an enlarged active site resulting in the loss of a hydrogen bond in the S3 subsite from Gly48 to P3 of SQV, as well as less favorable hydrophobic packing interactions between P1 Phe of SQV and the S1 subsite. PRG48T/L89M-SQV assumes a more open conformation relative to PRWT-SQV, as illustrated by the downward displacement of the fulcrum and elbows and weaker interatomic flap interactions. We also show that the Leu89Met mutation disrupts the hydrophobic sliding mechanism by causing a redistribution of van der Waals interactions in the hydrophobic core in PRG48T/L89M-SQV. Our mechanism for PRG48T/L89M-SQV drug resistance proposes that a defective hydrophobic sliding mechanism results in modified conformational dynamics of the protease. As a consequence, the protease is unable to achieve a fully closed conformation that results in an expanded active site and weaker inhibitor binding.

Breast cancer is the second leading cause of cancer related deaths among women in the United States. Despite the tremendous progress that has been made towards treating localized tumors, nearly 40,000 women die each year, predominantly from metastatic drug resistance. The tumor microenvironment plays a pivotal role in determining tumor growth, invasion, metastasis, and therapeutic success or failure. Therefore, a therapy targeting the tumor microenvironment is needed to sufficiently preserve the quality of life of cancer patients, by inhibiting metastasis. Elevated levels of Carbonic anhydrase IX (CAIX) expression in primary breast cancers is a marker for highly aggressive and metastatic tumors, especially of the triple negative subtype (TNBC). It is also associated with hypoxia, extracellular acidification, and poor prognosis. Low pH (values of ~6.5-6.8) is toxic to normal cells in the tumor microenvironment while enhancing cancer cell proliferation and tumor growth. Our goal was to compare the structure of a CAIX-mimic bound to ureidosulfonamide inhibitors with the biological activity of these inhibitors in triple negative and estrogen receptor positive (ER+) breast cancer cell lines. CAIX is a reversible enzyme and at low pH (high proton concentration), the enzyme will consume protons, raising pH. Our hypothesis is that CAIX inhibition, in the context of an acidic microenvironment, will dysregulate its ability to maintain the acidic pH preferred by cancer cells which favors their growth and migration. In this study, we have shown the interaction of sulfonamide-based inhibitors with a CAIX-mimic using X-ray crystallography. These structures show that the inhibitors make multiple contacts within the active site cavity. This is consistent with the inhibitor-induced decrease in CAIX activity and to some extent expression. We have also investigated the effect of CA inhibition on breast cancer cell growth, proliferation, activation of cell death pathways and migration. This reveals that, although CA inhibition with the sulfonamide-based compounds inhibits cell growth and migration, it does not activate apoptotic pathways. In total, these observations indicate that CAIX is a viable small molecular drug target for the treatment of metastatic breast cancer. Citation Format: Mam Y. Mboge, Zhijuan Chen, Brian P. Mahon, Chingkkuang Tu, Alyssa S. Wolff, John V. Mathias, Fabrizio Carta, Claudiu T. Supuran, Rob McKenna, Susan C. Frost. Targeting membrane-bound carbonic anhydrases in breast cancer to intervene in the metastatic phenotype [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2931. doi:10.1158/1538-7445.AM2017-2931

Carbonic anhydrase IX (CA IX) is an extracellular transmembrane homodimeric zinc metalloenzyme that has been validated as a prognostic marker and therapeutic target for several types of aggressive cancers. CA IX shares a close homology with other CA isoforms, making the design of CA IX isoform selective inhibitors challenging. In this paper, we describe the development of a new class of CA IX inhibitors that comprise a sulfamate as the zinc binding group, a variable linker, and a carbohydrate “tail” moiety. Seven compounds inhibited CA IX with low nM Ki values of 1–2 nM and also exhibited permeability profiles to preferentially target the binding of extracellular CA IX over cytosolic CAs. The crystal structures of two of these compounds in complex with a CA IX-mimic (a variant of CA II, with active site residues that mimic CA IX) and one compound in complex with CA II have been determined to 1.7 Å resolution or better and demonstrate a selective mechanism of binding between the hydrophilic and hydrophobic pockets of CA IX versus CA II. These compounds present promising candidates for anti-CA IX drugs and the treatment for several aggressive cancer types.

The microenvironment within a solid tumor is usually heterogeneous with certain regions being acidic and hypoxic. These acidic and hypoxic regions arise from rapidly proliferating cells combined with poor tumor perfusion. Cancer cells cope with these hostile changes in their microenvironment by expressing genes that are essential for survival. One of the coping mechanisms is an upregulation of pH regulatory factors, including carbonic anhydrase IX (CAIX). The action of CAIX helps to maintain physiological pH inside the cell (pHi) while regulating extracellular acidification (pHe). Extracellular acidification of the tumor microenvironment promotes local invasion, metastasis and decreases the effectiveness of adjuvant therapies, thus contributing to poor clinical outcome. Our goal was to compare the structure of a CAIX-mimic bound to ureidosulfonamide inhibitors with the biological activity of these inhibitors in a triple negative breast cancer cell line. CAIX is a reversible enzyme and at low pH (high proton concentration), the enzyme will consume protons, raising pH. Our hypothesis is that CAIX inhibition, in the context of an acidic microenvironment, will dysregulate its ability to maintain the acidic pH preferred by cancer cells which favors their growth and migration. In this study, we have shown the interaction of sulfonamide-based inhibitors using X-ray crystallography methods. These structures show the inhibitors make multiple contacts within the active site cavity. This is consistent with the inhibitor-induced decrease in CAIX activity measured as 18O exchange between H2O16 and H13C18O3 . We have also investigated the effect CAIX inhibition on cancer cell metabolism and extracellular acidification using Seahorse technology. This reveals that CAIX may contribute to the “non-glycolytic” acidification process. In total, these observations indicate that CAIX is a viable small molecular drug target and contribute to our understanding of the function of CAIX in modulating pH in cancer cells. Citation Format: Mam Y. Mboge, Zhijuan Chen, Brian P. Mahon, Nicole Lamas, Shingkuang Tu, Fabrizio Carta, Claudiu T. Superan, Robert McKenna, Susan C. Frost. Characterization, targeting, and modulation of carbonic anhydrase IX activity for the development of small-molecule inhibitors to treat triple-negative breast cancer. [abstract]. In: Proceedings of the AACR Special Conference: Function of Tumor Microenvironment in Cancer Progression; 2016 Jan 7–10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2016;76(15 Suppl):Abstract nr B25.