6 research outputs found
Development of Potent Carbonic Anhydrase Inhibitors Incorporating Both Sulfonamide and Sulfamide Groups
A series of compounds incorporating both sulfonamide
and sulfamide as zinc-binding groups (ZBGs) are reported as inhibitors
of the metalloenzyme carbonic anhydrase (CA, EC 4.2.1.1). Crystallographic
studies on the complex of hCA II with the lead compound of this series,
namely, 4-sulfamido-benzenesulfonamide, revealed the binding of two
molecules in the enzyme active site cavity, the first one canonically
coordinated to the zinc ion by means of the sulfonamide group and
the second one located at the entrance of the cavity. This observation
led to the design of elongated molecules incorporating these two ZBGs,
separated by a linker of proper length, to allow the simultaneous
binding to these different sites. The “long” inhibitors
indeed showed around 10 times better enzyme inhibitory properties
as compared to the shorter molecules against four physiologically
relevant human (h) isoforms, hCA I, II, IX, and XII
Disclosing the Interaction of Carbonic Anhydrase IX with Cullin-Associated NEDD8-Dissociated Protein 1 by Molecular Modeling and Integrated Binding Measurements
Human Carbonic Anhydrase
(hCA) IX is a membrane-associated member
of the CA enzyme family, involved in solid tumor acidification. This
enzyme is a marker of tumor hypoxia and a prognostic factor for several
human cancers. In a recent paper, we showed that CA IX interacts with
cullin-associated NEDD8-dissociated protein 1 (CAND1), a nuclear protein
involved in gene transcription and assembly of SCF ubiquitin ligase
complexes. A functional role for this interaction was also identified,
since lower CA IX levels were observed in cells with decreased CAND1
expression <i>via</i> shRNA-mediated interference. In this
paper, we describe the identification of the structural determinants
responsible for the CA IX/CAND1 interaction by means of a multidisciplinary
approach, consisting of binding assay measurements, molecular docking,
and site-directed mutagenesis. These data open a novel scenario in
the design of anticancer drugs targeting CA IX. Indeed, the knowledge
of the structural determinants responsible for the CAND1/CA IX interaction
provides the molecular basis to design molecules able to destabilize
it. Due to the proposed function of CAND1 in stabilizing CA IX, these
molecules could represent an efficient tool to lower the amount of
CA IX in hypoxic cancer cells, thus limiting its action in survival
and the metastatic spread of tumors
Probing Molecular Interactions between Human Carbonic Anhydrases (hCAs) and a Novel Class of Benzenesulfonamides
On the basis of X-ray crystallographic
studies of the complex of
hCA II with 4-(3,4-dihydro-1<i>H</i>-isoquinoline-2-carbonyl)Âbenzenesulfonamide
(<b>3</b>) (PDB code 4Z1J), a novel series of 4-(1-aryl-3,4-dihydro-1<i>H</i>-isoquinolin-2-carbonyl)Âbenzenesulfonamides (<b>23</b>–<b>33</b>) was designed. Specifically, our idea was
to improve the selectivity toward druggable isoforms through the introduction
of additional hydrophobic/hydrophilic functionalities. Among the synthesized
and tested compounds, the (<i>R</i>,<i>S</i>)-4-(6,7-dihydroxy-1-phenyl-3,4-tetrahydroisoquinoline-1<i>H</i>-2-carbonyl)Âbenzenesulfonamide (<b>30</b>) exhibited
a remarkable inhibition for the brain-expressed hCA VII (<i>K</i><sub>i</sub> = 0.20 nM) and selectivity over wider distributed hCA
I and hCA II isoforms. By enantioselective HPLC, we solved the racemic
mixture and ascertained that the two enantiomers (<b>30a</b> and <b>30b</b>) are equiactive inhibitors for hCA VII. Crystallographic
and docking studies revealed the main interactions of these inhibitors
into the carbonic anhydrase (CA) catalytic site, thus highlighting
the relevant role of nonpolar contacts for this class of hCA inhibitors
Characterization of Carbonic Anhydrase IX Interactome Reveals Proteins Assisting Its Nuclear Localization in Hypoxic Cells
Carbonic anhydrase IX (CA IX) is a transmembrane protein
affecting
pH regulation, cell migration/invasion, and survival in hypoxic tumors.
Although the pathways related to CA IX have begun to emerge, molecular
partners mediating its functions remain largely unknown. Here we characterize
the CA IX interactome in hypoxic HEK-293 cells. Most of the identified
CA IX-binding partners contain the HEAT/ARM repeat domain and belong
to the nuclear transport machinery. We show that the interaction with
two of these proteins, namely XPO1 exportin and TNPO1 importin, occurs
via the C-terminal region of CA IX and increases with protein phosphorylation.
We also demonstrate that nuclear CA IX is enriched in hypoxic cells
and is present in renal cell carcinoma tissues. These data place CA
IX among the cell-surface signal transducers undergoing nuclear translocation.
Accordingly, CA IX interactome involves also CAND1, which participates
in both gene transcription and assembly of SCF ubiquitin ligase complexes.
It is noteworthy that down-regulation of CAND1 leads to decreased
CA IX protein levels apparently via affecting its stability. Our findings
provide the first evidence that CA IX interacts with proteins involved
in nuclear/cytoplasmic transport, gene transcription, and protein
stability, and suggest the existence of nuclear CA IX protein subpopulations
with a potential intracellular function, distinct from the crucial
CA IX role at the cell surface
Characterization of Carbonic Anhydrase IX Interactome Reveals Proteins Assisting Its Nuclear Localization in Hypoxic Cells
Carbonic anhydrase IX (CA IX) is a transmembrane protein
affecting
pH regulation, cell migration/invasion, and survival in hypoxic tumors.
Although the pathways related to CA IX have begun to emerge, molecular
partners mediating its functions remain largely unknown. Here we characterize
the CA IX interactome in hypoxic HEK-293 cells. Most of the identified
CA IX-binding partners contain the HEAT/ARM repeat domain and belong
to the nuclear transport machinery. We show that the interaction with
two of these proteins, namely XPO1 exportin and TNPO1 importin, occurs
via the C-terminal region of CA IX and increases with protein phosphorylation.
We also demonstrate that nuclear CA IX is enriched in hypoxic cells
and is present in renal cell carcinoma tissues. These data place CA
IX among the cell-surface signal transducers undergoing nuclear translocation.
Accordingly, CA IX interactome involves also CAND1, which participates
in both gene transcription and assembly of SCF ubiquitin ligase complexes.
It is noteworthy that down-regulation of CAND1 leads to decreased
CA IX protein levels apparently via affecting its stability. Our findings
provide the first evidence that CA IX interacts with proteins involved
in nuclear/cytoplasmic transport, gene transcription, and protein
stability, and suggest the existence of nuclear CA IX protein subpopulations
with a potential intracellular function, distinct from the crucial
CA IX role at the cell surface
Characterization of Carbonic Anhydrase IX Interactome Reveals Proteins Assisting Its Nuclear Localization in Hypoxic Cells
Carbonic anhydrase IX (CA IX) is a transmembrane protein
affecting
pH regulation, cell migration/invasion, and survival in hypoxic tumors.
Although the pathways related to CA IX have begun to emerge, molecular
partners mediating its functions remain largely unknown. Here we characterize
the CA IX interactome in hypoxic HEK-293 cells. Most of the identified
CA IX-binding partners contain the HEAT/ARM repeat domain and belong
to the nuclear transport machinery. We show that the interaction with
two of these proteins, namely XPO1 exportin and TNPO1 importin, occurs
via the C-terminal region of CA IX and increases with protein phosphorylation.
We also demonstrate that nuclear CA IX is enriched in hypoxic cells
and is present in renal cell carcinoma tissues. These data place CA
IX among the cell-surface signal transducers undergoing nuclear translocation.
Accordingly, CA IX interactome involves also CAND1, which participates
in both gene transcription and assembly of SCF ubiquitin ligase complexes.
It is noteworthy that down-regulation of CAND1 leads to decreased
CA IX protein levels apparently via affecting its stability. Our findings
provide the first evidence that CA IX interacts with proteins involved
in nuclear/cytoplasmic transport, gene transcription, and protein
stability, and suggest the existence of nuclear CA IX protein subpopulations
with a potential intracellular function, distinct from the crucial
CA IX role at the cell surface