Egidijus Kazlauskas

Discovery and characterization of novel selective inhibitors of carbonic anhydrase IX.

Human carbonic anhydrase IX (CA IX) is highly expressed in tumor tissues, and its selective inhibition provides a potential target for the treatment of numerous cancers. Development of potent, highly selective inhibitors against this target remains an unmet need in anticancer therapeutics. A series of fluorinated benzenesulfonamides with substituents on the benzene ring was designed and synthesized. Several of these exhibited a highly potent and selective inhibition profile against CA IX. Three fluorine atoms significantly increased the affinity by withdrawing electrons and lowering the pKa of the benzenesulfonamide group. The bulky ortho substituents, such as cyclooctyl or even cyclododecyl groups, fit into the hydrophobic pocket in the active site of CA IX but not CA II, as shown by the compounds co-crystal structure with chimeric CA IX. The strongest inhibitor of recombinant human CA IXs catalytic domain in human cells achieved an affinity of 50 pM. However, the high affinity diminished the selectivity. The most selective compound for CA IX exhibited 10 nM affinity. The compound that showed the best balance between affinity and selectivity bound with 1 nM affinity. The inhibitors described in this work provide the basis for novel anticancer therapeutics targeting CA IX.

5-Aryl-4-(5-substituted-2,4-dihydroxyphenyl)-1,2,3-thiadiazoles as inhibitors of Hsp90 chaperone

A series of 5-aryl-4-(5-substituted-2,4-dihydroxyphenyl)-1,2,3-thiadiazoles were synthesized and their binding to several constructs of human Hsp90 chaperone measured by isothermal titration calorimetry (ITC). The most potent compound bound Hsp90 with the dissociation constant of about 5 nM.

Thermodynamics of aryl-dihydroxyphenyl-thiadiazole binding to human Hsp90.

The design of specific inhibitors against the Hsp90 chaperone and other enzyme relies on the detailed and correct understanding of both the thermodynamics of inhibitor binding and the structural features of the protein-inhibitor complex. Here we present a detailed thermodynamic study of binding of aryl-dihydroxyphenyl-thiadiazole inhibitor series to recombinant human Hsp90 alpha isozyme. The inhibitors are highly potent, with the intrinsic Kd approximately equal to 1 nM as determined by isothermal titration calorimetry (ITC) and thermal shift assay (TSA). Dissection of protonation contributions yielded the intrinsic thermodynamic parameters of binding, such as enthalpy, entropy, Gibbs free energy, and the heat capacity. The differences in binding thermodynamic parameters between the series of inhibitors revealed contributions of the functional groups, thus providing insight into molecular reasons for improved or diminished binding efficiency. The inhibitor binding to Hsp90 alpha primarily depended on a large favorable enthalpic contribution combined with the smaller favorable entropic contribution, thus suggesting that their binding was both enthalpically and entropically optimized. The enthalpy-entropy compensation phenomenon was highly evident when comparing the inhibitor binding enthalpies and entropies. This study illustrates how detailed thermodynamic analysis helps to understand energetic reasons for the binding efficiency and develop more potent inhibitors that could be applied for therapeutic use as Hsp90 inhibitors.

Co-crystalization and in vitro biological characterization of 5-Aryl-4-(5-substituted-2-4-dihydroxyphenyl)-1,2,3-thiadiazole Hsp90 inhibitors

A potential therapeutic strategy for targeting cancer that has gained much interest is the inhibition of the ATP binding and ATPase activity of the molecular chaperone Hsp90. We have determined the structure of the human Hsp90α N-terminal domain in complex with a series of 5-aryl-4-(5-substituted-2-4-dihydroxyphenyl)-1,2,3-thiadiazoles. The structures provide the molecular details for the activity of these inhibitors. One of these inhibitors, ICPD 34, causes a structural change that affects a mobile loop, which adopts a conformation similar to that seen in complexes with ADP, rather than the conformation generally seen with the pyrazole/isoxazole-resorcinol class of inhibitors. Competitive binding to the Hsp90 N-terminal domain was observed in a biochemical assay, and these compounds showed antiproliferative activity and induced apoptosis in the HCT116 human colon cancer cell line. These inhibitors also caused induction of the heat shock response with the upregulation of Hsp72 and Hsp27 protein expression and the depletion of Hsp90 clients, CRAF, ERBB2 and CDK4, thus confirming that antiproliferative activity was through the inhibition of Hsp90. The presence of increased levels of the cleavage product of PARP indicated apoptosis in response to Hsp90 inhibitors. This work provides a framework for the further optimization of thiadiazole inhibitors of Hsp90. Importantly, we demonstrate that the thiadiazole inhibitors display a more limited core set of interactions relative to the clinical trial candidate NVP-AUY922, and consequently may be less susceptible to resistance derived through mutations in Hsp90.

Hsp90 stabilizes Cdc25A and counteracts heat shock-mediated Cdc25A degradation and cell-cycle attenuation in pancreatic carcinoma cells

Pancreas cancer cells escape most treatment options. Heat shock protein (Hsp)90 is frequently over-expressed in pancreas carcinomas and protects a number of cell-cycle regulators such as the proto-oncogene Cdc25A. We show that inhibition of Hsp90 with geldanamycin (GD) destabilizes Cdc25A independent of Chk1/2, whereas the standard drug for pancreas carcinoma treatment, gemcitabine (GEM), causes Cdc25A degradation through the activation of Chk2. Both agents applied together additively inhibit the expression of Cdc25A and the proliferation of pancreas carcinoma cells thereby demonstrating that both Cdc25A-destabilizing/degrading pathways are separated. The role of Hsp90 as stabilizer of Cdc25A in pancreas carcinoma cells is further supported by two novel synthetic inhibitors 4-tosylcyclonovobiocic acid and 7-tosylcyclonovobiocic acid and specific Hsp90AB1 (Hsp90β) shRNA. Our data show that targeting Hsp90 reduced the resistance of pancreas carcinoma cells to treatment with GEM.

Interaction of the middle domains stabilizes Hsp90α dimer in a closed conformation with high affinity for p23

Abstract The human genome encodes two highly similar cytosolic Hsp90 proteins called isoforms Hsp90α and Hsp90β. Of the 300 client proteins for Hsp90 identified so far only a handful interact specifically with one Hsp90 isoform. Here we report for the first time that Hsp90 cochaperone p23 binds preferentially to Hsp90α and that this interaction is mediated by the middle domain of Hsp90α. Based on the homology modeling, we infer that the middle domains in the Hsp90α dimer bind stronger with each other than in the Hsp90β dimer. Therefore, compared to Hsp90β, Hsp90α may adopt closed conformation more easily. Hsp90 interacts with p23 in the closed conformation. Hsp90α binds human recombinant p23 about three times stronger than Hsp90β but with significantly smaller exothermic enthalpy as determined by isothermal titration calorimetry of direct binding between the purified proteins. As p23 binds to Hsp90 in a closed conformation, stabilization of the Hsp90α dimer in the closed conformation by its middle domains explains preference of p23 to this Hsp90 isoform.

Improving the Hsp90 Inhibitors Containing 4-(2,4-Dihydroxyphenyl)-1,2,3- thiadiazole Scaffold: Synthesis, Affinity and Effect on Cancer Cells

BACKGROUND Human Hsp90 chaperone inhibitors are known to be potential anticancer drugs. Previously we have shown a couple of 5-aryl-4-(2,4-dihydroxyphenyl)-1,2,3-thiadiazoles to be promising anticancer agents. OBJECTIVE To improve the compounds containing 4-(2,4-dihydroxyphenyl)-1,2,3-thiadiazole scaffold as human Hsp90 inhibitors. METHOD We employed chemical synthesis to obtain new compounds and assayed their binding to Hsp90 using the fluorescence thermal shift assay and used MTT assays to determine their effect on cancer cells. RESULTS A series of compounds containing the 4-(2,4-dihydroxyphenyl)-1,2,3-thiadiazole scaffold were synthesized as Hsp90 inhibitors. Analysis of their binding to the recombinant N-terminal domain of Hsp90 revealed that four of these compounds bound to Hsp90 with Kd of 0.6 to 0.8 nM. The compounds fully inhibited the growth of all tested cancer cell lines: A549 (lung adenocarcinoma), IGR39 (melanoma), and U87 (glioblastoma), with the effective antiproliferative concentration (EC50) of the compounds reaching 0.35 µM. CONCLUSION This series of 14 novel and efficient Hsp90 inhibitors provided additional information on the structure-activity relationship of Hsp90 inhibitors and may be further developed into drugs targeting Hsp90.