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Retroviral integrases (INs) contain two known metal binding domains. The N-terminal domain includes a zinc finger motif and has been shown to bind Zn2+, whereas the central catalytic core domain includes a triad of acidic amino acids that bind Mn2+ or Mg2+, the metal cofactors required for enzymatic activity. The integration reaction occurs in two distinct steps; the first is a specific endonucleolytic cleavage step called "processing," and the second is a polynucleotide transfer or "joining" step. Our previous results showed that the metal preference for in vitro activity of avian sarcoma virus IN is Mn2+ > Mg2+ and that a single cation of either metal is coordinated by two of the three critical active site residues (Asp-64 and Asp-121) in crystals of the isolated catalytic domain. Here, we report that Ca2+, Zn2+, and Cd2+ can also bind in the active site of the catalytic domain. Furthermore, two zinc and cadmium cations are bound at the active site, with all three residues of the active site triad (Asp-64, Asp-121, and Glu-157) contributing to their coordination. These results are consistent with a two-metal mechanism for catalysis by retroviral integrases. We also show that Zn2+ can serve as a cofactor for the endonucleolytic reactions catalyzed by either the full-length protein, a derivative lacking the N-terminal domain, or the isolated catalytic domain of avian sarcoma virus IN. However, polynucleotidyl transferase activities are severely impaired or undetectable in the presence of Zn2+. Thus, although the processing and joining steps of integrase employ a similar mechanism and the same active site triad, they can be clearly distinguished by their metal preferences.

Inhibitors of the DNA damage checkpoint kinase, Chk1, are highly effective as chemo- and radio-sensitizers in preclinical studies but are not well tolerated by patients. We took advantage of the promiscuous nature of kinase inhibitors and screened 9 clinically relevant kinase inhibitors for their ability to sensitize pancreatic cancer cells (Panc1) to a sub-lethal concentration of gemcitabine. bosutinib, dovitinib and BEZ-235, were identified as sensitizers that abrogated the DNA damage checkpoint. We further characterized bosutinib, an FDA approved Src / Abl inhibitor for the treatment of chronic myelogenous leukemia (CML). Other Src-family kinase inhibitors did not exhibit chemosensitization activity. Unbeknownst to us, the bosutinib we purchased was an isomer (Bos-I) that was unknowingly synthesized and sold to the research community as "authentic" bosutinib. The two compounds differed only in the arrangement of the same R groups around the aniline ring. Authentic bosutinib is designated 2, 4 dichloro, 5-methoxy, while Bosutinib isomer is 3, 5 dichloro, 4-methyoxy. Although our screen identified the isomer, we found that authentic bosutinib also had chemosensitization activity In vitro. Both bosutinib and Bos-I inhibited DNA damage checkpoint kinases Chk1 and Wee1 in vitro, with Bos-I showing greater potency. These inhibitors enhanced sensitivity to genotoxic agents that either arrest cells in S phase (gemcitabine, cisplatin) or G2 (doxorubicin). Live-cell imaging and immunofluorescence staining showed that Bos-I forced drug-treated cells to override the checkpoint arrest and entered an aberrant mitosis. The in vivo efficacy of bosutinib and Bos-I were validated using cells derived directly from a pancreatic cancer patient's tumor. Notably, the xenograft studies showed that the combination of gemcitabine and bosutinib or Bos-I were significantly more effective in suppressing tumor growth than any of the agents used alone. Consistent with the in vitro data that showed Bos-I was more potent checkpoint inhibitor than bosutinib, higher concentrations of bosutinib were needed to suppress tumor growth by gemcitabine. Molecular modeling of bosutinib and Bos-I bound to Chk1 and Wee1 indicate that the gatekeeper residues and the position of the methoxy group in the aniline ring may be critical determinants of drug binding. The methoxy group of bosutinib (position 5) clashes within the binding pocket particularly near the gatekeeper residues Leu and Asn, in Chk1 and Wee1 respectively. The methoxy group in the Bos-I (position 3) is shifted away from the gatekeeper and allows the inhibitor to bind deeper in the binding site for both kinases. The structural predictions are supported by relative binding energy calculations of bosutininb and Bos-I bound to these kinases. In addition, in vitro kinase assays that show that changing the gatekeeper residue in Wee1 reduced its sensitivity to Bos-I but enhanced its sensitivity to the authentic bosutinib. Our strategy to screen clinically relevant kinase inhibitors for off-target effects on cell cycle checkpoints is a promising approach to re-purpose drugs as chemosensitizers. Checkpoint override by bosutinib and its isomer maybe due to ability to dually inhibit Chk1 and Wee1 kinases. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):B57. Citation Format: Neil Beeharry, Eugenia Banina, Vladmir Khazak, Sean Deacon, Jeff Peterson, Mark Andrake, Igor Astsaturov, Tim J. Yen. Bosutinib and bosutinib-isomer are novel Chk1 and Wee1 kinase inhibitors that sensitize cells to DNA damaging agent by overriding cell cycle checkpoint arrest. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr B57.

In tumor but not normal cells, the stress-inducible heat shock protein 70 (HSP70) is abundantly expressed and becomes incorporated into lysosome membranes, where it serves to stabilize and protect lysosome function. Inhibition of HSP70 thus results in defective lysosome function, along with impaired autophagy. We previously reported that the HSP70 inhibitor phenylethynesulfonamide (PES) is a potent and effective autophagy inhibitor (1). PES shows marked cytotoxicity to tumor cells, but minimal effects in normal, non-transformed cells. We previously reported that PES binds to both HSP70 and to the constitutively-expressed family member HSC70; these are critical co-chaperones for HSP90, and we previously reported that treatment of tumor cells with PES leads to decreased function of HSP90 client proteins like HER2, AKT and CDK4 (2). We have recently performed a preliminary structure-activity relationship for PES. These studies revealed a novel PES analogue that we call PES-Cl, which shows 10-fold decreased IC50 for tumor cells, minimal cytotoxicity to normal cells, and a greatly enhanced ability to inhibit autophagy. In vitro we show that PES-Cl is cytotoxic to melanoma cells, including those with both intrinsic and acquired resistance to BRAF inhibitors, but shows minimal cytotoxicity to primary melanocytes. In a pre-clinical model for B-cell lymphoma (Eu-myc transgenic mouse), we show that PES-Cl demonstrates significant ability to extend the life of mice (p=0.000006), with no evidence for liver pathology or toxicity. We report that PES binds to the substrate-binding domain of HSP70, and requires the C-terminal helical ‘lid’ of this protein (amino acids 573-616) in order to bind. Using molecular modeling and in silico docking, we have identified a candidate binding site for PES in HSP70, and we identify point mutants that fail to interact with this compound. Our cell cycle analyses of PES-Cl-treated cells show that this compound induces G2/M arrest. Interestingly, we also show that this HSP70 inhibitor impairs the activity of the Anaphase Promoting Complex/Cyclosome (APC/C) in cell-free extracts. PES-Cl is thus a promising new HSP70 inhibitor that binds to the C-terminal ‘lid’ of HSP70, and multiple mechanisms of action: inhibition of autophagy, inhibition of HSP90 function, and inhibition of the Anaphase Promoting Complex/Cyclosome (APC/C). 1. Leu et al, Molecular Cell, 6(1):15-27, 2009 2. Leu et al, Molecular Cancer Research, 9(7):936-47, 2011 Citation Format: Gregor Balaburski, Julia I-Ju Leu, Neil Beeharry, Seth Hayik, Mark D. Andrake, Gao Zhang, Meenhard Herlyn, Jessie Villanueva, Roland L. Dunbrack, Tim Yen, Donna L. George, Maureen E. Murphy. Autophagy inhibition for cancer therapy. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1683. doi:10.1158/1538-7445.AM2013-1683

The stress-induced heat shock protein HSP70 is an ATP dependent molecular chaperone that plays a role in the folding of nascent peptides, assembly of multi-protein complexes, and transport of proteins across membranes and overall. Additionally, HSP70 has significant cytoprotective and anti-apoptotic functions. High levels of HSP70 can be found in many cancers, and are associated with poor therapeutic response and poor patient prognosis. These findings suggest that HSP70 may be a desirable molecular therapeutic target, in addition to being a useful tumor prognostic marker. Previously we demonstrated that the compound 2-phenylethynesulfonamide (PES) interacts with HSP70 and antagonizes HSP70 function by disrupting the interaction between HSP70 and its co-chaperones and client proteins. We showed that PES is toxic to cancer cells but not non-transformed cells. Moreover, PES was able to significantly extend the lifespan of mice in a Myc-driven model of lymphoma (p Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3793. doi:1538-7445.AM2012-3793

The stress induced molecular chaperone Hsp70 has a role in protein folding, assembly of multi-protein complexes and transport of proteins across membranes. Overall, this protein possesses significant cytoprotective and anti-apoptotic functions. High levels of Hsp70 can be found in many cancers, and these are associated with resistance to radiation and chemotherapy. Not surprisingly, overexpression of Hsp70 is generally a marker of poor patient prognosis. These combined data suggest that Hsp70, in addition to being a potential tumor prognostic marker, may also be a desirable molecular therapeutic target. 2-phenylethynesulfonamide (PES) was previously identified as a compound that inhibits apoptosis in thymocytes, by virtue of its ability to inhibit the direct p53 mitochondrial pathway. However, the mechanism of action of PES remained unknown. We recently demonstrated that PES specifically interacts with Hsp70, disrupts the interaction between Hsp70 and its co-chaperones and client proteins, and inhibits Hsp70 function at the lysosome. We further showed that PES exhibits selective toxicity to transformed cells by virtue of its ability to inhibit autophagy, a key survival pathway utilized by tumor cells. Here-in we report that we have used deletion mapping and molecular modeling to elucidate the domain of Hsp70 that interacts with PES. Additionally, to further elucidate the mechanism of action of PES, we generated and tested several analogues of PES. We find that the ability of PES to induce the death of tumor cells consistently correlates with its ability to inhibit autophagy. Further, we identified one analogue that has superior ability to induce programmed cell death in tumor cells. We are currently analyzing the efficacy and mechanism of action of this analogue in pre-clinical models. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3771. doi:10.1158/1538-7445.AM2011-3771

Biosynthesis of bacteriophage T4 DNA polymerase is autogenously regulated at the translational level. The enzyme, product of gene 43, represses its own translation by binding to its mRNA 5' to the initiator AUG at a 36-40 nucleotide segment that includes the Shine-Dalgarno sequence and a putative RNA hairpin structure consisting of a 5-base-pair stem and an 8-base loop. We constructed mutations that either disrupted the stem or altered specific loop residues of the hairpin and found that many of these mutations, including single-base changes in the loop sequence, diminished binding of purified T4 DNA polymerase to its RNA in vitro (as measured by a gel retardation assay) and derepressed synthesis of the enzyme in vivo (as measured in T4 infections and by recombinant-plasmid-mediated expression). In vitro effects, however, were not always congruent with in vivo effects. For example, stem pairing with a sequence other than wild-type resulted in normal protein binding in vitro but derepression of protein synthesis in vivo. Similarly, a C----A change in the loop had a small effect in vitro and a strong effect in vivo. In contrast, an A----U change near the base of the hairpin that was predicted to increase the length of the base-paired stem had small effects both in vitro and in vivo. The results suggest that interaction of T4 DNA polymerase with its structured RNA operator depends on the spatial arrangement of specific nucleotide residues and is subject to modulation in vivo.

SummaryThe nuclear lamina is a protein meshwork that lies under the inner nuclear membrane of metazoan cells. One function of the nuclear lamina is to organize heterochromatin at the inner nuclear periphery. However, very little is known about how heterochromatin attaches to the nuclear lamina and how such attachments are restored at mitotic exit. Here, we show that a previously unstudied human protein, PRR14, functions to tether heterochromatin to the nuclear periphery during interphase, through associations with heterochromatin protein 1 (HP1) and the nuclear lamina. During early mitosis, PRR14 is released from the nuclear lamina and chromatin and remains soluble. Strikingly, at the onset of anaphase, PRR14 is incorporated rapidly into chromatin through HP1 binding. Finally, in telophase, PRR14 relocalizes to the reforming nuclear lamina. This stepwise reassembly of PRR14 suggests a function in the selection of HP1-bound heterochromatin for reattachment to the nuclear lamina as cells exit mitosis.

In bacteriophage T4 the protein product of gene 43 (gp43) is a multifunctional DNA polymerase that is essential for replication of the phage genome. The protein harbors DNA-binding, deoxyribonucleotide-binding, DNA-synthesizing (polymerase) and 3'-exonucleolytic (editing) activities as well as a capacity to interact with several other T4-induced replication enzymes. In addition, the T4 gp43 is a repressor of its own synthesis in vivo. We show here that this protein is an autogenous repressor of translation, and we have localized its RNA-binding sequence (translational operator) to the translation initiation domain of gene 43 mRNA. This mechanism for regulation of T4 DNA polymerase expression underscores the ubiquity of translational repression in the control of T4 DNA replication. Many T4 DNA polymerase accessory proteins and nucleotide biosynthesis enzymes are regulated by the phage-induced translational repressor regA, while the T4 single-stranded DNA-binding protein (T4 gp32) is, like gp43, autogenously regulated at the translational level.