Edward T. Petri

Structure and Clinical Relevance of the Epidermal Growth Factor Receptor in Human Cancer

PURPOSE To review the recent advances in the atomic-level understanding of the epidermal growth factor receptor (EGFR) tyrosine kinase (TK). We aim to highlight the current and future importance of these studies for the understanding and treatment of malignancies where EGFR-TK is improperly activated. METHODS The analysis was conducted on published crystal structures deposited in the Protein Data Bank (www.pdb.org) using the program O. RESULTS In this review we emphasize how recent EGFR kinase domain crystal structures can explain the mechanisms of activation for L858R and other EGFR-TK mutations, and compare these distinct activating mechanisms with those recently described for the wild-type EGFR. We suggest an atomic-level mechanism for the poor efficacy of lapatinib against tumors with activating EGFR kinase domain point mutations compared with the efficacy of gefitinib and erlotinib, and demonstrate how structural insights help our understanding of acquired resistance to these agents. We also highlight how these new molecular-level structural data are expected to affect the development of EGFR-TK targeted small molecule kinase inhibitors. CONCLUSION There are now more crystal structures published for the EGFR-TK domain than for any other TK. This wealth of crystallographic information is beginning to describe the mechanisms by which proper regulation of EGFR-TK is lost in disease. These crystal structures are beginning to show how small molecules inhibit EGFR-TK activity and will aid development of EGFR-TK mutant targeted therapies.

Domain Mapping of the Polycystin-2 C-terminal Tail Using de Novo Molecular Modeling and Biophysical Analysis

In polycystic kidney disease (PKD), polycystin-2 (PC2) is frequently mutated or truncated in the C-terminal cytoplasmic tail (PC2-C). The currently accepted model of PC2-C consists of an EF-hand motif overlapping with a short coiled coil; however, this model fails to explain the mechanisms by which PC2 truncations C-terminal to this region lead to PKD. Moreover, direct PC2 binding to inositol 1,4,5-trisphosphate receptor, KIF3A, and TRPC1 requires residues in PC2-C outside this region. To address these discrepancies and investigate the role of PC2-C in PC2 function, we performed de novo molecular modeling and biophysical analysis. De novo molecular modeling of PC2-C using the ROBETTA server predicts two domains as follows: an EF-hand motif (PC2-EF) connected by a linker to a previously unidentified C-terminal coiled coil (PC2-CC). This model differs substantially from the current model and correlates with limited proteolysis, matrix-assisted laser desorption/ionization mass spectroscopy, N-terminal sequencing, and improved coiled coil prediction algorithms. PC2-C is elongated and oligomerizes through PC2-CC, as measured by analytical ultracentrifugation and size exclusion chromatography, whereas PC2-EF is globular and monomeric. We show that PC2-C and PC2-EF have micromolar affinity for calcium (Ca2+) by isothermal titration calorimetry and undergo Ca2+-induced conformational changes by circular dichroism. Mutation of predicted EF-hand loop residues in PC2 to alanine abolishes Ca2+ binding. Our results suggest that PC2-CC is involved in PC2 oligomerization, and PC2-EF is a Ca2+-sensitive switch. PKD-associated PC2 mutations are located in regions that may disrupt these functions, providing structural insight into how PC2 mutations lead to disease.

Structure of the EF-hand domain of polycystin-2 suggests a mechanism for Ca2+-dependent regulation of polycystin-2 channel activity

The C-terminal cytoplasmic tail of polycystin-2 (PC2/TRPP2), a Ca2+-permeable channel, is frequently mutated or truncated in autosomal dominant polycystic kidney disease. We have previously shown that this tail consists of three functional regions: an EF-hand domain (PC2-EF, 720–797), a flexible linker (798–827), and an oligomeric coiled coil domain (828–895). We found that PC2-EF binds Ca2+ at a single site and undergoes Ca2+-dependent conformational changes, suggesting it is an essential element of Ca2+-sensitive regulation of PC2 activity. Here we describe the NMR structure and dynamics of Ca2+-bound PC2-EF. Human PC2-EF contains a divergent non-Ca2+-binding helix-loop-helix (HLH) motif packed against a canonical Ca2+-binding EF-hand motif. This HLH motif may have evolved from a canonical EF-hand found in invertebrate PC2 homologs. Temperature-dependent steady-state NOE experiments and NMR R1 and R2 relaxation rates correlate with increased molecular motion in the EF-hand, possibly due to exchange between apo and Ca2+-bound states, consistent with a role for PC2-EF as a Ca2+-sensitive regulator. Structure-based sequence conservation analysis reveals a conserved hydrophobic surface in the same region, which may mediate Ca2+-dependent protein interactions. We propose that Ca2+-sensing by PC2-EF is responsible for the cooperative nature of PC2 channel activation and inhibition. Based on our results, we present a mechanism of regulation of the Ca2+ dependence of PC2 channel activity by PC2-EF.

Cyclin B and Cyclin a Confer Different Substrate Recognition Properties on Cdk2.

The transitions of the cell cycle are regulated by the cyclin dependent protein kinases(CDKs). The cyclins activate their respective CDKs and confer substrate recognitionproperties. We report the structure of phospho-CDK2/cyclin B and show that cyclin Bconfers M phase-like properties on CDK2, the kinase that is usually associated with S phase.Cyclin B produces an almost identical activated conformation of CDK2 as that produced bycyclin A. There are differences between cyclin A and cyclin B at the recruitment site, whichin cyclin A is used to recruit substrates containing an RXL motif. Because of sequencedifferences this site in cyclin B binds RXL motifs more weakly than in cyclin A. Despitesimilarity in kinase structures, phospho-CDK2/cyclin B phosphorylates substrates, such asnuclear lamin and a model peptide derived from p107, at sequences SPXX that differ fromthe canonical CDK2/cyclin A substrate recognition motif, SPXK. CDK2/cyclin Bphosphorylation at these non-canonical sites is not dependent on the presence of a RXLrecruitment motif. The p107 peptide contained two SP motifs each followed by a noncanonicalsequence of which only one site (Ser640) is phosphorylated by pCDK2/cyclin Awhile two sites are phosphorylated by pCDK2/cyclin B. The second site is too close to theRXL motif to allow the cyclin A recruitment site to be effective, as previous work has shownthat there must be at least 16 residues between the catalytic site serine and the RXL motif.Thus the cyclins A and B in addition to their role in promoting the activatory conformationalswitch in CDK2, also provide differential substrate specificity.

The crystal structure of human cyclin B.

Cyclin B is the key regulatory protein controlling mitosis in all eukaryotes, where it binds cyclin-dependent kinase, cdk1, forming a complex which initiates the mitotic program through phosphorylation of select proteins. Cyclin B regulates the activation, subcellular localization, and substrate specificity of cdk1, and destruction of cyclin B is necessary for mitotic exit. Overexpression of human cyclin B1 has been found in numerous cancers and has been associated with tumor aggressiveness. Here we report the crystal structure of human cyclin B1 to 2.9 Å. Comparison of the structure with cyclin A and cyclin E reveals remarkably similar N-terminal cyclin box motifs but significant differences among the C-terminal cyclin box lobes. Divergence in sequence gives rise to unique interaction surfaces at the proposed cyclin B/ cdk1 interface as well as the ‘RxL’ motif substrate binding site on cyclin B. Examination of the structure provides insight into the molecular basis for differential affinities of protein based cyclin/cdk inhibitors such as p27, substrate recognition, and cdk interaction.

Calcium-induced Conformational Changes in C-terminal Tail of Polycystin-2 Are Necessary for Channel Gating

Background: Polycystin-2, a calcium-permeable TRP channel, is mutated in autosomal dominant polycystic kidney disease. Results: Calcium binding by the polycystin-2 EF-hand domain induces discrete conformational and oligomerization state transitions that impact channel gating. Conclusion: Polycystin-2 channel activity is regulated by cytoplasmic calcium-induced conformational changes. Significance: These studies provide a structural and mechanistic understanding for the impact of calcium binding on channel regulation. Polycystin-2 (PC2) is a Ca2+-permeable transient receptor potential channel activated and regulated by changes in cytoplasmic Ca2+. PC2 mutations are responsible for ∼15% of autosomal dominant polycystic kidney disease. Although the C-terminal cytoplasmic tail of PC2 has been shown to contain a Ca2+-binding EF-hand domain, the molecular basis of PC2 channel gating by Ca2+ remains unknown. We propose that the PC2 EF-hand is a Ca2+ sensor required for channel gating. Consistent with this, Ca2+ binding causes a dramatic decrease in the radius of gyration (Rg) of the PC2 EF-hand by small angle x-ray scattering and significant conformational changes by NMR. Furthermore, increasing Ca2+ concentrations cause the C-terminal cytoplasmic tail to transition from a mixture of extended oligomers to a single compact dimer by analytical ultracentrifugation, coupled with a >30 Å decrease in maximum interatomic distance (Dmax) by small angle x-ray scattering. Mutant PC2 channels unable to bind Ca2+ via the EF-hand are inactive in single-channel planar lipid bilayers and inhibit Ca2+ release from ER stores upon overexpression in cells, suggesting dominant negative properties. Our results support a model where PC2 channels are gated by discrete conformational changes in the C-terminal cytoplasmic tail in response to changes in cytoplasmic Ca2+ levels. These properties of PC2 are lost in autosomal dominant polycystic kidney disease, emphasizing the importance of PC2 to kidney cell function. We speculate that PC2 and the Ca2+-dependent transient receptor potential channels in general are regulated by similar conformational changes in their cytoplasmic domains that are propagated to the channel pore.

Analysis of the cytoplasmic interaction between polycystin-1 and polycystin-2

Autosomal dominant polycystic kidney disease (ADPKD) arises following mutations of either Pkd1 or Pkd2. The proteins these genes encode, polycystin-1 (PC1) and polycystin-2 (PC2), form a signaling complex using direct intermolecular interactions. Two distinct domains in the C-terminal tail of PC2 have recently been identified, an EF-hand and a coiled-coil domain. Here, we show that the PC2 coiled-coil domain interacts with the C-terminal tail of PC1, but that the PC2 EF-hand domain does not. We measured the K0.5 of the interaction between the C-terminal tails of PC1 and PC2 and showed that the direct interaction of these proteins is abrogated by a PC1 point mutation that was identified in ADPKD patients. Finally, we showed that overexpression of the PC1 C-terminal tail in MDCK cells alters the Ca2+ response, but that overexpression of the PC1 C-terminal tail containing the disease mutation does not. These results allow a more detailed understanding of the mechanism of pathogenic mutations in the cytoplasmic regions of PC1 and PC2.

17(E)-picolinylidene androstane derivatives as potential inhibitors of prostate cancer cell growth: antiproliferative activity and molecular docking studies.

We report a rapid and efficient synthesis of A-ring modified 17α-picolyl and 17(E)-picolinylidene androstane derivatives from dehydroepiandrosterone. Compounds were validated spectroscopically and structurally characterized by X-ray crystallography. Virtual screening by molecular docking against clinical targets of steroidal anticancer drugs (ERα, AR, Aromatase and CYP17A1) suggests that 17(E)-picolinylidene, but not 17α-picolyl androstanes could specifically interact with CYP17A1 (17α-hydroxylase) with similar geometry and affinity as Abiraterone, a 17-pyridinyl androstane drug clinically used in the treatment of prostate cancer. In addition, several 17(E)-picolinylidene androstanes demonstrated selective antiproliferative activity against PC3 prostate cancer cells, which correlates with Abiraterone antiproliferative activity and predicted CYP17A1 binding affinities. Based on these preliminary results, 17(E)-picolinylidene androstane derivatives could be a promising starting point for the development of new compounds for the treatment of prostate cancer.

Disruption of the EGFR E884-R958 ion pair conserved in the human kinome differentially alters signaling and inhibitor sensitivity.

Targeted therapy against epidermal growth factor receptor (EGFR) represents a major therapeutic advance in lung cancer treatment. Somatic mutations of the EGFR gene, most commonly L858R (exon 21) and short in-frame exon 19 deletions, have been found to confer enhanced sensitivity toward the inhibitors gefitinib and erlotinib. We have recently identified an EGFR mutation E884K, in combination with L858R, in a patient with advanced lung cancer who progressed on erlotinib maintenance therapy, and subsequently had leptomeningeal metastases that responded to gefitinib. The somatic E884K substitution appears to be relatively infrequent and resulted in a mutant lysine residue that disrupts an ion pair with residue R958 in the EGFR kinase domain C-lobe, an interaction that is highly conserved within the human kinome as demonstrated by our sequence analysis and structure analysis. Our studies here, using COS-7 transfection model system, show that E884K works in concert with L858R in-cis, in a dominant manner, to change downstream signaling, differentially induce Mitogen-activated protein kinase (extracellular signaling-regulated kinase 1/2) signaling and associated cell proliferation and differentially alter sensitivity of EGFR phosphorylation inhibition by ERBB family inhibitors in an inhibitor-specific manner. Mutations of the conserved ion pair E884–R958 may result in conformational changes that alter kinase substrate recognition. The analogous E1271K-MET mutation conferred differential sensitivity toward preclinical MET inhibitors SU11274 (unchanged) and PHA665752 (more sensitive). Systematic bioinformatics analysis of the mutation catalog in the human kinome revealed the presence of cancer-associated mutations involving the conserved E884 homologous residue, and adjacent residues at the ion pair, in known proto-oncogenes (KIT, RET, MET and FAK) and tumor-suppressor gene (LKB1). Targeted therapy using small-molecule inhibitors should take into account potential cooperative effects of multiple kinase mutations, and their specific effects on downstream signaling and inhibitor sensitivity. Improved efficacy of targeted kinase inhibitors may be achieved by targeting the dominant activating mutations present.

Synthesis and anticancer cell potential of steroidal 16,17-seco-16,17a-dinitriles: Identification of a selective inhibitor of hormone-independent breast cancer cells

We report the synthesis of steroidal 16,17-seco-16,17a-dinitriles and investigate their antitumor cell properties. Compounds were evaluated for anticancer potential by in vitro antiproliferation studies, molecular docking and virtual screening. Several compounds inhibit the growth of breast and prostate cancer cell lines (MCF-7, MDA-MB-231 and PC3), and/or cervical cancer cells (HeLa). Supporting this, molecular docking predicts that steroidal 16,17-seco-16,17a-dinitriles could bind with high affinity to multiple molecular targets of breast and prostate cancer treatment (aromatase, estrogen receptor α, androgen receptor and 17α-hydroxylase) facilitated by D-seco flexibility and nitrile-mediated contacts. Thus, 16,17-seco-16,17a-dinitriles may be useful for the design of inhibitors of multiple steroidogenesis pathways. Strikingly, 10, a 1,4-dien-3-on derivative, displayed selective submicromolar antiproliferative activity against hormone-dependent (MCF-7) and -independent (MDA-MB-231) breast cancer cells (IC50 0.52, 0.11μM, respectively). Ligand-based 3D similarity searches suggest AKR1C, 17β-HSD and/or 3β-HSD subfamilies as responsible for this antiproliferative activity, while fast molecular docking identified AKR1C and ERβ as potential binders-both targets in the treatment of hormone-independent breast cancers.