Michael C. Cavalier

Structure of the STRA6 receptor for retinol uptake.

A window into the cell for vitamin A Vitamin A is an essential nutrient for mammals, and its metabolites affect diverse biological processes. It is carried in the bloodstream as retinol by retinol binding protein (RBP); a protein called STRA6 is implicated in facilitating retinol translocation across the cell membrane. Chen et al. determined the structure of zebrafish STRA6 to a resolution of 3.9 Å by electron microscopy. A lipophilic cleft is a likely binding site for RBP, and an opening in the cleft may allow retinol to diffuse into the membrane. Unexpectedly, the structure also includes bound calcium-modulated protein, but its function remains unclear. Science, this issue p. 887 The structure of a STRA6-calmodulin complex gives insight into how retinol (vitamin A) enters cells. INTRODUCTION Vitamin A is an essential nutrient for all mammals, being vital for vision and for transcription of a wide array of genes. Retinol (vitamin A alcohol) is the predominant circulating retinoid. In the fasting state, retinol from liver stores is mobilized bound to retinol-binding protein (RBP), which transports this highly hydrophobic molecule in the bloodstream. How retinol is released from RBP and internalized by target cells has been the subject of intense debate. The RBP receptor, STRA6, was cloned in 2007. STRA6 was predicted to be a 75-kDa multipass transmembrane (TM) protein without sequence similarity to any known transporter, channel, or receptor. STRA6 is expressed widely, with particular abundance in the eye and placenta. Mutations in the human STRA6 gene have been linked to Matthew-Wood syndrome, which presents with ocular abnormalities and developmental defects. RATIONALE Despite a wealth of biochemical work aimed at investigating how STRA6 mediates internalization of retinol from RBP, progress at the molecular level has been hindered by the absence of structural information. Purified STRA6 from zebrafish was a detergent-stable dimer in an unexpected association with calmodulin (CaM), forming a 180-kDa complex. RESULTS Using cryo-electron microscopy, we determined the structure of zebrafish STRA6 in complex with CaM to 3.9 Å resolution. The protein is assembled as an intricate dimer with a topology that includes 18 TM helices (nine per protomer) and two long horizontal intramembrane (IM) helices interacting at the dimer core. Each STRA6 protomer comprises an N-terminal domain (NTD) of the first five TM helices, connected by a linker containing the first CaM-binding peptide to a central domain at the dimer interface that includes TMs 6 to 9 and the IM helices, and a cytoplasmic C-terminal segment that interacts with CaM through two additional helices. Each protomer is compactly associated with one molecule of CaM, adopting an unconventional arrangement in which it is bound to three helical regions of STRA6. We characterized the STRA6-CaM interaction biophysically by isothermal titration calorimetry, showing that the affinity of CaM for one STRA6 peptide alone is subnanomolar, and structurally by x-ray crystallography. We also demonstrated that the STRA6-CaM association is physiological by performing immunoprecipitation experiments on native zebrafish tissue. Both the extra- and intracellular surfaces of the NTD feature conserved polar pockets. The outer NTD pocket spans half the bilayer. The central domain of STRA6 defines a large ~20,000 Å3 cleft on the extracellular side, which encompasses the space between previously characterized binding sites for RBP, ~25 Å above the membrane surface, and the IM helices located down at the mid-bilayer level. This outer cleft is hydrophobic, contains two ordered putative cholesterols, and is exposed to the membrane through two symmetry-related lateral windows defined by TMs 8 and 9 and the IM helices. CONCLUSIONS The structure of STRA6 suggests a mechanism for retinol release from RBP into the hydrophobic environment of the outer cleft and direct diffusion into the membrane through the lateral window. Our work also sets the basis for future experiments aimed at investigating how the system is regulated, whether STRA6 also has a role in signaling, and the functional relevance of its association with CaM. The structure of STRA6 in complex with CaM. The STRA6 dimer, drawn as a ribbon representation with one protomer in dark red and the other in black, is associated with two molecules of calmodulin, drawn in gray and gold. The internal volume of the outer cleft is represented as a semitransparent blue surface. Calcium ions are represented as green spheres. Vitamin A homeostasis is critical to normal cellular function. Retinol-binding protein (RBP) is the sole specific carrier in the bloodstream for hydrophobic retinol, the main form in which vitamin A is transported. The integral membrane receptor STRA6 mediates cellular uptake of vitamin A by recognizing RBP-retinol to trigger release and internalization of retinol. We present the structure of zebrafish STRA6 determined to 3.9-angstrom resolution by single-particle cryo-electron microscopy. STRA6 has one intramembrane and nine transmembrane helices in an intricate dimeric assembly. Unexpectedly, calmodulin is bound tightly to STRA6 in a noncanonical arrangement. Residues involved with RBP binding map to an archlike structure that covers a deep lipophilic cleft. This cleft is open to the membrane, suggesting a possible mode for internalization of retinol through direct diffusion into the lipid bilayer.

Covalent Small Molecule Inhibitors of Ca(2+)-Bound S100B.

Elevated levels of the tumor marker S100B are observed in malignant melanoma, and this EF-hand-containing protein was shown to directly bind wild-type (wt) p53 in a Ca2+-dependent manner, dissociate the p53 tetramer, and inhibit its tumor suppression functions. Likewise, inhibiting S100B with small interfering RNA (siRNAS100B) is sufficient to restore wild-type p53 levels and its downstream gene products and induce the arrest of cell growth and UV-dependent apoptosis in malignant melanoma. Therefore, it is a goal to develop S100B inhibitors (SBiXs) that inhibit the S100B–p53 complex and restore active p53 in this deadly cancer. Using a structure–activity relationship by nuclear magnetic resonance approach (SAR by NMR), three persistent binding pockets are found on S100B, termed sites 1–3. While inhibitors that simultaneously bind sites 2 and 3 are in place, no molecules that simultaneously bind all three persistent sites are available. For this purpose, Cys84 was used in this study as a potential means to bridge sites 1 and 2 because it is located in a small crevice between these two deeper pockets on the protein. Using a fluorescence polarization competition assay, several Cys84-modified S100B complexes were identified and examined further. For five such SBiX–S100B complexes, crystallographic structures confirmed their covalent binding to Cys84 near site 2 and thus present straightforward chemical biology strategies for bridging sites 1 and 3. Importantly, one such compound, SC1982, showed an S100B-dependent death response in assays with WM115 malignant melanoma cells, so it will be particularly useful for the design of SBiX molecules with improved affinity and specificity.

Molecular basis of the fructose-2,6-bisphosphatase reaction of PFKFB3: Transition state and the C-terminal function.

The molecular basis of fructose‐2,6‐bisphosphatase (F‐2,6‐P2ase) of 6‐phosphofructo‐2‐kinase/fructose‐2,6‐bisphosphatase (PFKFB) was investigated using the crystal structures of the human inducible form (PFKFB3) in a phospho‐enzyme intermediate state (PFKFB3‐P•F‐6‐P), in a transition state–analogous complex (PFKFB3•AlF4), and in a complex with pyrophosphate (PFKFB3•PPi) at resolutions of 2.45, 2.2, and 2.3 Å, respectively. Trapping the PFKFB3‐P•F‐6‐P intermediate was achieved by flash cooling the crystal during the reaction, and the PFKFB3•AlF4 and PFKFB3•PPi complexes were obtained by soaking. The PFKFB3•AlF4 and PFKFB3•PPi complexes resulted in removing F‐6‐P from the catalytic pocket. With these structures, the structures of the Michaelis complex and the transition state were extrapolated. For both the PFKFB3‐P formation and break down, the phosphoryl donor and the acceptor are located within ∼5.1 Å, and the pivotal point 2‐P is on the same line, suggesting an “in‐line” transfer with a direct inversion of phosphate configuration. The geometry suggests that NE2 of His253 undergoes a nucleophilic attack to form a covalent NP bond, breaking the 2OP bond in the substrate. The resulting high reactivity of the leaving group, 2O of F‐6‐P, is neutralized by a proton donated by Glu322. Negative charges on the equatorial oxygen of the transient bipyramidal phosphorane formed during the transfer are stabilized by Arg252, His387, and Asn259. The C‐terminal domain (residues 440–446) was rearranged in PFKFB3•PPi, implying that this domain plays a critical role in binding of substrate to and release of product from the F‐2,6‐P2ase catalytic pocket. These findings provide a new insight into the understanding of the phosphoryl transfer reaction. Proteins 2012;

Investigating combinatorial approaches in virtual screening on human inducible 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFKFB3): A case study for small molecule kinases ☆

Efforts toward improving the predictiveness in tier-based approaches to virtual screening (VS) have mainly focused on protein kinases. Despite their significance as drug targets, small molecule kinases have been rarely tested with these approaches. In this paper, we investigate the efficacy of a pharmacophore screening-combined structure-based docking approach on the human inducible 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase, an emerging target for cancer chemotherapy. Six out of a total 1364 compounds from NCIs Diversity Set II were selected as true actives via throughput screening. Using a database constructed from these compounds, five programs were tested for structure-based docking (SBD) performance, the MOE of which showed the highest enrichments and second highest screening rates. Separately, using the same database, pharmacophore screening was performed, reducing 1364 compounds to 287 with no loss in true actives, yielding an enrichment of 4.75. When SBD was retested with the pharmacophore filtered database, 4 of the 5 SBD programs showed significant improvements to enrichment rates at only 2.5% of the database, with a 7-fold decrease in an average VS time. Our results altogether suggest that combinatorial approaches of VS technologies are easily applicable to small molecule kinases and, moreover, that such methods can decrease the variability associated with single-method SBD approaches.

A natural human monoclonal antibody targeting Staphylococcus Protein A protects against Staphylococcus aureus bacteremia

Staphylococcus aureus can cause devastating and life-threatening infections. With the increase in multidrug resistant strains, novel therapies are needed. Limited success with active and passive immunization strategies have been attributed to S. aureus immune evasion. Here, we report on a monoclonal antibody, 514G3, that circumvents a key S. aureus evasion mechanism by targeting the cell wall moiety Protein A (SpA). SpA tightly binds most subclasses of immunoglobulins via their Fc region, neutralizing effector function. The organism can thus shield itself with a protective coat of serum antibodies and render humoral immunity ineffective. The present antibody reactivity was derived from an individual with natural anti-SpA antibody titers. The monoclonal antibody is of an IgG3 subclass, which differs critically from other immunoglobulin subclasses since its Fc is not bound by SpA. Moreover, it targets a unique epitope on SpA that allows it to bind in the presence of serum antibodies. Consequently, the antibody opsonizes S. aureus and maintains effector function to enable natural immune mediated clearance. The data presented here provide evidence that 514G3 antibody is able to successfully rescue mice from S. aureus mediated bacteremia.

Novel protein–inhibitor interactions in site 3 of Ca2+-bound S100B as discovered by X-ray crystallography

Structure-based drug discovery is under way to identify and develop small-molecule S100B inhibitors (SBiXs). Such inhibitors have therapeutic potential for treating malignant melanoma, since high levels of S100B downregulate wild-type p53 tumor suppressor function in this cancer. Computational and X-ray crystallographic studies of two S100B-SBiX complexes are described, and both compounds (apomorphine hydrochloride and ethidium bromide) occupy an area of the S100B hydrophobic cleft which is termed site 3. These data also reveal novel protein-inhibitor interactions which can be used in future drug-design studies to improve SBiX affinity and specificity. Of particular interest, apomorphine hydrochloride showed S100B-dependent killing in melanoma cell assays, although the efficacy exceeds its affinity for S100B and implicates possible off-target contributions. Because there are no structural data available for compounds occupying site 3 alone, these studies contribute towards the structure-based approach to targeting S100B by including interactions with residues in site 3 of S100B.

Abstract 1358: Structure-based drug design of 3-site binding, high affinity inhibitors of S100B in malignant melanoma

An especially predictive biomarker when used in combination with other diagnostic indicators, S100B is elevated in >90% of malignant melanoma (MM) patients and its protein level correlates directly with poor survival ( Citation Format: Michael C. Cavalier, Paul T. Wilder, Diane Luci, David J. Maloney, Lei Fang, Mohd. Imran Ansari, Alexander D. MacKerell, Andrew Coop, Ajit Jadhav, David J. Weber. Structure-based drug design of 3-site binding, high affinity inhibitors of S100B in malignant melanoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1358.

Abstract 2454: Targeting the translational enhancer complex MCT-1:DenR to disrupt NHL survival

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA Despite the vast number of therapeutic advances that have been made, Non-Hodgkin Lymphoma (NHL) remains a leading cause of cancer-related deaths in the United States. In order to increase the NHL cure rate, new targeted options are needed. One such option that has been the focus of much recent interest is the post-transcriptional regulation in anti-apoptotic phenotypes of NHL, particularly the translational enhancer complex of MCT-1 and DENR. Involved in translation initiation and ribosomal recycling for transcripts regulated by 5′-uORF’s, the MCT-1:DENR complex has been shown to increase cell proliferation and the translation of survival-related transcripts through the combination of two critical RNA-binding domains. The goal of this project has been to biophysically characterize the formation and activity of this complex and to develop a method for evaluating the effects of disrupting its formation in vivo through the combination of NMR, X-ray crystallography, cell-free and cell-based assays. Ultimately, the information gained from this study will be used to evaluate a library of perturbagens that interfere with dimer formation and the development of MCT-1 driven anti-apoptotic phenotypes. Citation Format: Sean D. Stowe, Michael C. Cavalier, Raquel Godoy-Ruiz, Kristen J. Varney, Paul T. Wilder, Ronald B. Gartenhaus, David J. Weber. Targeting the translational enhancer complex MCT-1:DenR to disrupt NHL survival. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2454. doi:10.1158/1538-7445.AM2015-2454

Abstract 3221: Covalent inhibitors of S100B (SBiXs) in malignant melanoma

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Summary: The calcium binding protein, S100B, has been directly linked to malignant melanoma (MM) and has long been used as a prognostic indicator. Studies have proven S100B to not only be a clinical marker within MM, but that it binds to the tumor suppressor p53, and promotes its degradation. p53 is typically found as wild-type in MM, and inhibition of S100B expression restores both p53 protein levels and normal transcriptional activation/apoptosis activities. Subsequently, we have made it our long-term goal to discover S100B inhibitors (SBiXs) in order to restore the p53 tumor suppression function in patients with MM, and have already identified many SBiXs. Purpose: It is our purpose to discover/synthesize and/or to improve existing inhibitors of S100B to restore p53 activity in human malignant melanoma. Amongst these SBiXs are compounds which covalently modify S100B. The effects of covalent SBiXs on S100B in Malignant Melanoma are to be investigated. Results and conclusions: The development of S100B inhibitors (SBiXs) is ongoing at the University of Maryland Schools of Pharmacy and Medicine with the goal of improving affinity and specificity towards inhibiting S100B. Fluorescent Polarization Completion Assays have identified compounds that bind S100B and inhibit melanoma cell growth. S100B binding by these SBiXs was confirmed using NMR spectroscopy. The crystallographic structures of the SBiX:S100B complexes were solved revealing covalent modifiers occupying persistent binding site 2. The compounds offer potential bridging scaffolds between sites 1 and 3. All five SBiXs demonstrated the ability to kill malignant melanoma with some killing in a S100B dependent manner. TheS100B-SBiX complexes presented will act as the basis for the design of improved SBiXs. SBiXs may also have therapeutic value for treating other cancers with elevated S100B and wt p53 such as astrocytoma, renal tumors, and some forms of leukemia. Citation Format: Michael C. Cavalier. Covalent inhibitors of S100B (SBiXs) in malignant melanoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3221. doi:10.1158/1538-7445.AM2014-3221

Abstract B103: Inhibitors of S100B (SBiXs) in malignant melanoma.

Summary: In malignant melanoma (MM), elevated levels of the tumor marker, S100B, binds directly to wild-type (wt) p53, dissociates the p53 tetramer, and down-regulates p53-dependent tumor suppression functions; therefore, it is the goal to develop S100B inhibitors (SBiXs; X=compound number) to restore active p53 in this deadly cancer. As a proof of principle for a drug design project, inhibiting S100B with small interfering antisense RNA (siRNAS100B) and several SBiXs was shown to restore wild-type p53 levels and its downstream gene products, as necessary to induce cell growth arrest and apoptosis in malignant melanoma. Purpose: It is our purpose to discover/synthesize and/or to improve existing inhibitors of S100B to restore p53 activity in human malignant melanoma. Such compounds can then be examined for in vivo efficacy. Results and conclusions: Development of S100B inhibitors (SBiXs) is ongoing at the University of Maryland Schools of Pharmacy and Medicine with the goal of obtaining higher affinity and better specificity towards inhibiting S100B. Computer aided drug design (CADD) followed by synthesis and experimental assays (i.e. NMR, binding, and cellular assays) have identified compounds that bind S100B and inhibit melanoma cell growth. Available binding data, IC50 data, and 3D structures of S100B-SBiX complexes will be presented that will act as the basis for the design of improved SBiXs. SBiXs may also have therapeutic value for treating other cancers with elevated S100B and wt p53 such as astrocytoma, renal tumors, and some forms of leukemia. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):B103. Citation Format: Michael Cavalier, Paul T. Wilder, Andrew Coop, Alexander MacKerell, David J. Weber. Inhibitors of S100B (SBiXs) in malignant melanoma. [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 B103.