Elvin D. de Araujo

The first nucleotide binding domain of the sulfonylurea receptor 2A contains regulatory elements and is folded and functions as an independent module.

The sulfonylurea receptor 2A (SUR2A) is an ATP-binding cassette (ABC) protein that forms the regulatory subunit of ATP-sensitive potassium (K(ATP)) channels in the heart. ATP binding and hydrolysis at the SUR2A nucleotide binding domains (NBDs) control gating of K(ATP) channels, and mutations in the NBDs that affect ATP hydrolysis and cellular trafficking cause cardiovascular disorders. To date, there is limited information on the SUR2A NBDs and the effects of disease-causing mutations on their structure and interactions. Structural and biophysical studies of NBDs, especially from eukaryotic ABC proteins like SUR2A, have been hindered by low solubility of the isolated domains. We hypothesized that the solubility of heterologously expressed SUR2A NBDs depends on the precise definition of the domain boundaries. Putative boundaries of SUR2A NBD1 were identified by structure-based sequence alignments and subsequently tested by exploring the solubility of SUR2A NBD1 constructs with different N and C termini. We have determined boundaries of SUR2A NBD1 that allow for soluble heterologous expression of the protein, producing a folded domain with ATP binding activity. Surprisingly, our alignment and screening data indicate that SUR2A NBD1 contains two putative, previously unidentified, regulatory elements: a large insert within the β-sheet subdomain and a C-terminal extension. Our approach, which combines the use of structure-based sequence alignments and predictions of disordered regions combined with biochemical and biophysical studies, may be applied as a general method for developing suitable constructs of other NBDs of ABC proteins.

Disarming an Electrophilic Warhead: Retaining Potency in Tyrosine Kinase Inhibitor (TKI)‐Resistant CML Lines While Circumventing Pharmacokinetic Liabilities

Pharmacologic blockade of the activation of signal transducer and activator of transcription 3 (STAT3) in tyrosine kinase inhibitor (TKI)‐resistant chronic myeloid leukemia (CML) cell lines characterized by kinase‐independent resistance was shown to re‐sensitize CML cells to TKI therapy, suggesting that STAT3 inhibitors in combination with TKIs are an effective combinatorial therapeutic for the treatment of CML. Benzoic acid‐ and hydroxamic acid‐based STAT3 inhibitors SH‐4‐054 and SH‐5‐007, developed previously in our laboratory, demonstrated promising activity against these resistant CML cell lines. However, pharmacokinetic studies in murine models (CD‐1 mice) revealed that both SH‐4‐054 and SH‐5‐007 are susceptible to glutathione conjugation at the para position of the pentafluorophenyl group via nucleophilic aromatic substitution (SNAr). To determine whether the electrophilicity of the pentafluorophenyl sulfonamide could be tempered, an in‐depth structure–activity relationship (SAR) study of the SH‐4‐054 scaffold was conducted. These studies revealed that AM‐1‐124, possessing a 2,3,5,6‐tetrafluorophenylsulfonamide group, retained STAT3 protein affinity (Ki=15 μm), as well as selectivity over STAT1 (Ki>250 μm). Moreover, in both hepatocytes and in in vivo pharmacokinetic studies (CD‐1 mice), AM‐1‐124 was found to be dramatically more stable than SH‐4‐054 (t1/2=1.42 h cf. 10 min, respectively). AM‐1‐124 is a promising STAT3‐targeting inhibitor with demonstrated bioavailability, suitable for evaluation in preclinical cancer models.

Phosphorylation-dependent Changes in Nucleotide Binding, Conformation, and Dynamics of the First Nucleotide Binding Domain (NBD1) of the Sulfonylurea Receptor 2B (SUR2B)

Background: Phosphorylation of SUR2B NBD1 activates ATP-sensitive K+ (KATP) channels. Results: Phosphorylation-dependent changes in NBD1 conformation and nucleotide binding are mimicked by removing the N-terminal tail that contains the phosphorylation sites. Conclusion: Phosphorylation disrupts interactions of the N-terminal tail with the NBD1 core, leading to increased nucleotide binding. Significance: These data provide insights into the molecular basis by which NBD1 phosphorylation activates KATP channels. The sulfonylurea receptor 2B (SUR2B) forms the regulatory subunit of ATP-sensitive potassium (KATP) channels in vascular smooth muscle. Phosphorylation of the SUR2B nucleotide binding domains (NBD1 and NBD2) by protein kinase A results in increased channel open probability. Here, we investigate the effects of phosphorylation on the structure and nucleotide binding properties of NBD1. Phosphorylation sites in SUR2B NBD1 are located in an N-terminal tail that is disordered. Nuclear magnetic resonance (NMR) data indicate that phosphorylation of the N-terminal tail affects multiple residues in NBD1, including residues in the NBD2-binding site, and results in altered conformation and dynamics of NBD1. NMR spectra of NBD1 lacking the N-terminal tail, NBD1-ΔN, suggest that phosphorylation disrupts interactions of the N-terminal tail with the core of NBD1, a model supported by dynamic light scattering. Increased nucleotide binding of phosphorylated NBD1 and NBD1-ΔN, compared with non-phosphorylated NBD1, suggests that by disrupting the interaction of the NBD core with the N-terminal tail, phosphorylation also exposes the MgATP-binding site on NBD1. These data provide insights into the molecular basis by which phosphorylation of SUR2B NBD1 activates KATP channels.

Successful development and use of a thermodynamic stability screen for optimizing the yield of nucleotide binding domains

ATP sensitive potassium (KATP) channels consist of four copies of a pore-forming inward rectifying potassium channel (Kir6.1 or Kir6.2) and four copies of a sulfonylurea receptor (SUR1, SUR2A, or SUR2B). SUR proteins are members of the ATP-binding cassette superfamily of proteins. Binding of ATP to the Kir6.x subunit mediates channel inhibition, whereas MgATP binding and hydrolysis at the SUR NBDs results in channel opening. Mutations in SUR1 and SUR2A NBDs cause diseases of insulin secretion and cardiac disorders, respectively, underlying the importance of studying the NBDs. Although purification of SUR2A NBD1 in a soluble form is possible, the lack of long-term sample stability of the protein in a concentrated form has precluded detailed studies of the protein aimed at gaining a molecular-level understanding of how SUR mutations cause disease. Here we use a convenient and cost-effective thermodynamic screening method to probe stabilizing conditions for SUR2A NBD1. Results from the screen are used to alter the purification protocol to allow for significantly increased yields of the purified protein. In addition, the screen provides strategies for long-term storage of NBD1 and generating NBD1 samples at high concentrations suitable for NMR studies. NMR spectra of NBD1 with MgAMP-PNP are of higher quality compared to using MgATP, indicating that MgAMP-PNP be used as the ligand in future NMR studies. The screen presented here can be expanded to using different additives and can be employed to enhance purification yields, sample life times, and storage of other low stability nucleotide binding domains, such as GTPases.

NMR and fluorescence studies of drug binding to the first nucleotide binding domain of SUR2A.

ATP sensitive potassium (K(ATP)) channels are composed of four copies of a pore-forming inward rectifying potassium channel (Kir6.1 or Kir6.2) and four copies of a sulfonylurea receptor (SUR1, SUR2A, or SUR2B) that surround the pore. SUR proteins are members of the ATP-binding cassette (ABC) superfamily of proteins. Binding of MgATP at the SUR nucleotide binding domains (NBDs) results in NBD dimerization, and hydrolysis of MgATP at the NBDs leads to channel opening. The SUR proteins also mediate interactions with K(ATP) channel openers (KCOs) that activate the channel, with KCO binding and/or activation involving residues in the transmembrane helices and cytoplasmic loops of the SUR proteins. Because the cytoplasmic loops make extensive interactions with the NBDs, we hypothesized that the NBDs may also be involved in KCO binding. Here, we report nuclear magnetic resonance (NMR) spectroscopy studies that demonstrate a specific interaction of the KCO pinacidil with the first nucleotide binding domain (NBD1) from SUR2A, the regulatory SUR protein in cardiac K(ATP) channels. Intrinsic tryptophan fluorescence titrations also demonstrate binding of pinacidil to SUR2A NBD1, and fluorescent nucleotide binding studies show that pinacidil binding increases the affinity of SUR2A NBD1 for ATP. In contrast, the KCO diazoxide does not interact with SUR2A NBD1 under the same conditions. NMR relaxation experiments and size exclusion chromatography indicate that SUR2A NBD1 is monomeric under the conditions used in drug binding studies. These studies identify additional binding sites for commonly used KCOs and provide a foundation for testing binding of drugs to the SUR NBDs.

High-throughput thermofluor-based assays for inhibitor screening of STAT SH2 domains

&NA; The development of STAT protein‐specific inhibitors has been the focus of a number of drug discovery programs. STAT activation occurs through phosphorylation at the STAT SH2 domain, resulting in dimerization, translocation to the nucleus, and transcription of proliferative genes. Due to the functional significance of the SH2 domain in mediating multiple components of the STAT signalling cascade, many libraries of inhibitors have been designed to target the SH2 domain. This has triggered the requirement for effective high‐throughput screening platforms for analyzing binding by larger chemical libraries to STAT proteins. Herein, we present strategies for the development of a high‐throughput thermal denaturation‐based assay for identifying STAT inhibitors as well as high‐yielding recombinant expression and purification of untagged STAT1, STAT3, and STAT5 proteins. This assay reports changes in the fluorescence of a labelled peptide bound to the STAT protein as a function of increasing temperature. STAT inhibitors which displace the labelled peptide elicit a change in the melt profile, which is quantitatively determined as a change in the area under the curve. This assay offers an alternative, but complimentary, high‐throughput screening strategy for identifying new inhibitors of STAT proteins as well as characterizing further, the mode of inhibition by existing libraries of compounds. Graphical abstract Figure. No caption available. HighlightsThermal denaturation of STAT protein: peptide complexes can identify inhibitors.Chemical libraries (>100 compounds) can be rapidly screened against STAT proteins.The thermofluor assay was validated using previously identified STAT inhibitors.STAT1, STAT3 and STAT5 were expressed in E. coli and purified at >15 mg per liter.

Strategies for over-expression and purification of recombinant full length STAT5B in Escherichia coli

STAT5B, a ubiquitious transcription factor, has been implicated in the onset and progression of several cancers. Since the inhibition of STAT activity holds significant therapeutic potential, there is a need to develop high-throughput biophysical screening platforms to rapidly identify high affinity binders of STATs. Biophysical assays would benefit from the efficient and cost-effective production of high purity, full-length STAT proteins. Herein, we have sampled a large region of protein expression and purification space that has substantially increased recombinant STAT5B protein yields from Escherichia coli. The identity of STAT5B was confirmed by Western blotting analysis, while the results of a fluorescence polarization assay indicated that the purified protein is correctly folded and functional. A thermal shift assay was employed to assess the effect of various osmolytes on the stability of the protein. The protein expression conditions identified in this study allowed for more efficient and higher recovery of soluble STAT5B protein, which will enable a broad range of biophysical studies and facilitate high-throughput STAT5B drug screening.

Pharmacologic inhibition of STAT5 in acute myeloid leukemia

The transcription factor STAT5 is an essential downstream mediator of many tyrosine kinases (TKs), particularly in hematopoietic cancers. STAT5 is activated by FLT3-ITD, which is a constitutively active TK driving the pathogenesis of acute myeloid leukemia (AML). Since STAT5 is a critical mediator of diverse malignant properties of AML cells, direct targeting of STAT5 is of significant clinical value. Here, we describe the development and preclinical evaluation of a novel, potent STAT5 SH2 domain inhibitor, AC-4–130, which can efficiently block pathological levels of STAT5 activity in AML. AC-4–130 directly binds to STAT5 and disrupts STAT5 activation, dimerization, nuclear translocation, and STAT5-dependent gene transcription. Notably, AC-4–130 substantially impaired the proliferation and clonogenic growth of human AML cell lines and primary FLT3-ITD+ AML patient cells in vitro and in vivo. Furthermore, AC-4–130 synergistically increased the cytotoxicity of the JAK1/2 inhibitor Ruxolitinib and the p300/pCAF inhibitor Garcinol. Overall, the synergistic effects of AC-4–130 with TK inhibitors (TKIs) as well as emerging treatment strategies provide new therapeutic opportunities for leukemia and potentially other cancers.

Regulating the Master Regulator: Controlling Ubiquitination by Thinking Outside the Active Site

The labeling of proteins with ubiquitin/ubiquitin-like (Ubl) proteins is crucial for several physiological processes and in the onset of various diseases. Recently, targeting ubiquitin protein labeling has shifted toward the use of allosteric mechanisms over classical activity-based approaches. Allosteric enzyme regulation offers the potential for greater selectivity and has demonstrated less susceptibility to acquired resistance often associated with active site inhibitors. Furthermore, the isoform diversity among E1 activating, E2 conjugating, E3 ligase, and deubiquitinating (DUB) enzymes offers an ideal platform for modulating activity via allostery. Herein, we have reviewed allosteric inhibitors of the ubiquitin E1-E2-E3 and DUB enzymatic cascade developed over the past decade with a focus on their mechanisms of action. We have highlighted the advantages as well as the challenges associated with designing allosteric modulators of the ubiquitin labeling machinery, and the future promise in targeting these systems using allosteric approaches.

A functional in vitro assay for screening inhibitors of STAT5B phosphorylation

&NA; Inhibition of STAT phosphorylation is recognized as a viable therapeutic strategy for disrupting tumorigenesis. Constitutive STAT phosphorylation is found with high frequency in a number of primary tumor types, while non‐cancer cells exhibit low basal activity, providing an exploitable therapeutic window. STAT activation involves phosphorylation of the SH2 domain by a number of tyrosine kinases followed by STAT dimerization and translocation to the nucleus. By blocking the cognate binding site, STAT SH2‐domain inhibitors can impede kinase‐mediated de novo STAT phosphorylation. Assessing for inhibitors of STAT phosphorylation has previously been conducted exclusively in cellulo using Western blot analysis. However, while providing useful in cellulo efficacy, it is not possible to conclude that inhibition is due to a direct blockade of STAT protein. Here we developed a functional assay that directly reports the blockade of phosphorylation as a result of inhibitor interaction with STAT proteins. We have optimized reaction conditions for the functional assay and validated the assay against known STAT5B ligands, including peptides and small molecule inhibitors. As part of the study, we have also identified several sites of STAT5B phosphorylation by Abl kinase. This assay will serve to delineate the functional mechanism of STAT binders in vitro and deconvolute the mechanism of phospho‐STAT inhibition observed in Western blot analysis. Graphical abstract Figure. No caption available. HighlightsSTAT5 phosphorylation by an in vivo kinase (cAbl) was adapted as a functional inhibitor screening assay.Assay parameters were optimized to determine ideal protein, buffer and reaction conditions.Predicted in silico STAT5 phosphorylation sites were confirmed by mass spectrometry.The assay was validated with known STAT5 ligands including peptides and inhibitors.