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Structure-based drug design (SBDD) is a prominent method in rational drug development and has traditionally benefitted from the atomic models of protein targets obtained using X-ray crystallography at cryogenic temperatures. In this perspective, we highlight recent advances in the development of structural techniques that are capable of probing dynamic information about protein targets. First, we discuss advances in the field of X-ray crystallography including serial room-temperature crystallography as a method for obtaining high-resolution conformational dynamics of protein-inhibitor complexes. Next, we look at cryogenic electron microscopy (cryoEM), another high-resolution technique that has recently been used to study proteins and protein complexes that are too difficult to crystallize. Finally, we present small angle X-ray scattering (SAXS) as a potential high-throughput screening tool to identify inhibitors that target protein complexes and protein oligomerization.

Physio-chemical and spectroscopic data of GA11 and MF7

IHC of BM and LM of human TNBC patients

PDF file - 128K, Two mechanistically-distinct mTOR inhibitors, rapamycin and INK-128, show differential abilities to inhibit signaling components upstream of mTORC1. SKBR3 cells were serum-starved for 40-48 h. Rapamycin (50 nM) or INK (50 nM) was added to the cells for 30 minutes prior to the additional treatment of cells with or without 1 nM HRG for 30 min. Cells were then harvested, lysed, and then lysates were analyzed by Western blotting for relative levels of phospho-mTOR (S2448), phospho-TSC2 (T1462), phopsho-AKT (T308), phospho-AKT (S473) and the corresponding total proteins.

Cell viability assay using ALDH1A3 inhibitors and ALDH1A3 expression in scRNA-seq data

PDF file - 136K, HRG-dependent stimulation of mTORC1-related signaling activities in SKBR3 cells is blocked by pre-treatment with the ErbB2-specific kinase inhibitor, CP-724,714. SKBR3 cells were serum-starved followed by treatment with or without 5 microM CP-724,714 for 30 minutes prior to stimulation with 1 nM HRG for an additional 30 minutes. Cells were collected and lysed, and the lysates were analyzed by Western blotting using specific antibodies to probe for phospho-ErbB2 (Y1248), phospho-mTOR (S2448), phospho-TSC2 (T1462), phospho-AKT (T308), phospho-AKT (S473), phospho-ribosomal S6 (S235/236), the corresponding pan-specific antibodies, or actin.

PDF file, 48KB, Graph showing 968 potency at multiple doses when 968 is added to GAC before or after inorganic phosphate.

PDF file, 50KB, Time dependence of two compounds, 35 and 40, showing that their activity decreases when incubated with GAC for 10 or 20 minutes prior to beginning the enzymatic reaction in the assay.

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Kaplan Meier survival analysis of mice using MDA-468 cells

PDF file - 62K, Ectopically-expressed components of both mTORC1 and mTORC2 in HEK 293T cells can interact with TSC2 as well as mTOR. HEK 293T cells were transfected with either Myc-Raptor or Myc-Rictor. Cells were then lysed in lysis buffer containing 0.3% CHAPS and subjected to immunoprecipitation using anti-Myc antibody to precipitate Raptor or Rictor. The precipitated samples were subjected to Western blotting probing for mTOR, TSC2, and Myc.

PDF file - 201K, HRGalpha, as well as HRGbeta, is capable of stimulating the ability of SKBR3 cells to form colonies and activate components of the mTORC1 signaling pathway. A, SKBR3 cells were seeded in 0.3% agarose-containing complete medium with the addition of 1 nM HRGalpha or 1 nM HRGbeta. Cells were fed every three days with the growth factor-containing medium and colonies were counted on day 13. The experiment was performed in triplicate and the results were averaged and graphed. B, SKBR3 cells were serum-starved for 40-48 h, and were then challenged with HRGalpha for the indicated times. Cell lysates were then collected and analyzed by Western blotting to determine relative levels of phospho-mTOR (S2448), phospho-TSC2 (T1462), phospho-AKT (T308), phospho-AKT (S473), phospho-ribosomal S6 (S235/236), actin, as well as for the total levels of the respective proteins.

Elevation of ALDH1A3 expression in BM tissues and cells

PDF file, 294KB, Complete list of compounds examined in the recombinant GAC assay, and % inhibition determined against the enzyme for each compound.

DOC file, 32.5KB.

One hallmark of cancer cells is their adaptation to rely upon an altered metabolic scheme that includes changes in the glycolytic pathway, known as the Warburg effect, and elevated glutamine metabolism. Glutaminase, a mitochondrial enzyme, plays a key role in the metabolism of glutamine in cancer cells, and its inhibition could significantly impact malignant transformation. The small molecule 968, a dibenzophenanthridine, was recently shown to inhibit recombinantly expressed glutaminase C, to block the proliferation and anchorage-independent colony formation of human cancer cells in culture, and to inhibit tumor formation in mouse xenograft models. Here, we examine the structure–activity relationship that leads to 968-based inhibition of glutaminase and cancer cell proliferation, focusing upon a “hot-spot” ring previously identified as critical to 968 activity. We find that the hot-spot ring must be substituted with a large, nonplanar functionality (e.g., a t-butyl group) to bestow activity to the series, leading us to a model whereby the molecule binds glutaminase at a previously undescribed allosteric site. We conduct docking studies to locate potential 968-binding sites and proceed to test a specific set of docking solutions via site-directed mutagenesis. We verify the results from our initial assay of 968 and its analogues by cellular studies using MDA-MB-231 breast cancer cells. Mol Cancer Ther; 11(6); 1269–78. ©2012 AACR.

The development of efficacious therapies targeting metastatic spread of breast cancer to the brain represents an unmet clinical need. Accordingly, an improved understanding of the molecular underpinnings of central nervous system spread and progression of breast cancer brain metastases (BCBM) is required. In this study, the clinical burden of disease in BCBM was investigated, as well as the role of aldehyde dehydrogenase 1A3 (ALDH1A3) in the metastatic cascade leading to BCBM development. Initial analysis of clinical survival trends for breast cancer and BCBM determined improvement of breast cancer survival rates; however, this has failed to positively affect the prognostic milestones of triple-negative breast cancer (TNBC) brain metastases (BM). ALDH1A3 and a representative epithelial–mesenchymal transition (EMT) gene signature (mesenchymal markers, CD44 or Vimentin) were compared in tumors derived from BM, lung metastases (LM), or bone metastases (BoM) of patients as well as mice after injection of TNBC cells. Selective elevation of the EMT signature and ALDH1A3 were observed in BM, unlike LM and BoM, especially in the tumor edge. Furthermore, ALDH1A3 was determined to play a role in BCBM establishment via regulation of circulating tumor cell adhesion and migration phases in the BCBM cascade. Validation through genetic and pharmacologic inhibition of ALDH1A3 via lentiviral shRNA knockdown and a novel small-molecule inhibitor demonstrated selective inhibition of BCBM formation with prolonged survival of tumor-bearing mice. Given the survival benefits via targeting ALDH1A3, it may prove an effective therapeutic strategy for BCBM prevention and/or treatment.

PDF file - 42K, Attenuation of HRG-stimulated AKT phosphorylation by the knock-down of Rictor can be rescued by the expression of an shRNA-insensitive Rictor construct. SKBR3 cells were infected either with control or Rictor shRNA-containing virus twice, one day apart, and selected with 2 microg/mL puromycin for 48 h. Cells were then subjected to transfection either with mock (no DNA) or 5 microg of Rictor plasmid for 3 hours, followed by serum-starvation for 48 h. On the day of the harvest, cells were treated with or without 1 nM HRG. Whole cell lysates were collected and subjected to Western blotting probing for Rictor, phospho-AKT (T308), phospho-AKT (S473), and total AKT.

Adhesion assay and migration assay using shALDH1A3 in MDA-468 cells

PDF file, 118KB, Graph showing dependence of 968 activity upon order in which GAC is exposed to assay reagents.

Synthesis steps of MF-7 and GA11

Survival analysis of breast cancer patients

IHC for ALDH1A3 in a mouse BM tissues of MDA-468 cells

Tissue transglutaminase (tTG) is a rather unique GTP-binding/protein crosslinking enzyme that has been shown to play important roles in a number of cellular processes that impact both normal physiology and disease states. This is especially the case in the context of aggressive brain tumors, such as glioblastoma. The diverse roles played by tTG in cancer survival and progression have led to significant interest in recent years in using tTG as a therapeutic target. In this review, we provide a brief overview of the transglutaminase family, and then discuss the primary biochemical activities exhibited by tTG with an emphasis on the role it plays in glioblastoma progression. Finally, we consider current approaches to target tTG which might eventually have clinical impact.

The mitochondrial glutaminase enzymes initiate glutaminolysis by catalyzing the hydrolysis of glutamine to glutamate, satisfying the metabolic requirements of aggressive cancers and thus representing potential therapeutic targets. However, the mechanisms underlying their allosteric regulation are poorly understood. It has been suggested that glutaminases form oligomeric filament-like structures essential for their activation. Here, we provide structural evidence for the ability of the glutaminase enzymes to form filaments upon substrate binding, and present the first cryo-EM structures of the human full-length glutaminase isozyme GLS2 that offer an unprecedented view of the mechanism responsible for catalyzing glutamine hydrolysis. The GLS2 structures reveal that the ‘activation loop’, a motif previously identified to regulate enzymatic activity, assumes a unique conformation and works together with a ‘lid’ that closes over the active site to ‘lock in’ the substrate glutamine. Tyrosine 251 of the GLS2 activation loop forms a cation-π interaction with Lysine 222 in the active site, which in turn enables a key catalytic residue, Serine 219, to undergo deprotonation for nucleophilic attack on the substrate. These findings further suggest that allosteric glutaminase inhibitors disrupt this interaction, which is critical for catalysis, while activators stabilize it. The GLS2 structures also show how the ankyrin repeats regulate different glutaminase isozymes.

Cancer cells, both within a developing tumor and during metastatic spread, encounter many stresses that require adaptive mechanisms to survive and maintain malignant progression. Here we describe a signaling complex involving the small GTPase Cdc42 and Dock7, a Cdc42/Rac GEF and unique Cdc42-effector, that has a previously unappreciated role in regulating AKT, mTOR, and other mTOR signaling and regulatory partners including the TSC1/TCS2 complex and S6K during serum starvation. Dock7 is highly expressed in triple-negative breast cancers and is essential for the malignant properties in nutrient-deprived growth conditions of several cancer cell lines. We find that Dock7 interacts with phosphorylated AKT to maintain a low, but critical activation of a rapamycin-sensitive and Raptor-independent mTORC1-like activity required for survival during nutrient stress. Following the knock-out of Dock7 from cancer cells, interactions between AKT and the phosphatase PHLPP increased while phosphorylation of AKT at Ser473 decreased, suggesting Dock7 protects AKT from dephosphorylation. The DHR1 domain of Dock7, previously of unknown function, is responsible for maintaining AKT Ser473 phosphorylation during serum starvation through an interaction requiring its C2-like motif. Together, these findings indicate that Dock7 protects and maintains the phosphorylation of AKT to sustain a tonic mTOR/S6K activity in cancer cells necessary for their resistance to anoikis and to prevent them from undergoing apoptosis during stressful conditions.

Metabolic reprogramming is a major hallmark of malignant transformation in cancer, and part of the so-called Warburg effect, in which the upregulation of glutamine catabolism plays a major role. The glutaminase enzymes convert glutamine to glutamate, which initiates this pathway. Inhibition of different forms of glutaminase (KGA, GAC, or LGA) demonstrated potential as an emerging anti-cancer therapeutic strategy. The regulation of these enzymes, and the molecular basis for their inhibition, have been the focus of much recent research. This review will explore the recent progress in understanding the molecular basis for activation and inhibition of different forms of glutaminase, as well as the recent focus on combination therapies of glutaminase inhibitors with other anti-cancer drugs.

IGF2BP2 binds to a number of RNA transcripts and has been suggested to function as a tumor promoter, although little is known regarding the mechanisms that regulate its roles in RNA metabolism. Here we demonstrate that IGF2BP2 binds to the 3′ untranslated region of the transcript encoding ATP6V1A, a catalytic subunit of the vacuolar ATPase (v-ATPase), and serves as a substrate for the NAD + -dependent deacetylase SIRT1, which regulates how IGF2BP2 affects the stability of the ATP6V1A transcript. When sufficient levels of SIRT1 are expressed, it catalyzes the deacetylation of IGF2BP2, which can bind to the ATP6V1A transcript but does not mediate its degradation. However, when SIRT1 expression is low, the acetylated form of IGF2BP2 accumulates, and upon binding to the ATP6V1A transcript recruits the XRN2 nuclease, which catalyzes transcript degradation. Thus, the stability of the ATP6V1A transcript is significantly compromised in breast cancer cells when SIRT1 expression is low or knocked-down. This leads to a reduction in the expression of functional v-ATPase complexes in cancer cells and to an impairment in their lysosomal activity, resulting in the production of a cellular secretome consisting of increased numbers of exosomes enriched in ubiquitinated protein cargo and soluble hydrolases, including cathepsins, that together combine to promote tumor cell survival and invasiveness. These findings describe a previously unrecognized role for IGF2BP2 in mediating the degradation of a messenger RNA transcript essential for lysosomal function and highlight how its sirtuin-regulated acetylation state can have significant biological and disease consequences.

Tumor-initiating cells contained within the aggressive brain tumor glioma (glioma stem cells, GSCs) promote radioresistance and disease recurrence. However, mechanisms of resistance are not well understood. Herein, we show that the proteome-level regulation occurring upon radiation treatment of several patient-derived GSC lines predicts their resistance status, whereas glioma transcriptional subtypes do not. We identify a mechanism of radioresistance mediated by the transfer of the metabolic enzyme NAMPT to radiosensitive cells through microvesicles (NAMPT-high MVs) shed by resistant GSCs. NAMPT-high MVs rescue the proliferation of radiosensitive GSCs and fibroblasts upon irradiation, and upon treatment with a radiomimetic drug or low serum, and increase intracellular NAD(H) levels. Finally, we show that the presence of NAMPT within the MVs and its enzymatic activity in recipient cells are necessary to mediate these effects. Collectively, we demonstrate that the proteome of GSCs provides unique information as it predicts the ability of glioma to resist radiation treatment. Furthermore, we establish NAMPT transfer via MVs as a mechanism for rescuing the proliferation of radiosensitive cells upon irradiation.

SIRT5 is a member of the sirtuin family of NAD(+)-dependent protein lysine deacylases implicated in a variety of physiological processes. SIRT5 removes negatively charged malonyl, succinyl, and glutaryl groups from lysine residues and thereby regulates multiple enzymes involved in cellular metabolism and other biological processes. SIRT5 is overexpressed in human breast cancers and other malignancies, but little is known about the therapeutic potential of SIRT5 inhibition for treating cancer. Here we report that genetic SIRT5 disruption in breast cancer cell lines and mouse models caused increased succinylation of IDH2 and other metabolic enzymes, increased oxidative stress, and impaired transformation and tumorigenesis. We, therefore, developed potent, selective, and cell-permeable small-molecule SIRT5 inhibitors. SIRT5 inhibition suppressed the transformed properties of cultured breast cancer cells and significantly reduced mammary tumor growth in vivo, in both genetically engineered and xenotransplant mouse models. Considering that Sirt5 knockout mice are generally normal, with only mild phenotypes observed, these data establish SIRT5 as a promising target for treating breast cancer. The new SIRT5 inhibitors provide useful probes for future investigations of SIRT5 and an avenue for targeting SIRT5 as a therapeutic strategy.

The small GTPase CDC42 plays essential roles in neurogenesis and brain development. Previously, we showed that a CDC42 splice variant that has a ubiquitous tissue distribution specifically stimulates the formation of neural progenitor cells, whereas a brain-specific CDC42 variant, CDC42b, is essential for promoting the transition of neural progenitor cells to neurons. These specific roles of CDC42 and CDC42b in neurogenesis are ascribed to their opposing effects on mTORC1 activity. Specifically, the ubiquitous form of CDC42 stimulates mTORC1 activity and thereby upregulates tissue-specific transcription factors that are essential for neuroprogenitor formation, whereas CDC42b works together with activated CDC42-associated kinase (ACK) to downregulate mTOR expression. Here, we demonstrate that the EGF receptor (EGFR) is an additional and important target of CDC42b and ACK, which is downregulated by their combined actions in promoting neurogenesis. The activation status of the EGFR determines the timing by which neural progenitor cells derived from P19 embryonal carcinoma terminally differentiate into neurons. By promoting EGFR degradation, we found that CDC42b and ACK stimulate autophagy, which protects emerging neurons from apoptosis and helps trigger neural progenitor cells to differentiate into neurons. Moreover, our results reveal that CDC42b is localized in phosphatidylinositol (3,4,5)-triphosphate-enriched microdomains on the plasma membrane, mediated through its polybasic sequence

Stem cells use a variety of mechanisms to help maintain their pluripotency and promote self-renewal, as well as, at the appropriate time, to differentiate into specialized cells. One such mechanism that is attracting significant attention from the stem cell, development, and regenerative medicine research communities involves a form of intercellular communication, specifically, the ability of cells to form and release nontraditional membrane-enclosed structures, referred to as extracellular vesicles (EVs). There are two major classes of EVs, microvesicles (MVs), which are generated through the outward budding and fission of the plasma membrane, and exosomes, which are formed as multivesicular bodies (MVBs) in the endo-lysosomal pathway that fuse with the cell surface to release their contents. Although they differ in how they are formed, both MVs and exosomes have been shown to contain a diverse array of bioactive cargo, such as proteins, RNA transcripts, microRNAs, and even DNA, which can be transferred to other cells and promote phenotypic changes. Here, we will describe what is currently known regarding EVs and the roles they play in stem cell biology and different aspects of early development. We will also highlight how the EVs produced by stem cells are being aggressively pursued for clinical applications, including their potential use as therapeutic delivery systems and for their regenerative capabilities.

Glioma is a malignant brain tumor that is highly resistant to radiation and chemotherapy, where patients survive on average only 15 months after diagnosis. Furthering the understanding of mechanisms leading to radiation resistance of glioma is paramount to identify novel therapeutic targets. Previous studies have shown that glioma stem cells (GSCs) play an important role in promoting radiation resistance and disease recurrence. Herein we analyze the proteomic alterations occurring in patient-derived GSCs upon radiation treatment in order to identify molecular drivers of resistance. We show that proteome changes upon radiation accurately predict the resistance status of the cells, whereas resistance to radiation does not correlate with glioma transcriptional subtypes. We further show that the radio-resistant GSC-267 cell line sheds microvesicles (MVs) enriched in the metabolic enzyme nicotinamide phosphoribosyltransferase (NAMPT). These MVs can be transferred to recipient fibroblasts and radio-sensitive GSCs, enhancing their intracellular total NAD+ and NADH level, and their ability to proliferate when cultured in low serum, treated with a radio-mimetic drug or irradiated. The NAMPT enzymatic inhibitor FK-866 blocked the ability of MVs from GSC-267 cells to mediate these effects. Similarly, GSC-267 cells where NAMPT was knocked-down using shRNA, which produced MVs depleted of this enzyme, were unable to promote cell proliferation. Collectively, our findings demonstrates that proteome-level regulation can accurately predict the radio-resistance status of GSCs, and identifies NAMPT transfer via MVs as a mechanism for spreading radiation resistance within the glioma tumor microenvironment.SignificanceThe highly aggressive and deadly brain cancer glioma is commonly resistant to standard chemo- and radio-therapy. We used systems biology approaches to study patient-derived glioma stem cells (GSCs), which are known to be responsible for therapeutic resistance, and cell-to-cell communication mediated by extracellular vesicles (EVs), which plays an important role in tumor progression. Analysis of the proteome of GSCs and of the EVs they release led us to determine that the EV-mediated transfer of the metabolic enzyme nicotinamide phosphorybosyltransferase (NAMPT) from radio-resistant to less aggressive cells confers resistance to radiation. Our findings identify a mechanism of therapy resistance in glioma, and suggest that NAMPT inhibition could enhance the efficacy of radiation for the treatment of glioma.