Thermal Stability Assays Research Articles

Abstract 1633: ADT-030, a novel dual-acting RAS and β-catenin inhibitor with robust antitumor activity

Abstract BACKGROUND: Previous publications have reported that the antineoplastic activity of sulindac occurs by a cyclooxygenase independent mechanism, possibly involving direct inhibition of RAS. Suppression of β-catenin/TCF transcription has also been reported by inhibiting cyclic guanosine monophosphate (cGMP) phosphodiesterase (PDE) and the activation of cGMP-dependent protein kinase (PKG). Here we characterize a potent and selective non-COX inhibitory sulindac derivative, ADT-030, that inhibits cancer cell growth by blocking RAS and β-catenin signaling. METHODS AND RESULTS: ADT-030 inhibited the proliferation of lung cancer cells expressing the PDE10A isozyme, while normal airway cells lacking PDE10A displayed reduced sensitivity. ADT-030 inhibited recombinant PDE10 activity at concentrations that inhibit lung cancer cells proliferation. ADT-030 induced cell cycle arrest, apoptosis, and morphological characteristics of autophagy blockage. A cellular thermal stability assay showed that ADT-030 binds both RAS and PDE10 in lysates from cancer cells with nanomolar affinity. Consistent with its PDE10 inhibitory activity, ADT-030 activated PKG and phosphorylated β-catenin on amino acid residues known to induce proteasomal degradation, and reduced levels of the unphosphorylated (oncogenic stabilized) form of β-catenin. Consistent with its ability to directly bind RAS, ADT-030 also inhibited EGF-stimulated MAPK/AKT signaling. Oral administration of ADT-030 inhibited tumor growth in an orthotopic xenograft mouse model of lung cancer, providing a durable survival response with 25% tumor-free mice at dosages having no discernable toxicity. ADT-030 was also highly efficacious in two different chemical-induced mouse models of lung tumorigenesis suppressing the carcinogen-activation of RAS, as well as in multiple mouse subcutaneous tumor models, including highly aggressive models of metastasis. CONCLUSIONS: These results support further development of ADT-030, a novel inhibitor of RAS and β-catenin signaling for the treatment of lung cancer and other RAS driven cancers, including those with co-occurring mutations in the Wnt/β-catenin pathway. Supported by NIH grants R01 CA238514, R01 CA197147, R01 CA254197. Citation Format: Veronica Ramirez-Alcantara, Xi Chen, Michele A. Schuler, Dennis Otali, William Grizzle, Kristy Berry, Antonio Ward, Ivan Babic, Elmar Nurmemmedov, Gang Zhou, Chengguo Xing, Greg Gorman, Lori Coward, Yulia Maxuitenko, Adam B. Keeton, Gary A. Piazza. ADT-030, a novel dual-acting RAS and β-catenin inhibitor with robust antitumor activity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1633.

Structures of Ric-8B in complex with Gα protein folding clients reveal isoform specificity mechanisms

Mammalian Ric-8 proteins act as chaperones to regulate the cellular abundance of heterotrimeric G protein α subunits. The Ric-8A isoform chaperones Gαi/o, Gα12/13, and Gαq/11 subunits, while Ric-8B acts on Gαs/olf subunits. Here, we determined cryoelectron microscopy (cryo-EM) structures of Ric-8B in complex with Gαs and Gαolf, revealing isoform differences in the relative positioning and contacts between the C-terminal α5 helix of Gα within the concave pocket formed by Ric-8 α-helical repeat elements. Despite the overall architectural similarity with our earlier structures of Ric-8A complexed to Gαq and Gαi1, Ric-8B distinctly accommodates an extended loop found only in Gαs/olf proteins. The structures, along with results from Ric-8 protein thermal stability assays and cell-based Gαolf folding assays, support a requirement for the Gα C-terminal region for binding specificity, and highlight that multiple structural elements impart specificity for Ric-8/G protein binding.

An intrinsic purine metabolite AICAR blocks lung tumour growth by targeting oncoprotein mucin 1

BackgroundLung cancer cells overexpress mucin 1 (MUC1) and active subunit MUC1-CT. Although a peptide blocks MUC1 signalling, metabolites targeting MUC1 are not well studied. AICAR is a purine biosynthesis intermediate.MethodsCell viability and apoptosis were measured in AICAR-treated EGFR-mutant and wild-type lung cells. AICAR-binding proteins were evaluated by in silico and thermal stability assays. Protein–protein interactions were visualised by dual-immunofluorescence staining and proximity ligation assay. AICAR-induced whole transcriptomic profile was determined by RNA sequencing. EGFR-TL transgenic mice-derived lung tissues were analysed for MUC1 expression. Organoids and tumours from patients and transgenic mice were treated with AICAR alone or in combination with JAK and EGFR inhibitors to evaluate treatment effects.ResultsAICAR reduced EGFR-mutant tumour cell growth by inducing DNA damage and apoptosis. MUC1 was one of the leading AICAR-binding and degrading proteins. AICAR negatively regulated JAK signalling and JAK1-MUC1-CT interaction. Activated EGFR upregulated MUC1-CT expression in EGFR-TL-induced lung tumour tissues. AICAR reduced EGFR-mutant cell line-derived tumour formation in vivo. Co-treating patient and transgenic mouse lung-tissue-derived tumour organoids with AICAR and JAK1 and EGFR inhibitors reduced their growth.ConclusionsAICAR represses the MUC1 activity in EGFR-mutant lung cancer, disrupting protein–protein interactions between MUC1-CT and JAK1 and EGFR.

CHalf: Folding Stability Made Simple.

The structure of a protein defines its function and integrity and correlates with the protein folding stability (PFS). Quantifying PFS allows researchers to assess differential stability of proteins in different disease or ligand binding states, providing insight into protein efficacy and potentially serving as a metric of protein quality. There are a number of mass spectrometry (MS)-based methods to assess PFS, such as Thermal Protein Profiling (TPP), Stability of Proteins from Rates of Oxidation (SPROX), and Iodination Protein Stability Assay (IPSA). Despite the critical value that PFS studies add to the understanding of mechanisms of disease and treatment development, proteomics research is still primarily dominated by concentration-based studies. We found that a major reason for the lack of PFS studies is the lack of a user-friendly data processing tool. Here we present the first user-friendly software, CHalf, with a graphical user interface for calculating PFS. Besides calculating site-specific PFS of a given protein from chemical denature folding stability assays, CHalf is also compatible with thermal denature folding stability assays. CHalf also includes a set of data visualization tools to help identify changes in PFS across protein sequences and in between different treatment conditions. We expect the introduction of CHalf to lower the barrier of entry for researchers to investigate PFS, promoting the usage of PFS in studies. In the long run, we expect this increase in PFS research to accelerate our understanding of the pathogenesis and pathophysiology of disease.

Visible-light-driven super-active Sn and GO single- and Sn/Cu Co-doped nanophotocatalysts for phenol degradation: Thin-film printability, thermal stability, and cytotoxicity assay

Sn- and graphene oxide (GO) single-doped and Sn/Cu co-doped titanium dioxide (TiO2) nanoparticles were synthesized as printed-layer nano-photocatalysts for degradation of phenol under visible-light. The doped TiO2 nanoparticles (Sn-doped-TiO2, Cu/Sn-doped-TiO2, and GO-doped-TiO2) were synthesized via sol–gel method and calcinated at a temperature of 450 °C. X-ray diffraction (XRD), UV–Visible DRS analysis, Raman analyses, and Energy-dispersive X-ray spectroscopy (EDXS) were utilized to evaluate the crystalline structure and photocatalytic activities of doped-TiO2 nanoparticles. Different screen-printing pastes were separately formulated with 26% w/w doped-TiO2 nanoparticles (Paste1-to-Paste3) for printing onto microscope glass slides. Photocatalytic printed film was sustainable filter for unused suspension of nanoparticles in wastewater. The printed pasts were sintered at varying temperatures (100–500 °C), then three-layer printed nanoparticles sintered at 500 °C examined for photocatalysis. Photocatalytic activity of single-layer-printed GO-doped-TiO2 nanoparticles was extraordinary (80% degradation of 10 ppm phenol solution). Hydroquinone, catechol, benzoquinone and acetic acid were observed to be produced at different concentrations over different time intervals as a result of phenol degradation for single-layer-printed GO-doped-TiO2 nanoparticles sintered at 500 °C. Bandgap of GO-doped-TiO2 nanoparticles was 2 eV, meaningfully lower than anatase TiO2 (3.2 eV). The cytotoxicity assay applied for normal and cancer cells demonstrated cytocompatibility besides photodegradability. This novel process is a targeted, clean, cost-effective and on-demand approach, taking a big step toward decontamination of the environmental pollution.

Thermal Shift Assay for Small GTPase Stability Screening: Evaluation and Suitability.

Thermal unfolding methods are commonly used as a predictive technique by tracking the protein’s physical properties. Inherent protein thermal stability and unfolding profiles of biotherapeutics can help to screen or study potential drugs and to find stabilizing or destabilizing conditions. Differential scanning calorimetry (DSC) is a ‘Gold Standard’ for thermal stability assays (TSA), but there are also a multitude of other methodologies, such as differential scanning fluorimetry (DSF). The use of an external probe increases the assay throughput, making it more suitable for screening studies, but the current methodologies suffer from relatively low sensitivity. While DSF is an effective tool for screening, interpretation and comparison of the results is often complicated. To overcome these challenges, we compared three thermal stability probes in small GTPase stability studies: SYPRO Orange, 8-anilino-1-naphthalenesulfonic acid (ANS), and the Protein-Probe. We studied mainly KRAS, as a proof of principle to obtain biochemical knowledge through TSA profiles. We showed that the Protein-Probe can work at lower concentration than the other dyes, and its sensitivity enables effective studies with non-covalent and covalent drugs at the nanomolar level. Using examples, we describe the parameters, which must be taken into account when characterizing the effect of drug candidates, of both small molecules and Designed Ankyrin Repeat Proteins.

Characterizing BfpU, an Essential Protein in the Type IV Pilus of Enteropathogenic Escherichia coli

Type IV pili (T4Ps) are a family of surface appendages that are important for adhesion, colonization, biofilm formation, virulence, twitching motility and many other functions. BfpU is an essential periplasmic protein of the T4P of Enteropathogenic Escherichia coli. Previous crosslinking and localization studies have shown that BfpU might interact with the outer membrane secretin BfpB, which is also essential for T4P biogenesis. To date, BfpU has no known homologs and its function is unknown. To purify BfpU, we performed affinity and size exclusion chromatography. Preliminary characterization of BfpU via thermal stability assay and circular dichroism suggests a melting temperature of 80 °C, and its secondary structure is comprised of ~40% anti‐parallel β‐sheet and ~45% random coil. The secondary structure profile is reminiscent of PilP, an essential periplasmic protein anchored in the membrane that interacts with secretins in other T4P systems. Additionally, random mutagenesis is in progress to assess essential residues of BfpU that may be involved in protein interactions within this T4P system. Single residue mutations from this mutagenesis screening will be used to confirm interaction sites within BfpU. The results of these studies will help clarify the role of BfpU in T4P biogenesis and its interactions with BfpB.

The NSAID glafenine rescues class 2 CFTR mutants via cyclooxygenase 2 inhibition of the arachidonic acid pathway

Most cases of cystic fibrosis (CF) are caused by class 2 mutations in the cystic fibrosis transmembrane regulator (CFTR). These proteins preserve some channel function but are retained in the endoplasmic reticulum (ER). Partial rescue of the most common CFTR class 2 mutant, F508del-CFTR, has been achieved through the development of pharmacological chaperones (Tezacaftor and Elexacaftor) that bind CFTR directly. However, it is not clear whether these drugs will rescue all class 2 CFTR mutants to a medically relevant level. We have previously shown that the nonsteroidal anti-inflammatory drug (NSAID) ibuprofen can correct F508del-CFTR trafficking. Here, we utilized RNAi and pharmacological inhibitors to determine the mechanism of action of the NSAID glafenine. Using cellular thermal stability assays (CETSAs), we show that it is a proteostasis modulator. Using medicinal chemistry, we identified a derivative with a fourfold increase in CFTR corrector potency. Furthermore, we show that these novel arachidonic acid pathway inhibitors can rescue difficult-to-correct class 2 mutants, such as G85E-CFTR > 13%, that of non-CF cells in well-differentiated HBE cells. Thus, the results suggest that targeting the arachidonic acid pathway may be a profitable way of developing correctors of certain previously hard-to-correct class 2 CFTR mutations.

Characterization of two chicken origin highly pathogenic H7N9 viruses isolated in northern China

Since the national vaccination program was implemented with the H5/H7 bivalent vaccine in poultry in September 2017, the prevalence of H7N9 avian influenza viruses (AIVs) has been controlled effectively in China. However, highly pathogenic H7N9 viruses still exist, causing sporadic outbreaks especially in some regions of northern China. During our routine surveillance in poultry in 2020, we isolated two strains of H7N9 subtype AIV from breeder layer farms in northern China. We found that these two chicken-origin H7N9 isolates were both highly pathogenic (HP) with a four-amino-acid (KRTA) insertion and an I326V mutation (H3 numbering) in the cleavage site of HA to make the motif PEVPKRKRTAR↓GLF. Molecular markers associated with antigenic drift and enhanced pathogenicity in mammals and interspecies transmission were detected in both isolates. Remarkably, both strains gained the F102V and N157D mutations in their HA genes, which have never been reported before. Solid-phase direct binding assay showed that these two isolates both had dual-receptor binding characteristics, while thermal and acid stability assays indicated that they were relatively stable in high-temperature or acidic conditions. In addition, the animal experiments demonstrated that both strains were highly pathogenic to chickens but low pathogenic to mice. These results suggested that the evolution of H7N9 subtype AIV is still continuing, and they pose a potential threat to poultry and public health. Thus, attention should be paid to the importance of continual surveillance of the H7N9 AIVs.

Clemastine Inhibits the Biofilm and Hemolytic of Staphylococcus aureus through the GdpP Protein.

ABSTRACTStaphylococcus aureus poses a significant threat to human health due to its virulence and multidrug resistance. In addition, recalcitrant biofilm formation of S. aureus often results in chronic infection and the treatment tolerance toward the traditional antibiotics. Thus, the development of novel antimicrobial agents capable to inhibit or eradicate S. aureus biofilm formation does matter. Here, we demonstrated that clemastine showed slight bacteriostatic activity and enhanced the antibacterial activity of oxacillin against S. aureus. Moreover, the dramatic inhibition of biofilm formation was found in clinical S. aureus strains by clemastine. Clemastine inhibited the release of eDNA during the biofilm formation and decreased the S. aureus hemolytic activity. Moreover, the S. aureus SA113 treated with clemastine displayed the decreased transcriptional level of the biofilm formation relevant genes (fnbB, icaA, and icaB), virulence genes (hlg, hld, lukde, lukpvl, beta-PSM, delta-PSM, and cap5A), and the regulatory genes agrA. The proteomics analysis of SA113 treated with clemastine demonstrated the significant changes in levels of biofilm-related proteins (stress response regulators ClpB and GroS, ATP-binding proteins, and urease metabolism), virulence-related proteins (SspA, superantigen, and VWbp), and methicillin resistance-related proteins (glutamine metabolism). The genetic mutations on gdpP (cyclic di-AMP phosphodiesterase) were found in the clemastine-induced tolerant derivative isolate by whole-genome sequencing. Furthermore, the interaction between clemastine and GdpP protein was demonstrated by the molecular docking, gdpP overexpression experiment, and thermal stability assay. Conclusively, clemastine might exert its inhibitory effects against the biofilm formation and hemolysis in S. aureus through targeting GdpP protein.IMPORTANCE The biofilm formation, which protects bacteria from stresses, including antibiotics and host immune responses, can be commonly found in clinical S. aureus isolates worldwide. Treatment failure of traditional antibiotics in biofilm-associated S. aureus infections remains a serious challenge. The novel anti-biofilm drug is urgently needed to address the looming crisis. In this study, clemastine, which is a histamine receptor H1 (HRH1) antagonist, was found to have a novel role of the significant inhibition against the biofilm formation and hemolytic activity of S. aureus and enhanced antibacterial activity against S. aureus when used in combination with oxacillin by targeting the GdpP protein. The discovery of this study identified novel use and mechanism of action of clemastine as a potential anti-biofilm drug for clinical application for S. aureus infectious.

Universal platform for the generation of thermostabilized GPCRs that crystallize in LCP.

Structural studies of G-protein-coupled receptors (GPCRs) are often limited by difficulties in obtaining well-diffracting crystals suitable for high-resolution structure determination. During the past decade, crystallization in lipidic cubic phase (LCP) has become the most successful and widely used technique for obtaining such crystals. Despite often intense efforts, many GPCRs remain refractory to crystallization, even if receptors can be purified in sufficient amounts. To address this issue, we have developed a highly efficient screening and stabilization strategy for GPCRs, based on a fluorescence thermal stability assay readout, which seems to correlate particularly well with those GPCR constructs that remain native during incorporation into the LCP. Detailed protocols are provided for rapid and cost-efficient mutant and construct generation using sequence- and ligation-independent cloning, high-throughput magnetic bead-based protein purification from small-scale expressions in mammalian cells, the screening and optimal combination of mutations for increased receptor thermostability and the rapid identification of suitable chimeric fusion protein constructs for successful crystallization in LCP. We exemplify the method on three receptors from two different classes: the neurokinin 1 receptor, the oxytocin receptor and the parathyroid hormone 1 receptor.

Single-Cellular Biological Effects of Cholesterol-Catabolic Bile Acid-Based Nano/Micro Capsules as Anti-Inflammatory Cell Protective Systems.

Recent studies in our laboratories have shown promising effects of bile acids in ➀ drug encapsulation for oral targeted delivery (via capsule stabilization) particularly when encapsulated with Eudragit NM30D® and ➁ viable-cell encapsulation and delivery (via supporting cell viability and biological activities, postencapsulation). Accordingly, this study aimed to investigate applications of bile acid-Eudragit NM30D® capsules in viable-cell encapsulation ready for delivery. Mouse-cloned pancreatic β-cell line was cultured and cells encapsulated using bile acid-Eudragit NM30D® capsules, and capsules’ images, viability, inflammation, and bioenergetics of encapsulated cells assessed. The capsules’ thermal and chemical stability assays were also assessed to ascertain an association between capsules’ stability and cellular biological activities. Bile acid-Eudragit NM30D® capsules showed improved cell viability (e.g., F1 < F2 & F8; p < 0.05), insulin, inflammatory profile, and bioenergetics as well as thermal and chemical stability, compared with control. These effects were formulation-dependent and suggest, overall, that changes in ratios of bile acids to Eudragit NM30D® can change the microenvironment of the capsules and subsequent cellular biological activities.

Structural insights into the binding behavior of NiO with myoglobin

Valuable and useful information on the biotechnological and medical applications of nanoparticles can be achieved through the interactions of proteins with nanoparticles and the structural changes exerted by nanoparticles on these biomolecules. Nickel oxide (NiO) nanoparticles have widespread industrial and therapeutic applications in the medical and biological sciences. Therefore, structural changes and stability exerted by NiO nanoparticles on myoglobin as a globular protein sample were examined at a pH of 7.4 using UV–Vis spectroscopy and fluorescence emission techniques. Myoglobin is a monomeric protein that functions as oxygen storage in the skeletal muscle of vertebrates. Theoretical molecular docking technique was also applied to predict the interactions between myoglobin and NiO nanoparticle complex. Results showed that binding of NiO nanoparticles to myoglobin quenched its emission signals through a static quenching mechanism. Also, electrostatic interaction was predominating in the complex formation relative to other non-covalent interactions. Besides, the spontaneous bonding of NiO nanoparticles was confirmed by negative Gibbs free energy (ΔG°). Measurements performed by protein thermal stability assay showed that thermal stability of myoglobin increased with rising concentrations of NiO nanoparticles. CD spectroscopic studies showed that in the presence of NiO nanoparticles, the content of α-helix protein increased and the content of other secondary structures decreased. Accordingly, it is clearly evident that the structure and stability of myoglobin are altered based on the experimental and theoretical data obtained under the influence of NiO nanoparticles.

In vitro pharmacological profiling of selected small molecules compound using recombinant human iduronate-2-sulphatase

Background: Mucopolysaccharidoses type II (MPS II) is an X-linked lysosomal storage disease (LSD). It is due to mutation in IDS gene encoding iduronate-2-sulphatase (IDS) involved in the catabolism of dermatan sulphate and heparan sulphate. Currently, the treatments for MPS II patients are enzyme replacement therapy (ERT) and bone marrow transplantation (BMT). However, ERT is not effectively reducing the central nervous system manifestation and finding the suitable donor maybe quite challenging in BMT. Over the past decades, pharmacological chaperone has been an alternative approach for management of MPS II patient. Here, we described the in vitro profiling of small molecules in group of chondroitin/dermatan (CD) sulphate disaccharide, heparin oligosaccharides, unsaturated heparin disaccharides and 6-O-desulphated heparin oligosaccharide, using recombinant human iduronate-2-sulphatase (rhIDS). Twenty-one small molecule compounds with several concentrations were each screened by inhibition and thermal stability assays.&#x0D; Results: Our study revealed that condroitin dermatan trisulphate (CD3S), heparin tetrasaccharide (H4Sac), heparin octasaccharide (H8Sac) and heparin octadecasaccharide (H18Sac) showed high inhibition constant, Ki and low inhibition concentration, IC50 in comparison to others. In the thermal stability study, only rhIDS incubated with CD3S was found to preserve enzyme activity (20%) after incubated at 67oC.&#x0D; Conclusion: Overall, our experiments discovered that CD3S was able to bind, inhibit and chaperone rhIDS. These features suggest a potential pharmacological chaperone for MPS II.

A Small Molecule, ACAi-028, with Anti-HIV-1 Activity Targets a Novel Hydrophobic Pocket on HIV-1 Capsid.

The human immunodeficiency virus type 1 (HIV-1) capsid (CA) is an essential viral component of HIV-1 infection and an attractive therapeutic target for antivirals. Here, we report that a small molecule, ACAi-028, inhibits HIV-1 replication by targeting a hydrophobic pocket in the N-terminal domain of CA (CA-NTD). ACAi-028 is 1 of more than 40 candidate anti-HIV-1 compounds identified by in silico screening and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays. Our binding model showed that ACAi-028 interacts with the Q13, S16, and T19 amino acid residues, via hydrogen bonds, in the targeting pocket of CA-NTD. Using recombinant fusion methods, TZM-bl, time-of-addition, and colorimetric reverse transcriptase (RT) assays, the compound was found to exert anti-HIV-1 activity in the early stage between reverse transcription and proviral DNA integration, without any effect on RT activity in vitro, suggesting that this compound may affect HIV-1 core disassembly (uncoating) as well as a CA inhibitor, PF74. Moreover, electrospray ionization mass spectrometry (ESI-MS) also showed that the compound binds directly and noncovalently to the CA monomer. CA multimerization and thermal stability assays showed that ACAi-028 decreased CA multimerization and thermal stability via S16 or T19 residues. These results indicate that ACAi-028 is a new CA inhibitor by binding to the novel hydrophobic pocket in CA-NTD. This study demonstrates that a compound, ACAi-028, targeting the hydrophobic pocket should be a promising anti-HIV-1 inhibitor.

Characterization of Two NMN Deamidase Mutants as Possible Probes for an NMN Biosensor.

Nicotinamide mononucleotide (NMN) is a key intermediate in the nicotinamide adenine dinucleotide (NAD+) biosynthesis. Its supplementation has demonstrated beneficial effects on several diseases. The aim of this study was to characterize NMN deamidase (PncC) inactive mutants to use as possible molecular recognition elements (MREs) for an NMN-specific biosensor. Thermal stability assays and steady-state fluorescence spectroscopy measurements were used to study the binding of NMN and related metabolites (NaMN, Na, Nam, NR, NAD, NADP, and NaAD) to the PncC mutated variants. In particular, the S29A PncC and K61Q PncC variant forms were selected since they still preserve the ability to bind NMN in the micromolar range, but they are not able to catalyze the enzymatic reaction. While S29A PncC shows a similar affinity also for NaMN (the product of the PncC catalyzed reaction), K61Q PncC does not interact significantly with it. Thus, PncC K61Q mutant seems to be a promising candidate to use as specific probe for an NMN biosensor.

Targeting Interleukin‐1 Receptor‐Associated Kinase 1 (IRAK1) In Ovarian Cancer

Ovarian cancer (OvCa) is the leading cause of gynecologic cancer-related deaths, due largely to late-stage diagnoses and the development of chemoresistant disease. Nearly 70% of affected women develop recurrent disease, characterized by resistance to standard chemotherapy despite initial response. Cancer stem cells (CSCs) are a subset of cells that have an innate ability to self-renew and enter a dormant state and have been further implicated in resistance to chemotherapy due to high expression of the ATP-binding Cassette (ABC) transporters. To understand the pathways that may be implicated in enhancing this stem-like phenotype, RNA sequencing was performed on cisplatin sensitive and isogenic cisplatin-resistant OvCa cell lines, as well as cells grown in 2- dimensional (2-D) or 3-dimensional (3-D) cultures. OvCa cells grown as spheroids, a 3-D growth model, are enriched with CSCs, and therefore represent a surrogate for stemness. Interestingly, the Toll-like (TLR) or interleukin-1 (IL-1) receptor pathways were highly enriched in resistant cancer cells and spheroid cultures. A critical factor in these signaling pathways is the interleukin receptor associated kinase 1 (IRAK1). IRAK1 is also highly expressed in chemoresistant OvCa and is responsible for activating numerous downstream pathways involved in tumor progression, such as NF-kB, P38/MAPK, and C-Jun. High expression of IRAK1 is associated with poor overall survival based on analysis of the Cancer Genome Atlas (TCGA) publicly available datasets. Due to the important role IRAK1 has in OvCa growth signaling and patient outcomes, IRAK1 represents an attractive target for CSC-targeted drug therapy. The present study is aimed at identifying small molecule inhibitors of IRAK1 by using in-silico molecular docking-based screening. FDA-approved compound libraries were screened using Autodock Vina software. Compounds were assessed for binding within the c-terminal kinase domain. The compounds are ranked-based on their binding energies (BE) and hydrogen bonding patterns. Our screen identified multiple compounds, one of which included mebendazole, an anthelminthic with a BE of -10.3 kCal/mol. This suggests potent interaction with the kinase domain of IRAK1. To confirm the binding in vitro, we performed cellular thermal shift stability assays (CETSA). We observed that mebendazole treatment protected IRAK1 protein from thermal denaturation, confirming the in silico docking data. Lastly, we performed hexosaminidase proliferation assays to assess the effectiveness of mebendazole on inhibiting cancer cell growth. Cisplatin sensitive OvCar3, A1847, A2780 OvCa and cisplatin resistant C30 OvCa cell lines were treated with increasing doses of mebendazole. The inhibitory concentration 50 (IC50) was determined to be approximately 300 nM at 72h for all cell lines. This suggests that mebendazole has potent anti-cancer activity that may be, in part, mediated through the inhibition of IRAK1.

A Novel Thermostable Keratinase from Deinococcus geothermalis with Potential Application in Feather Degradation

Keratinase can specifically attack disulfide bridges in keratin to convert them from complex to simplified forms. Keratinase thermal stability has drawn attention to various biotechnological industries. In this study, a keratinase DgeKer was identified from a slightly thermophilic species, D. geothermalis. The in silico analysis showed that DgeKer is composed of signal peptide, N-terminal propeptide, mature domain, and C-terminal extension. DgeKer and its C-terminal extension-truncated enzyme (DgeKer-C) were cloned and expressed in E. coli. The purified DgeKer and DgeKer-C showed maximum activity at 70 °C and pH 9–The thermal stability assay (60 °C) showed that the half-life value of DgeKer and DgeKer-C were 103.45 min and 169.10 min, respectively. DgeKer and DgeKer-C were stable at the range of pH from 9 to 11 and showed good tolerance to some metal ions, surfactants and organic solvent. Furthermore, DgeKer could degrade feathers at 70 °C for 60 min. However, the medium became turbid with obvious softening of barbules after being treated with DgeKer-C, which might be due to C-terminal extension. In summary, a thermostable keratinase DgeKer with high efficiency degradation of feathers may have great potential in industry.

Production and characterization of lipase from Penicillium aurantiogriseum under solid-state fermentation using sunflower pulp

Solid-State Fermentation (SSF) defined as a system in which the growth of microorganisms occurs on a moist solid substrate, shows tremendous potential in applications of microbial enzyme production as a low-cost, low-energy option. In this work, the production of lipase from Penicillium aurantiogriseum by using SSF was investigated. For this purpose, the sunflower pulp, an agro-industrial by-product, was utilized as a solid substrate in the SSF method. The optimization and characterization studies for lipase extract were performed by using the p-nitrophenyl palmitate (pNPP) (C16) as substrate. The lipase activity was found as 29.6 ± 0.11 U/mg protein at optimum conditions (50 mM of Tris-HCl buffer, pH 7.0, 40 °C). In thermal stability assay, the catalytic activities of lipase which are kept at 40, 50 and, 60 °C in a water bath for 1 h, were calculated as 100%, 60.48 ± 1.3%, 32.67 ± 0.2%, respectively. Also, the enzyme lost rapidly its activity within 15 min at 70 °C. The kinetic data, K m and V max , were determined as 0.17 mM and 2.9 µM/min, respectively. Also, 10 mM of Ba+2 ion had a slight activating effect (15%) showed on lipase. Lipase kept about 50% of its activity at 4 °C at the end of the 30 days. The hydrolytic activities of lipase for commercial olive oil and sunflower oil were found as 3.52 ± 0.1 U/mg and 3.90 ± 0.12 U/mg, respectively. The SSF method had obvious potential for the sustainable and cost-effective production of lipase from P. aurantiogriseum. Also, these results confirmed that the sunflower pulp could be used as an alternative substrate to produce enzymes in SSF conditions.

Polymer Modification of Lipases, Substrate Interactions, and Potential Inhibition.

An industrially important enzyme, Candida antarctica lipase B (CalB), was modified with a range of functional polymers including hydrophilic, hydrophobic, anionic, and cationic character using a "grafting to" approach. We determined the impact of polymer chain length on CalB activity by synthesizing biohybrids of CalB with each polymer at three different chain lengths, using reversible addition-fragmentation chain transfer (RAFT) polymerization. The activity of CalB in both aqueous and aqueous-organic media mixtures was significantly enhanced for acrylamide (Am) and N,N-dimethyl acrylamide (DMAm) conjugates, with activity remaining approximately constant in 25 and 50% ethanol solvent systems. Interestingly, the activity of N,N-dimethylaminopropyl-acrylamide (DMAPA) conjugates increased gradually with increasing organic solvent content in the system. Contrary to other literature reports, our study showed significantly diminished activity for hydrophobic polymer-protein conjugates. Functional thermal stability assays also displayed a considerable enhancement of retained activity of Am, DMAm, and DMAPA conjugates compared to the native CalB enzyme. Thus, this study provides an insight into possible advances in lipase production, which can lead to new improved lipase bioconjugates with increased activity and stability.