Hydrophobic Substitutions Research Articles

Distinct Mechanisms of Receptor Activation Are Associated with CSF3R Gain-of-Function Mutations

Activating mutations in granulocyte colony-stimulating factor receptor (CSF3R), are found in the extracellular, the membrane proximal, the transmembrane and the cytoplasmic domain of the receptor in various leukemia. To expand the spectrum of CSF3R mutations that can arise to transform Ba/F3 cells initially transfected with wild type CSF3R, we performed a spontaneous cellular transformation assay. We identified several new CSF3R activating mutations. These mutations are located in the extracellular, the membrane proximal, and the transmembrane domains of CSF3R. We further characterized that these mutations transform cells via four distinct mechanisms: (1) cysteine and disulfide bond mediated dimerization, leading to increased STAT3 activation (S581C); (2) polar, non-charged amino acid substitution at the transmembrane helix dimer interface T640, inducing increased STAT3 and ERK activation; (3) increased internalization and STAT3 activation by an E524 substitution; and (4) hydrophobic amino acid substitutions in the membrane proximal O glycosylation sites (T612, T615 and T618), leading to enhanced activation of STAT3 and ERK. Furthermore, we showed that the distinct signaling activation is related to different localization of CSF3R mutants. To this end, we have expanded the region of the CSF3R extracellular and transmembrane domain in which missense mutations exhibit leukemogenic capacity and further elucidated the mechanistic underpinnings that underlie distinct signaling activation and leukemogenic mechanisms. Cumulatively, these findings will facilitate improved evaluation of the biological and clinical relevance of CSF3R mutations identified in patients. [Display omitted] DisclosuresTyner:Agios Pharmaceuticals: Research Funding; Gilead: Research Funding; Syros: Research Funding; Genentech: Research Funding; Constellation Pharmaceuticals: Research Funding; AstraZeneca: Research Funding; Seattle Genetics: Research Funding; Janssen Pharmaceutica: Research Funding; Incyte Corporation: Research Funding; Array Biopharma: Research Funding; Takeda Pharmaceutical Company: Research Funding; Aptose Biosciences: Research Funding; Leap Oncology: Consultancy.

The Iodide Transport Defect-Causing Y348D Mutation in the Na+/I- Symporter Renders the Protein Intrinsically Inactive and Impairs Its Targeting to the Plasma Membrane.

Background: The sodium/iodide (Na+/I-) symporter (NIS) mediates active transport of I- into the thyroid gland. Mutations in the SLC5A5 gene, which encodes NIS, cause I- transport defects (ITDs)-which, if left untreated, lead to congenital hypothyroidism and consequent cognitive and developmental deficiencies. The ITD-causing NIS mutation Y348D, located in transmembrane segment (TMS) 9, was reported in three Sudanese patients. Methods: We generated cDNAs coding for Y348D NIS and mutants with other hydrophilic and hydrophobic amino acid substitutions at position 348 and transfected them into cells. The activity of the resulting mutants was quantitated by radioiodide transport assays. NIS glycosylation was investigated by Western blotting after endoglycosidase H (Endo H) and PNGase-F glycosidase treatment. Subcellular localization of the mutant proteins was ascertained by flow cytometry analysis, cell surface biotinylation, and immunofluorescence. The intrinsic activity of Y348D was studied by measuring radioiodide transport in membrane vesicles prepared from Y348D-NIS-expressing cells. Our NIS homology models and molecular dynamics simulations were used to identify residues that interact with Y348 and investigate possible interactions between Y348 and the membrane. The sequences of several Slc5 family transporters were aligned, and a phylogenetic tree was generated in ClustalX. Results: Cells expressing Y348D NIS transport no I-. Furthermore, Y348D NIS is only partially glycosylated, is retained intracellularly, and is intrinsically inactive. Hydrophilic residues other than Asp at position 348 also yield NIS proteins that fail to be targeted to the plasma membrane (PM), whereas hydrophobic residues at this position, which we show do not interact with the membrane, rescue PM targeting and function. Conclusions: Y348D NIS does not reach the PM and is intrinsically inactive. Hydrophobic amino acid substitutions at position 348, however, preserve NIS activity. Our findings are consistent with our homology model's prediction that Y348 should face the side opposite the TMS9 residues that coordinate Na+ and participate in Na+ transport, and with the notion that Y348 interacts only with hydrophobic residues. Hydrophilic or charged residues at position 348 have deleterious effects on NIS PM targeting and activity, whereas a hydrophobic residue at this position rescues NIS activity.

Investigation of water-insoluble hydrophobic polyethylenimines as RNAi vehicles in chronic myeloid leukemia therapy.

The discovery of RNA interference (RNAi) more than two decades ago opened avenues for avant-garde cancer treatments that possess the ability to evade issues hampering current chemotherapeutic strategies, owing to its specific gene sequence-driven mechanism of action. A potent short interfering RNA (siRNA) delivery vehicle designed to overcome physiological barriers is imperative for successful RNAi therapy. For this purpose, this study explored the characteristics and therapeutic efficacy of low-molecular weight (MW) polyethylenimine (PEI) with high cholesterol substitution, yielding water-insoluble polymers, in chronic myeloid leukemia (CML) K562 cells. A strong impact of cholesterol grafting on the physicochemical attributes of the resultant polymers and their corresponding complexes with siRNA was observed, with the siRNA binding capacity of polymers increasing and complex dissociation sensitivity decreasing with increase in cholesterol content of the polymers. The modified polymer complexes were significantly smaller in size and possessed higher cationic charge compared to the parent polymer. The interaction with anionic heparan sulfate preoteoglycans present on the cell surface was significant in cellular uptake of the complexes. The therapeutic efficacy of siRNA/polymer complexes was reflected in their ability to effectively silence the reporter green fluorescent protein gene and endogenous CML oncogene BCR-ABL as well as significantly inhibit colony formation by K562 cells post BCR-ABL silencing. The results of this study demonstrated beneficial effects of high levels of hydrophobic substitution on low MW PEI on their functional performance bestowing them the potential to be potent RNAi agents for CML therapy.

Potent ABA-independent activation of engineered PYL3.

Abscisic acid (ABA) plays a vital role in many developmental processes and the response to adaptive stress in plants. Under drought stress, plants enhance levels of ABA and activate ABA receptors, but under harsh environmental stress, plants usually cannot efficiently synthesize and release sufficient quantities of ABA. The response of plants to harsh environmental stress may be improved through ABA‐independent activation of ABA receptors. The molecular basis of ABA‐independent inhibition of group A protein phosphatases type 2C (PP2Cs) by pyrabactin resistance/Pyr1‐like (PYR1/PYLs) is not yet clear. Here, we used our previously reported structures of PYL3 to first obtain the monomeric PYL3 mutant and then to introduce bulky hydrophobic residue substitutions to promote the closure of the Gate/L6/CL2 loop, thereby mimicking the conformation of ABA occupancy. Through structure‐guided mutagenesis and biochemical characterization, we investigated the mechanism of ABA‐independent activation of PYL3. Two types of PYL3 mutants were obtained: (a) PYL3 V108K V107L V192F can bind to ABA and effectively inhibit HAB1 without ABA; (b) PYL3 V108K V107F V192F, PYL3 V108K V107L V192F L111F and PYL3 V108K V107F V192F L111F cannot recognize ABA but can greatly inhibit HAB1 without ABA. Intriguingly, the ability of PYL3 mutants to bind to ABA was severely compromised if any two of three variable residues (V107, V192 and L111) were mutated into a bulky hydrophobic residue. The introduction of PYL3 mutants into transgenic plants will help elucidate the functionality of PYL3 in vivo and may facilitate the future production of transgenic crops with high yield and tolerance of abiotic stresses.

Hydrophobic modifications of hydroxyethyl cellulose polymers: Their influence on the acute toxicity to aquatic biota

The hydrophobic substitution (HS) of cationic cellulose derivatives may be tuned, promoting their efficiency. This work studied the influence of HS on the acute ecotoxicity of quaternized hydroxyethyl cellulose polymers (SL) to aquatic biota. The ecotoxicity of four SL with different HS (SL-5, SL-30, SL-60, SL-100) was assessed for seven species: Vibrio fischeri, Raphidocelis subcapitata, Chlorella vulgaris, Daphnia magna, Brachionus calyciflorus, Heterocypris incongruens, and Danio rerio. The computed median effective concentrations were used to derive hazard concentrations, by using species sensitive distribution curves. All SL suspensions were characterized for particle size, zeta potential and rheological properties. Results indicated instability of the SL in suspension due to their relatively low zeta potential. Raphidocelis subcapitata, C. vulgaris and B. calyciflorus were the most sensitive to the four SL, suggesting that exposure to these compounds may imbalance the lowest trophic levels. Also, HS influenced the toxicity of SL, with the lowest HS (SL-5) revealing lower ecotoxicity. The maximum acceptable concentrations were 14.0, 2.9, 3.9 and 1.4 mg L−1 for SL-5, SL-30, SL-60, and SL-100, respectively. Accordingly, SL-5 is suggested as the eco-friendliest and is recommended to be used in the production of care products, in detriment of the other three tested variants.

Enhancing the activity and thermal stability of a phthalate-degrading hydrolase by random mutagenesis

Our previous work has reported that EstJ6 was a phthalate-degrading hydrolase. In the study, a random mutant library was constructed by two rounds of error-prone PCR, three mutants (ET1.1, ET2.1, and ET2.2) with enhanced hydrolytic activity against dibutyl phthalate (DBP) were obtained. The best mutant ET2.2, accumulated three amino acid substitutions (Thr91Met, Ala67Val, and Val249Ile) and exhibited 2.8-fold increase enzyme activity and 2.3-fold higher expression level. Meanwhile, compared with EstJ6, ET2.2 showed over 50% improvement in thermostability (at 50 °C for 1 h) and 1.2-fold increase in 50% methanol tolerance. Kinetic parameters analysis revealed that the Km value for ET2.2 decreased by 60% and the kcat/Km value increased by 166%. The molecular docking indicated that the shortening of hydrogen bond between Ser146-OH and DBP-CO, which may led to an increase in enzyme activity and catalytic efficiency, the enhancement of hydrophobicity of hydrophobic pocket was related to the improvement of organic solvents tolerance, and three hydrophobic amino acid substitutions Thr91Met, Ala67Val, and Val249Ile facilitated to improve the thermal stability and organic solvents tolerance. These results confirmed that random mutagenesis was an effective tool for improving enzyme properties and lay a foundation for practical applications of phthalate-degrading hydrolase in biotechnology and industrial fields.

Feleucin-K3 Analogue with an α-(4-Pentenyl)-Ala Substitution at the Key Site Has More Potent Antimicrobial and Antibiofilm Activities in Vitro and in Vivo.

The development of antimicrobial compounds is now regarded as an urgent problem. Antimicrobial peptides (AMPs) have great potential to become novel antimicrobial drugs. Feleucin-K3 is an α-helical cationic AMP isolated from the skin secretion of the Asian bombinid toad species Bombina orientalis and has antimicrobial activity. In our previous studies, amino acid scanning of Feleucin-K3 was performed to determine the key site affecting its activity. In this study, we investigated and synthesized a series of analogues that have either a natural or an unnatural hydrophobic amino acid substitution at the fourth amino acid residue of Feleucin-K3. Among these analogues, Feleucin-K59 (K59), which has an α-(4-pentenyl)-Ala substitution, was shown to have increased antimicrobial activity against both standard and drug-resistant strains of clinical common bacteria, improved stability, no hemolytic activity at antimicrobial concentrations, and no resistance. In addition, K59 has potent antibiofilm activity in vitro. More importantly, K59 showed better antimicrobial and antibiofilm activities against drug-resistant bacteria in in vivo experiments in mice than traditional antibiotics. In this preliminary study of the mechanism of action, we found that K59 could rapidly kill bacteria by a dual-action mechanism of disrupting the cell membrane and binding to intracellular DNA, thus making it difficult for bacteria to develop resistance.

A hydrophobic ratchet entrenches molecular complexes.

Most proteins assemble into multisubunit complexes1. The persistence of these complexes across evolutionary time is usually explained as the result of natural selection for functional properties that depend upon multimerization, like intersubunit allostery or the capacity to do mechanical work2. In many complexes, however, multimerization does not enable any known function3. An alternative explanation is that multimers could become entrenched if substitutions accumulate that are neutral in multimers but deleterious in monomers; purifying selection would then prevent reversion to the unassembled form, even if assembly per se does not enhance biological function3–7. Here we show that a hydrophobic mutational ratchet systematically entrenches molecular complexes. By applying ancestral protein reconstruction and biochemical assays to the evolution of steroid hormone receptors (SRs), we show that an ancient hydrophobic interface, conserved for hundreds of millions of years, is entrenched because exposing this interface to solvent reduces protein stability and causes aggregation, despite making no detectable contribution to function. Using structural bioinformatics, we show that a universal mutational propensity drives sites that are buried in multimeric interfaces to accumulate hydrophobic substitutions to levels not tolerated in monomers. In a database of hundreds of families of multimers, the majority show signatures of long-term hydrophobic entrenchment. It is therefore likely that many protein complexes persist because a simple ratchet-like mechanism entrenches them across evolutionary time, even when they are functionally gratuitous.

Flexible RSV Prefusogenic Fusion Glycoprotein Exposes Multiple Neutralizing Epitopes that May Collectively Contribute to Protective Immunity.

Human respiratory syncytial virus (RSV) is a cause of lower respiratory tract infection in infants, young children, and older adults. There is no licensed vaccine and prophylactic treatment options are limited. The RSV fusion (F) glycoprotein is a target of host immunity and thus a focus for vaccine development. F-trimers are metastable and undergo significant rearrangements from the prefusion to a stable postfusion structure with neutralizing epitopes on intermediate structures. We hypothesize that vaccine strategies that recapitulate the breathable F quaternary structure, and provide accessibility of B-cells to epitopes on intermediate conformations, may collectively contribute to protective immunity, while rigid prefusion F structures restrict access to key protective epitopes. To test this hypothesis, we used the near full-length prefusogenic F as a backbone to construct three prefusion F variants with substitutions in the hydrophobic head cavity: (1) disulfide bond mutant (DS), (2) space filling hydrophobic amino acid substitutions (Cav1), and (3) DS, Cav1 double mutant (DS-Cav1). In this study, we compared the immunogenicity of prefusogenic F to prefusion F variants in two animal models. Native prefusogenic F was significantly more immunogenic, producing high titer antibodies to prefusogenic, prefusion, and postfusion F structures, while animals immunized with DS or DS-Cav1 produced antibodies to prefusion F. Importantly, prefusogenic F elicited antibodies that target neutralizing epitopes including prefusion-specific site zero (Ø) and V and conformation-independent neutralizing sites II and IV. Immunization with DS or DS-Cav1 elicited antibodies primarily to prefusion-specific sites Ø and V with little or no antibodies to other key neutralizing sites. Animals immunized with prefusogenic F also had significantly higher levels of antibodies that cross-neutralized RSV A and B subtypes, while immunization with DS or DS-Cav1 produced antibodies primarily to the A subtype. We conclude that breathable trimeric vaccines that closely mimic the native F-structure, and incorporate strategies for B-cell accessibility to protective epitopes, are important considerations for vaccine design. F structures locked in a single conformation restrict access to neutralizing epitopes that may collectively contribute to destabilizing F-trimers important for broad protection. These results also have implications for vaccine strategies targeting other type 1 integral membrane proteins.

Effect of Non-natural Hydrophobic Amino Acids on the Efficacy and Properties of the Antimicrobial Peptide C18G.

Antimicrobial peptides (AMPs) have been an area of great interest, due to the high selectivity of these molecules toward bacterial targets over host cells and the limited development of bacterial resistance to these molecules through evolution. The peptides are known to selectively bind to bacterial cell surfaces through electrostatic interactions, and subsequently, the peptides insert into the cell membrane and cause local disruptions of membrane integrity leading to cell death. Previous experiments showed that replacing the Leu residues in the AMP C18G with other naturally occurring hydrophobic residues resulted in side-chain-dependent activities. This work extends the investigation to non-natural hydrophobic amino acids and the effect on peptide activity. Minimal inhibitory concentration (MIC) results demonstrated that amino acid substitutions containing long flexible carbon chains maintained or increased antimicrobial activity compared to natural analogues. In solution, the peptide showed aggregation only with the most hydrophobic non-natural amino acid substitutions. Binding assays using Trp fluorescence confirm a binding preference for anionic lipids while quenching experiments demonstrated that the more hydrophobic peptides are more deeply buried in the anionic lipid bilayers compared to the zwitterionic bilayers. The most effective peptides at killing bacteria were also those which showed some level of disruption of bacterial membranes; however, one peptide sequence exhibited very strong activity and very low levels of red blood cell hemolysis, yielding a promising target for future development.

Molecular-Level Understanding of the Somatostatin Receptor 1 (SSTR1)-Ligand Binding: A Structural Biology Study Based on Computational Methods.

Somatostatin receptor 1 (SSTR1), a subtype of somatostatin receptors, is involved in various signaling mechanisms in different parts of the human body. Like most of the G-protein-coupled receptors (GPCRs), the available information on the structural features of SSTR1 responsible for the biological activity is scarce. In this study, we report a molecular-level understanding of SSTR1–ligand binding, which could be helpful in solving the structural complexities involved in SSTR1 functioning. Based on a three-dimensional quantitative structure–activity relationship (3D-QSAR) study using comparative molecular field analysis (CoMFA) and comparative molecular similarity index analysis (CoMSIA), we have identified that an electronegative, less-bulkier, and hydrophobic atom substitution can substantially increase the biological activity of SSTR1 ligands. A density functional theory (DFT) study has been followed to study the electron-related properties of the SSTR1 ligands and to validate the results obtained via the 3D-QSAR study. 3D models of SSTR1–ligand systems have been embedded in lipid–lipid bilayer membranes to perform molecular dynamics (MD) simulations. Analysis of the MD trajectories reveals important information about the crucial residues involved in SSTR1–ligand binding and various conformational changes in the protein that occur after ligand binding. Additionally, we have identified the probable ligand-binding site of SSTR1 and validated it using MD. We have also studied the favorable conditions that are essential for forming the most stable and lowest-energy bioactive conformation of the ligands inside the binding site. The results of the study could be useful in constructing more potent and novel SSTR1 antagonists and agonists.

Preparation of Cholesteryl-Modified Aminated Pullulan Nanoparticles to Evaluate Nanoparticle of Hydrophobic Degree on Drug Release and Cytotoxicity

The formation of nanoparticles (NPs) and surface properties such as size and charge are affected by the amphiphilic property of polymer, which is vital for evaluating their function. Here, we synthesized cholesteryl-modified aminated pullulan polymers (CHPNs) with different amounts of cholesterol succinate (CHS). We characterized the three hydrophobically modified polymers (CHPN1, CHPN2, and CHPN3) (CHS: Pu‐NH2=1/5,2/5,3/5) by Fourier transform infrared spectrometry. Dynamic light scattering (DLS) was used to measure particle size and zeta potential of CHPN NPs. The particle sizes of the three NPs CHPN1, CHPN2, and CHPN3 were 178.0, 144.4, and 97.8 nm, respectively. The particle size was related to the cholesteryl substitution of polymers to a certain extent: the stronger the hydrophobicity, the smaller the particle size. In 48 h, the drug release for CHPN3 and CHPN1 NPs was 57.8% and 72.7%. Thus, the NPs showed good sustained drug release: the greater the degree of hydrophobic substitution, the better the sustained release. The cytotoxicity findings were reversed: CHPN1 NPs, with low hydrophobic substitution, showed the best inhibition of Lewis lung cancer cells.

4-O-methylascochlorin stabilizes hypoxia-inducible factor-1 in a manner different from hydroxylase inhibition by iron chelating or substrate competition.

Hypoxia-inducible factor-1 (HIF-1) is a transcription factor that plays essential roles in human diseases including cancer. The synthetic ascochlorin derivative 4-O-methylascochlorin stabilizes HIF-1α protein, and activates its transcriptional activity, resulting to induce gene expression of its downstream targets such as VEGF and GLUT-1. Here, we quantified protein level of HIF-1α in human osteosarcoma U2OS cells treated with ascochlorin-related compounds and typical HIF-1α stabilizers to characterize properties of HIF-1α stabilization by 4-O-methylascochlorin. Structure-activity relationship studies suggested that the aromatic moiety and hydrophobic substitution of the 4'-hydroxyl group are important for HIF-1α stabilization by ascochlorin-related compounds. 4-O-Methylascochlorin-induced HIF-1α stabilization was suppressed by ascorbic acid and compound C, but not by Fe(II), whereas ascorbic acid only suppressed HIF-1α stabilization by dimethyloxaloylglycine, an analog of the HIF-1 hydroxylase substrate. Fe(II) completely suppressed iron chelator-induced stabilization. These results suggest that ascochlorin-related compounds stabilize HIF-1α in a manner distinct from iron chelating or substrate competition.

Hydrophobic ring substitution on 9-O position of berberine act as a selective fluorescent sensor for the recognition of bovine serum albumin

Current research on the detection of serum albumin (SA) by fluorescence-based sensing probe has earned massive interest due to its various proficiencies. In this belief, we report a cost-effective fluorescence probe BRDR4 from well-known medicinal herb berberine for distinguishing BSA from HSA selectively. The probe itself has very weak fluorescence intensity which was increased ~269 fold with the addition of 47 μM of BSA (detection limit 5.9 nM). Interestingly under the same condition, HSA remains silent towards the studied probe. To establish the sensing mechanism and the fluorometric sensing potency we further compare characteristics of the interaction with structurally comparable four more berberine derivatives (BRDR1, BRDR2, BDRD3 and BRDR5) with BSA. Furthermore, to reveal the mechanistic insight of BSA sensing a systematic binding investigation was performed on all BRDR series using various spectroscopic techniques. The binding affinity of all BRDR with BSA was evaluated using absorbance and fluorescence spectroscopy. The calculated binding constants and quenching constants suggests highest binding affinity of BRDR4 towards BSA. On the other hand, fluorescence anisotropy, FT-IR spectroscopic study and 3D fluorometric analysis indicates the change of hydrophobic environment and α-helix content of BSA during the interaction. The fluorescence lifetime and FRET theory recommended that the stable ground state complex formation phenomenon was only responsible for BSA sensing. All the approached experimental evidences successfully assessed that the probe BRDR4 sufficiently altered the native structure of BSA which helps it to become a delicate BSA sensor.

Protein Translocation Inhibitors Overcome Cytokine-Induced Glucocorticoid Resistance in T-Cell Acute Lymphoblastic Leukemia

Glucocorticoids (GCs) are central to the treatment of T-cell acute lymphoblastic leukemia (T-ALL), and upfront resistance to GCs is a poor prognostic factor. We previously demonstrated that over one-third of primary patient T-ALLs are resistant to the GC dexamethasone (DEX) when cultured in the presence of interleukin-7 (IL7), a cytokine that is abundant in the microenvironment of leukemic blasts and that plays a well-established role in leukemogenesis. Mechanistically, we demonstrated that GCs paradoxically induce their own resistance by promoting the upregulation of IL7 receptor (IL7R) expression. In the presence of IL7, this augments signal transduction through the JAK/STAT5 axis, ultimately leading to increased STAT5 transcriptional output. This promotes the upregulation of the pro-survival protein BCL-2, which opposes DEX-induced apoptosis. Given that IL7-induced GC resistance depends on de novo synthesis of IL7R in response to DEX, and that newly synthesized IL7R reaches the cell surface via trafficking through the secretory pathway, we hypothesized that inhibiting the translocation of nascent IL7R peptide into the secretory pathway would effectively overcome IL7-induced DEX resistance. Sec61 is a protein-conducting channel in the membrane of the endoplasmic reticulum (ER) that is required for the cotranslational insertion of nascent polypeptides into the ER upon recognition of the signal sequence on secreted and cell surface proteins. To test the hypothesis that Sec61 inhibition could overcome IL7-induced DEX resistance, we utilized the human T-ALL cell line CCRF-CEM, which recapitulates the resistance phenotype observed in primary patient samples. Using a series of structurally distinct small molecule inhibitors of the Sec61 translocon, we demonstrated that Sec61 inhibition effectively overcomes the increase in cell surface IL7R expression in response to DEX. This occurs despite a persistent elevation in IL7R transcript expression following DEX exposure, confirming that Sec61 inhibitors act post-transcriptionally to attenuate cell surface IL7R expression. To determine whether the sensitivity of IL7R to Sec61 inhibitors is due specifically to the interaction between the IL7R signal sequence and Sec61 inhibitors, we generated IL7R constructs containing hydrophobic amino acid substitutions in the signal sequence, which are predicted to confer resistance to Sec61 inhibitors. Upon transient transfection of these constructs into HEK293T cells, we found that these mutations rendered IL7R resistant to the effects of Sec61 inhibition, confirming that the IL7R signal sequence confers sensitivity to these inhibitors. Using the Bliss independence model of synergy in CCRF-CEM cells, we demonstrated that Sec61 inhibitors potently synergize with DEX to overcome IL7-induced DEX resistance. Importantly, at concentrations at which synergy occurs, Sec61 inhibitors demonstrate no single-agent effect on cell survival, suggesting that these effects are not due to an overall reduction in secretory and membrane protein biogenesis. Furthermore, Sec61 inhibitors failed to sensitize CCRF-CEM cells to other chemotherapies used in T-ALL, none of which demonstrate IL7-induced resistance, thereby suggesting that these effects on DEX sensitivity are due specifically to the reduction in cell surface IL7R. To determine if Sec61 inhibitors prevent the DEX-induced increase in STAT5 transcription, we analyzed BCL-2 expression in cells exposed to DEX and IL7, and found that Sec61 inhibitors attenuate the increase in BCL-2 expression in a dose-dependent manner. We next analyzed a cohort of 34 primary patient T-ALL samples. As in CCRF-CEM cells, we found that specifically in those samples with IL7-induced DEX resistance, Sec61 inhibitors synergized with DEX to induce cell death in the presence of IL7. This effect occurred concomitantly with a reduction in cell surface IL7R expression and BCL-2 expression. Taken together, these data demonstrate the efficacy and feasibility of Sec61 inhibition as a novel and rational therapeutic strategy to overcome the IL7-induced DEX resistance phenotype that affects over one-third of newly diagnosed T-ALL patients. Disclosures Sharp: Kezar Life Sciences: Patents & Royalties. McMinn:Kezar Life Sciences: Employment, Equity Ownership. Kirk:Kezar Life Sciences: Employment, Equity Ownership. Taunton:Global Blood Therapeutics: Equity Ownership; Principia Biopharma: Equity Ownership, Patents & Royalties; Cedilla Therapeutics: Consultancy, Equity Ownership; Pfizer: Research Funding; Kezar Life Sciences: Equity Ownership, Patents & Royalties, Research Funding.

Designing of novel ERRγ inverse agonists by molecular modeling studies of docking and 3D-QSAR on hydroxytamoxifen derivatives

ERRγinverse agonist is a powerful therapeutic target for the treatment of cancers and certain metabolic disorders. Until now, only GSK5814 was reported as selective ERRγinverse agonist. So 60 newly hydroxytamoxifen analogues were selected to perform molecular docking and 3D-QSAR study to design more selective inverse agonist of ERRγ. Both established CoMFA and CoMSIA models obtained high predictive and satisfactory value, demonstrated that bulky, hydrophobic and negative electrostatic substitutions are preferred at R2 position, and introducing hydrophilic and H-bond donor and acceptor groups at R1 and R4 positions is greatly important for improving binding activities. The obtained information will be useful to provide clues for rationally designing novel and high potency ERRγinverse agonists.

Structure-Based Mutagenesis of Phycobiliprotein smURFP for Optoacoustic Imaging

Photo- or optoacoustics (OA) imaging is increasingly being used as a non-invasive imaging method that can simultaneously reveal structure and function in deep tissue. However, the most frequent transgenic OA labels are current fluorescent proteins that are not optimized for OA imaging. Thus, they lack OA signal strength, and their absorption maxima are positioned at short wavelengths, thus giving small penetration depths and strong background signals. Here, we apply insights from our recent determination of the structure of the fluorescent phycobiliprotein smURFP to mutate a range of residues to promote the nonradiative decay pathway that generates the OA signal. We identified hydrophobic and aromatic substitutions within the chromophore-binding pocket that substantially increase the intensity of the OA signal and red-shift the absorption. Our results demonstrate the feasibility of structure-based mutagenesis to repurpose fluorescent probes for OA imaging, and they may provide structure-function insights for de novo engineering of transgenic OA probes.

Interaction of the Mechanosensitive Channel, MscS, with the Membrane Bilayer through Lipid Intercalation into Grooves and Pockets

All membrane proteins have dynamic and intimate relationships with the lipids of the bilayer that may determine their activity. Mechanosensitive channels sense tension through their interaction with the lipids of the membrane. We have proposed a mechanism for the bacterial channel of small conductance, MscS, that envisages variable occupancy of pockets in the channel by lipid chains. Here, we analyze protein–lipid interactions for MscS by quenching of tryptophan fluorescence with brominated lipids. By this strategy, we define the limits of the bilayer for TM1, which is the most lipid exposed helix of this protein. In addition, we show that residues deep in the pockets, created by the oligomeric assembly, interact with lipid chains. On the cytoplasmic side, lipids penetrate as far as the pore-lining helices and lipid molecules can align along TM3b perpendicular to lipids in the bilayer. Cardiolipin, free fatty acids, and branched lipids can access the pockets where the latter have a distinct effect on function. Cholesterol is excluded from the pockets. We demonstrate that introduction of hydrophilic residues into TM3b severely impairs channel function and that even “conservative” hydrophobic substitutions can modulate the stability of the open pore. The data provide important insights into the interactions between phospholipids and MscS and are discussed in the light of recent developments in the study of Piezo1 and TrpV4.

Identifying the molecular target sites for CFTR potentiators GLPG1837 and VX-770.

The past two decades have witnessed major breakthroughs in developing compounds that target the chloride channel CFTR for the treatment of patients with cystic fibrosis. However, further improvement in affinity and efficacy for these CFTR modulators will require insights into the molecular interactions between CFTR modulators and their binding targets. In this study, we use in silico molecular docking to identify potential binding sites for GLPG1837, a CFTR potentiator that may share a common mechanism and binding site with VX-770, the FDA-approved drug for patients carrying mutations with gating defects. Among the five binding sites predicted by docking, the two top-scoring sites are located at the interface between CFTR's two transmembrane domains: site I consists of D924, N1138, and S1141, and site IIN includes F229, F236, Y304, F312, and F931. Using mutagenesis to probe the importance of these sites for GLPG187 binding, we find that disruption of predicted hydrogen-bonding interactions by mutation of D924 decreases apparent affinity, while hydrophobic amino acids substitutions at N1138 and introduction of positively charged amino acids at S1141 improve the apparent affinity for GLPG1837. Alanine substitutions at Y304, F312, and F931 (site IIN) decrease the affinity for GLPG1837, whereas alanine substitutions at F229 and F236 (also site IIN), or at residues in the other three lower-scoring sites, have little effect. In addition, current relaxation analysis to assess the apparent dissociation rate of VX-770 yields results consistent with the dose-response experiments for GLPG8137, with the dissociation rate of VX-770 accelerated by D924N, F236A, Y304A, and F312A, but decelerated by N1138L and S1141K mutations. Collectively, these data identify two potential binding sites for GLPG1837 and VX-770 in CFTR. We discuss the pros and cons of evidence for these two loci and the implications for future drug design.

Engineering Order and Cooperativity in a Disordered Protein.

Structural disorder in proteins arises from a complex interplay between weak hydrophobicity and unfavorable electrostatic interactions. The extent to which the hydrophobic effect contributes to the unique and compact native state of proteins is, however, confounded by large compensation between multiple entropic and energetic terms. Here we show that protein structural order and cooperativity arise as emergent properties upon hydrophobic substitutions in a disordered system with non-intuitive effects on folding and function. Aided by sequence-structure analysis, equilibrium, and kinetic spectroscopic studies, we engineer two hydrophobic mutations in the disordered DNA-binding domain of CytR that act synergistically, but not in isolation, to promote structure, compactness, and stability. The double mutant, with properties of a fully ordered domain, exhibits weak cooperativity with a complex and rugged conformational landscape. The mutant, however, binds cognate DNA with an affinity only marginally higher than that of the wild type, though nontrivial differences are observed in the binding to noncognate DNA. Our work provides direct experimental evidence of the dominant role of non-additive hydrophobic effects in shaping the molecular evolution of order in disordered proteins and vice versa, which could be generalized to even folded proteins with implications for protein design and functional manipulation.