KRAS-mutant lung cancer patients, who have a poor prognosis and no targeted therapy available yet. Although several ATP-competitive inhibitors of CDK4 have been developed for anticancer therapeutics, they suffer from limited specificity and efficacy.CDK4/cyclin D kinase constitutes an attractive pharmacological target for development of anticancer therapeutics, in particular in Methods: As an alternative to ATP-competitive inhibitors we have designed a stapled peptide to target the main interface between CDK4 and cyclin D, and have characterized its physico-chemical properties and affinity to bind cyclin D1.Results: We have validated a positive correlation between CDK4/cyclin D level and KRAS mutation in lung cancer patients. The stapled peptide enters cells rapidly and efficiently, and inhibits CDK4 kinase activity and proliferation in lung cancer cells. Its intrapulmonary administration in mice enables its retention in orthotopic lung tumours and complete inhibition of their growth when co-administered with Abemaciclib.Conclusion: The stapled peptide targeting the main interface between CDK4 and cyclin D provides promising therapeutic perspectives for patients with lung cancer. Lung cancer is the leading cause of cancer-related death worldwide in men and women INK4a, all of which lead to hyperactivation of this kinase. CDK4/cyclin D activity coordinates exit from quiescence and growth factor-stimulated entry into and progression through G1 through phosphorylation of the Retinoblastoma tumour suppressor proteins . Cyclin D expression and its association with CDK4 are induced by mitogenic signals, notably via Ras signaling pathway. Amplification of the cyclin D1 locus is observed in 5-30% of NSCLC and high levels of cyclin D1 protein are found in 18-76% of invasive NSCLC KRAS-driven NSCLC is particularly dependent on CDK4 activity. Targeting this kinase in NSCLC has therefore been proposed as a therapeutic strategy in KRAS-mutant lung cancer that is resistant to conventional and targeted therapies CDKs are heterodimeric protein kinases formed through association of a CDK catalytic subunit with a cyclin regulatory partner  pRb, p10, p130. CA large number of CDK/cyclin inhibitors have been identified from natural substances, in high throughput screening assays, or through structure-guided approaches . Especially, all-hydrocarbon stapled α-helical peptides have emerged as suitable pharmacological drug candidates with a large number of studies demonstrating their therapeutic potential Targeting protein/protein interactions (PPIs) that are essential for enzyme function represents a particularly attractive alternative to ATP-competitive inhibitors. However, the modulation of PPIs with selectivity and potency represents a major challenge, given the highly conserved structural features of protein kinases. In this respect constrained peptides and peptidomimetics constitute privileged structures for the controlled display of key functional groups that interact with target surface hotspot residues, by virtue of their stable, well-defined and predictable conformation, and therefore constitute particularly attractive and tractable biomolecules for development of targeted therapeutics 22The primary interface between CDK and cyclin partners is mediated by the C helix of the CDK and the α5 helix of the cyclin partner Lung adenocarcinoma and squamous cell carcinoma samples were obtained form surgical rsections and retrieved from the Biological Resource Center of Grenoble University hospital (CRB) (authorized by Ministry of Higher Education and Research - Accreditation number AC 2017-2949- BRIF BB-0033-00069). All tumours were classified according to the 2015 WHO classification Immunohistostainings were performed on 3 µm formalin-fixed paraffin-embedded tissue sections on Benchmark Autostrainer . Sections were incubated with cyclin D1 rabbit mAb , CDK4 rabbit mAb  and rabbit phospho-Rb (Ser807/811) mAb . Antigen retrieving was performed for cyclin D1 64 min in CC1 buffer , for pRB 60 min in CC1 buffer, and for CDK4 60 min in Novolink citrate buffer. The Ventana DAB detection kit  was used according to the manufacturer's instructions. Omission of the primary antibody and/or incubation with same species and isotype IgG at the same concentration of the primary antibody served as negative controls. Pathologist blinded to clinico-pathological variables, mutation status and treatment response, independently evaluated the levels of expression using the percentage of positive tumour cells, with a cut off of positivity of >10% of stained cells.A 3 µm tissue section was stained with H&E (hematoxylin and eosin) and examined by light microscopy to assess the quality of all the samples and to determine areas containing more than 70% of tumour cells for microdissection before DNA extraction. DNA was isolated  from 3 µm FFPE sections. The genotyping and mutation analyses were performed by using an accredited pyrosequencing method from Grenoble University Hospital clinical laboratory. The detailed methodology for tumor DNA sequencing has been previously described 2O/TFA 0.1%) to 100% (CH3CN/TFA 0.1%) in 5 min; flow rate 4 mL/min; detection at 214 nm (conditions B). LC/MS analyses were recorded on a Quattro micro ESI triple quadrupole mass spectrometer  equipped with a Chromolith Speed Rod RP-C18 185 Pm column  and an Alliance HPLC System ; gradient from 100% (H2O/HCO2H 0.1%) to 100% (CH3CN/HCO2H 0.1%) in 3 min; flowrate 3 mL/min; UV detection at 214 nm. High-Resolution Mass Spectrometric analyses were performed with a time-of-flight (TOF) mass spectrometer fitted with an Electrospray Ionisation source (ESI) in positive ion mode.All reagents and solvents were obtained from commercial sources and used without further purification. Analytical HPLC analyses were run on an Agilent Technology 1220 Infinity LC equipped with a Chromolith Speed Rod RP-C18 185 Pm column  with a gradient from 100%  for 10 min and filtered. For each coupling reaction, 5 equivalents (eq.) of Fmoc-Amino Acid, 5 eq. of Hexafluorophosphate Azabenzotriazole Tetramethyl Uronium (HATU) and 10 eq. of N,N-Diisopropylethylamine (DIEA) were added to a fritted reaction vessel and stirred in N,N-dimethylformamide (DMF) (2 x 5 min at room temperature). For the coupling of olefinic amino acid, 3 eq. of Fmoc-(S)-pentenylalanine, 3 eq. of HATU and 5 eq. of DIEA were added to the reactor and stirred in DMF (2 x 30 min). Deprotection of the Fmoc group at the N-terminus was performed using a 20% piperidine/DMF solution (2 x 5 min at room temperature). After each coupling and deprotection step, resin was washed three times with DMF. After removal of the Fmoc group of the last amino acid, peptides P1, P2, P3, P4, MSI2, peptide control and P2short were acetylated at the N-terminus with a DIEA/Ac2O/DMF 1/1/8 vvv solution (2 x 10 min at room temperature).Solid phase peptide synthesis were performed on an AmphispheresThe molecular stapling chemistry was first developed by Miller, Blackwell and Grubbs2O 95/2.5/2.5 vvv solution (2 × 90 min at r.t.). Resins were washed  and filtrates were evaporated under reduced pressure. Compounds were precipitated by addition of 50 mL of diethyl ether and centrifuged . Crude peptides were then solubilized in acetonitrile/water 1/1 vv solution and purified by preparative RP-HPLC on a Waters system controller equipped with a C18 Waters Delta-Pack column ; flow rate 50 mL/min; UV detection at 214 nm using a Waters 486 Tunable Absorbance Detector and a linear gradient of A = H2O (0.1% TFA) and B = CH3CN (0.1% TFA). Peptides were recovered as TFA salts. Final compound purity was assessed by LC-MS analyses.Peptides were then cleaved from the resin with a TFA/TIS/HCD experiments were carried out using a Jasco J815 spectropolarimeter. Spectra were recorded with 100 μM of peptides dissolved in phosphate buffer 50 mM, pH 7, using a 1 mm pathlength CD cuvette at 20 °C, over a wavelength range of 190-260 nm. Continuous scanning mode was used, with a response of 1.0 s with 0.1 nm steps and a bandwidth of 2 nm. The signal to noise ratio was improved by acquiring each spectrum over an average of two scans. Baseline was corrected by subtracting the background from the sample spectrum.obs * 100)/(-39500 * (1-2.57/n), where ([θ])obs is the mean residue ellipticity at 222 nm and n the number of peptide bonds.Alpha helical content was determined using the following equation: % Helix = ([θ])Synthesis of Cy5.5-labelled P2short: 1.8 mg (1.5 eq.) of Cyanine 5.5 NHS-ester was solubilized in 0.5 mL DMF (pH 12.0) and added to 3 mg (1 eq.) of P2shortA solubilized in 0.5 mL DMF (pH 12.0). The mixture was stirred for 2 h at room temperature and then purified by preparative RP-HPLC as described previously. DMF (pH 12.0) solution was prepared by adding 5 µL Et3N to 10 mL DMF.Synthesis of TAMRA-labelled P2short: 1.035 mg (1.6 eq.) of TAMRA maleimide was solubilized in 0.1 mL DMF and added to 3 mg (1 eq.) of P2short solubilized in 0.7 mL Phosphate buffer . The mixture was stirred for 2 h at room temperature and then purified by preparative RP-HPLC as described previously.TP2-Rho-labelled peptides: peptides were labelled on a unique cysteine residue with a five-fold molar excess of TP2-Rho-maleimide overnight, and then purified from free dye on NAP-5 columns , as described previously E. coli by IPTG induction and purified by FPLC chromatography as described previously Recombinant GST-CDK4 and GST-cyclin D1 were expressed in E. coli (BL21 DEA3) following induction with 1 mM IPTG overnight at 20 °C and purified by FPLC chromatography on a GSTrap HP 5 mL column  equilibrated in buffer A . GST-tagged proteins were eluted with buffer A containing 50 mM glutathione (Euromedex), and then further injected onto a desalting column  equilibrated in buffer A to eliminate free glutathione. All proteins were expressed and purified freshly and their purety was verified by SDS-PAGE.Recombinant GST-cyclin A, GST-cyclin E and GST-cyclin Y were expressed in Fluorescence titration assays were performed in 96-well plates in a thermostated chamber  at 30 °C in 200 μL phosphate buffer saline using 200 nM fluorescently-labelled peptides or proteins. The fluorescence emission intensity of TP2-Rho-labelled peptides was recorded at 614 nm following excitation at 510 nm. The fluorescence emission intensity of mant-ATP was measured at 356 nm following excitation at 448 nm. Data analysis was performed using the GraFit Software (Erathicus Ltd) and curve fitting was performed using a quadratic equation, as described previously 2.Non-small cell lung cancer A549, H358 and PC9 cell lines used in this study. Cell culture media, serum and antibiotics were purchased from Life Technologies. All cell lines were cultured in RPMI + Glutamax supplemented with 10% FCS, 100 units/mL penicillin  and 100 µg/mL streptomycin at 37 °C in an atmosphere containing 5% COCell extracts were prepared in PBS lysis buffer containing PBS (Sigma), pH 7.4, 150 mM NaCl, 0.2% NP40, 1 mM EDTA, 2 mM PMSF, CompleteTM protease inhibitors (Roche), and normalized following spectrophotometric dosage at 280 nm.For cell proliferation/viability assays, cells were seeded in 96-well plates at 4,000 cells/well in 100 µL medium. 24 h later, cells were treated in quadruplicate with different concentrations of small molecule inhibitors or peptides (from 10 nM to 20 µM). Stock solutions of drugs were prepared in DMSO and freshly diluted in PBS to the desired concentration prior to use, then added onto unsynchronized cells cultured to subconfluency (60-70%), which were then further incubated for 24, 48 or 72 h. Cell proliferation was determined by crystal violet assay following treatment with drugs. Cells were washed with PBS, fixed with 3.7% formaldehyde for 10 min and then incubated with 0.1% crystal violet dye for 30 min. After rinsing, crystals were dissolved in 10% acetic acid and viability was determined by measuring absorbance at 595 nm.Cell extracts (30 µg) were separated on 12.5 % polyacrylamide gels, then electrotransferred onto PVDF membranes for Western blotting. Membranes were blocked with 5% BSA/TBS and then probed over night at 4 °C with antibodies. Anti-phospho-Rb (S807/811-D20B12), anti-CDK4 (D9G3E) and anti-actin (4967S) antibodies were purchased from Cell Signalling. Anti-cyclin D1 (sc-8396), anti-CDK4 (sc56361), anti-cyclin A (sc-751), anti-cyclin B (GNS1), and anti-cyclin E (sc-247) were purchased from Santa Cruz. Secondary anti-mouse (NXA931) and anti-rabbit (NA934) peroxidase conjugated antibodies were from Amersham.The P2shortA stapled peptide was immobilized on activated CNBr resin (10-6 mole of peptide/0.3 g resin)  then saturated with PBS/1% BSA for 1 h at 4 °C prior to incubation with A549 cell extracts (60 µg/10 µL) for 1 h at 4 °C. The resin was then washed three times with PBS and boiled in Laemmli buffer for SDS-PAGE and Western blotting.2O (1 μg/mL) for 5 min, and cells were washed in water and coverslips were mounted on glass slides in Mowiol. Fluorescent cells were observed with a Leica DM6000 microscope equipped with a CoolSnap HQ2 camera and piloted by MetaMorph software. Excitation band / dichroic / emission band filters for imaging fluorescent signals were as follows: Hoechst: 340-380/400/450-490 nm; TAMRA using the Rhodamine filters: Cyanine 5.5 using the Cy5 filters 590-650/660/662-737 nm; Alexa488-conjugated antibodies using the YFP filters.TAMRA or Cyanine 5.5-labelled peptides were overlaid onto cultured cells grown to 50-60% confluency in DMEM 10% serum. Cells were then extensively washed with PBS and fixed with 3.7% formaldehyde/PBS for 10 min, washed twice and stained with Hoechst 33342 (Sigma) and either directly mounted onto glass slides in Mowiol for microscopic analysis or processed for indirect immunofluorescence. Following fixation, cells were incubated for 2 h in blocking buffer  and indirect immunofluorescence was performed by incubating cells overnight at 4 °C with anti-CDK4  or anti-cyclin D1  diluted 1/500 in blocking buffer. Cells were washed four times with PBS for 5 min and then incubated for 1 h at room temperature with Alexa488-conjugated anti-rabbit antibody  (diluted 1/500) followed by one wash in PBS for 5 min. Finally nuclei were stained with Hoechst 33342 (Sigma) diluted in HCells were trypsinized and fixed in 70% ice cold ethanol, washed in PBS and resuspended in PBS-1% BSA-50 mM citrate with 25 µg/mL RNase A for 15 min at 37 °C, supplemented with 25 µg/mL of propidium iodide for 30 min at room temperature. For each condition 20000 events were analysed by Gallios (Beckman Coulter) and data analysis was performed using FlowJo Software.All animal experiments were performed in agreement with the European Economic Community guidelines and the “Principles of Laboratory Animal Care” (NIH publication N 86-23 revised 1985). Animal experiment studies were approved through institutional guidelines and by the European Community for the use of experimental animals (authorization to experiment APAFIS#5738-2016062010163562 v3).6 H358 or A549 cells in 1X PBS. Tumour size was measured twice a week using a caliper, and the tumour volume was calculated as follows: length×(width)2×0.4.Female NMRI nude mice  were injected subcutaneously in the flank with 10×106 cells/50 µL. Six-week-old female NMRI nude mice  were anesthetized (isoflurane/air 4% for induction and 1.5% thereafter), and cells were inoculated in the lungs via the airways as previously described in vivo bioluminescence imaging  was used 10 min after the intraperitoneal injection of 10 mg/mL D-Luciferin  to monitor the quality of the implantation and to follow orthotopic growth Luciferase-modified human H358 NSCLC cells (H358-Luc cells) were suspended in medium with 10 mM EDTA and 5 mg/mL Matrigel (BD Biosciences) at 5.103, or to mice with orthotopic bioluminescent H358 lung tumours detected 5 weeks after implantation. Anesthetized mice (isoflurane/air 4% for induction and 1.5% thereafter) were injected intraperitoneally or intravenously via the tail vein with 200 µL of 8 µM (A549) or 10 µM (H358) Cy5.5-labeled P2-short in 5% glucose and 1% DMSO. Intrapulmonary administration of Cy5.5-labeled P2-short was performed using a nebulizing IA-1C Microspayer  connected to a FMJ-250-high-pressure syringe  containing 50 µL of 10 µM Cy5.5-labeled P2-short in 5% glucose and 1% DMSO, and as previously described Cy5.5-labeled P2-short was administered to healthy mice, or mice bearing subcutaneous H358 or A549 tumours when they reached ~250 mm50 μL of blood were sampled from the tail vein of healthy mice before Cy5.5-labeled P2-short administration and at different times after, centrifuged  and 10 µL of plasma were used for fluorescence imaging.3D-fluorescence acquisitions were performed 24 h after intravenous or intrapulmonary administration, with the continuous-wave fluorescence-enhanced diffuse optical tomography system TM  that excites fluorescence at 680 nm and detects the emitted light at wavelengths greater than 700 nm 2D-fluorescence images were acquired after administration using the Fluobeam 700ex vivo imaging using Optima Photon Imager optical imaging system (Biospace Lab). Semiquantitative data were obtained using the Wasabi® software  by drawing regions of interest (ROIs) on the different organs and were expressed as the number of Relative Light Units per pixel per unit of exposure time and relative to the fluorescence signal in the skin.Mice were sacrificed at different times, and some organs, plasma and tumours were collected for via the tail vein three times a week for three weeks under anesthesia (isoflurane/air 4% for induction and 1.5% thereafter) with 200 µL at 0.9 mg/kg, or administered intrapulmonary two times a week for three weeks under anesthesia with 50 µL at 0.45 mg/kg. Intrapulmonary administration of P2-short was performed using a catheter connected to a FMJ-250-high-pressure syringe  and introduced into the trachea of the animals using a dedicated laryngoscope. Mice were observed and weighed three times a week. Tumour growth was followed by in vivo bioluminescence imaging (IVIS Kinetic). After 3 weeks, mice were sacrificed, and blood samples were collected by cardiac puncture for biochemistry analysis (Vet16MScan II). Lungs were collected for immunohistochemistry analyses.Five weeks after the inoculation of H358-Luc cells, when bioluminescent orthotopic tumours were detected, nude mice were randomly divided into 6 groups. Mice were treated with vehicle , Abemaciclib (n = 10), or P2-short administered intravenously (n = 10) or intrapulmonary (n = 10), or treated with a combination of Abemaciclib and P2-short . Mice were daily orally treated for three weeks with 10 mg/kg Abemaciclib. P2-short was either administered Lungs were frozen and sections of a 7 μm thickness were stained with hematoxylin and eosin (HE) or fixed for immunohistochemical staining.Lung frozen sections were fixed with acetone and incubated with Ki67 antibody  for 2 h à room temperature, followed by incubation with goat anti-rabbit-HRP antibody (Dako) and DAB substrate (Dako). To determine proliferative index, total and Ki67 positive tumour epithelial cells were scored on two to eight fields per mouse in three or four mice per group, and reported as percentage of Ki67 positive cells. Results are reported as proliferative index in each field and each treatment group.TM HRP Polymer anti-rabbit IgG and ImmPACTTM NovaREDTM diluents (Vector Laboratories). Lung sections were imaged on an Olympus BX41 microscope. Total and p-RB positive tumour epithelial cells were scored on two to twenty-nine fields per mouse in three or four mice per group, and reported as percentage of p-RB positive cells in each field and each treatment group.For p-RB staining, lung sections were fixed with 4% paraformaldehyde and heat mediated antigen retrieval with Tris/EDTA buffer, pH 9.0 was performed. Phospho-RB S807 antibody  was incubated overnight at 4 °C, followed by incubations with ImmPRESSposthocs tests. P values ≤ 0.05 were considered statistically significant. All analyses were performed using the Graphpad Prism software.Statistical comparisons were made using Friedman test or Kruskall-Wallis test, with Dunn's multiple comparisons KRAS mutants and CDK4, we asked whether expression of cyclin D1 and CDK4 might be associated with the KRAS mutation in lung cancer patients. To this aim we first performed cyclin D1, CDK4 and pRb immunohistochemistry in a series of 215 NSCLC surgical resections. Clinical and molecular data are listed in Table S1.Since a synthetic lethal interaction has been reported between Figure  andTable S1). There was however no significant difference in CDK4 expression between ADC and SCC samples . A concomittant positive cyclin D1 and CDK4 expression, defined both by more than 10% of positive cells, was observed in 76% of the cases (Table S2), with a strong correlation . pRb expression was observed in 24% of the cyclin D1 positive cases and 30% of CDK4 positive cases (Table S2). A strong correlation between CDK4 and pRb , and a trend between cyclin D1 and pRb expression in ADC , but no significant correlation in SCC  were observed. Interestingly, a high level of cyclin D1 was significantly associated with KRAS exon 2 mutation: 89% of the KRAS mutant cases  highly expressed cyclin D1 . Taken together these data highlight a correlation between cyclin D1/CDK4 expression and KRAS mutation in lung ADC, suggesting enhanced activation of CDK4/Cyclin D in KRAS mutant lung cancer.High cyclin D1 expression was predominantly observed in ADC, whereas high pRb levels were more frequent in SCC  (48L49P50I51S52T53V54R55E56V57A58L59L60R61R62L63) . This peptide is disordered in phosphate buffer, but become partially helical upon addition of 25% TFE (helicity of 46%), indicating its propensity to fold into an α-helix in the appropriate environment .The crystal structure of CDK4/cyclin D1 shows that the primary interaction between these partner proteins is mediated by the C helix in the N-terminal lobe of CDK4, also known as the PISTVRE helix, and the complementary α5-helix of cyclin D1  and implementation of the Drugscore PPI webserver 49, I51, V54, R55V57 and R61 as critical residues positioned along the face of the C helix involved in the interaction with cyclin D1 . Residues S52, E56, L60 and E64, that lie on the opposite side of the C helix and point towards the core of CDK4 , were therefore substituted by unnatural alkenyl amino acids at the position , followed by a ring closing metathesis reaction using ruthenium as catalyst to form the staple, to generate four stapled peptides spanning residues 48-67 . P1, P2 and P3 comprise a unique staple, whereas P4 comprises two staples. Stapled peptide P3 was poorly soluble and was not investigated any further. Stapled peptides P1, P2 and P4 displayed typical α-helix CD profiles in phosphate buffer (two negative bands at 208 nm and 222 nm and a positive band centered at 195 nm), indicating that incorporation of staples in linear CDK4 C peptide induced α-helical conformation .To generate a stable and well-structured peptide derived from the CDK4 C helix, which would mimick its conformation in its native protein environment, and therefore constrain the peptide into an α-helix preserving the key residues required for interaction with Cyclin D1, we selected amino acids that were not involved in its interaction with cyclin D1 to introduce hydrocarbon staples. Close analysis of the crystal structure of CDK4/cyclin D1 complex Figure ). We further synthesized a shorter derivative of P2, named P2short, which lacks the four C-terminal residues (E64A65F66E67), and bears an N-terminal cysteine so as to enable site-specific fluorescent labelling for titration and cellular internalization experiments , P2shortA, in which the cysteine was substituted by an alanine to prevent peptide dimerization through cysteine oxidation, and an unstapled analog, CDK4 Chelix short . Both P2short and P2shortA displayed characteristic α-helical CD signatures, with a significant increase in helical content compared to P2, with 77% and 74% helical content, respectively, associated with deletion of the C-terminal tetrapeptide acidic patch in P2. As expected, substitution of the N-terminal cysteine by an alanine residue had no significant structural effect. CDK4 Chelix short peptide was disordered in solution . Finally, to address the specificity of the interaction between CDK4-derived peptides and their target, as well as their anti-proliferative effect, two unrelated control peptides were synthesized: a stapled peptide of the same length as P2short (Ctrl stapled peptide) and an unstapled peptide (Ctrl peptide).Stapled peptide P2 displayed the strongest tendency to form a helix (55%) compared to P1 (16%) and P4 (26%) . In comparison, P2 displayed 2-3 fold weaker affinity for C4D, with an average Kd of 245 +/- 84 nM, whereas P2short peptide exhibited 2-3 greater affinity for the C4D peptide with an average Kd of 56 +/- 19 nM, . Taken together, these results indicate that stapling did not improve the affinity of peptides derived from the CDK4 C helix for the complementary peptide derived from α5 helix of cyclin D1, but that removal of the C-terminal acidic patch improved the affinity of the stapled P2short.We first labelled a peptide derived from the alpha 5 helix of cyclin D1 (C4D peptide) to investigate its interactions with the peptides derived from CDK4 C helix. Titration of the TP2-Rho-labelled C4D peptide with unstapled CDK4 C helix peptide and stapled peptides P1, P2, P2short and P4 induced significant fluorescence enhancement in all cases and curve fitting and calculation of dissociation constants (Kd) yielded similar values for the CDK4 C helix peptide, P1 and P4 peptides  . Interestingly titration of TP2-Rho-P2short peptide with GST-cyclin D1 induced only 3-fold fluorescence enhancement inferring that positioning of the probe conjugated to the constrained peptide upon binding to the recombinant cyclin most likely differed from that of the probe conjugated to the unstructured CDK4 C helix peptide. Curve fitting and calculation of dissociation constants revealed that the affinity of the P2short peptide for GST-cyclin D1 was four times greater than that of CDK4 C helix peptide, with average Kd values of 12 +/- 6 nM and 43 +/- 24 nM, respectively . In contrast, TP2-Rho labelled control stapled peptide, did not induce any significant change in fluorescence upon titration with GST-cyclin D1.Conversely, the CDK4 C helix peptide and the P2short peptide were both labelled with TP2-Rho on their N-terminal cysteine. Fluorescence titration of the unstapled peptide with GST-cyclin D1 induced significant fluorescence enhancement (8 fold) indicating that it bound GST-cyclin D1, as expected, whereas GST alone had no significant incidence . Taken together these experiments show that P2short peptide, which is derived from a highly conserved region in CDKs, recognizes an equally highly conserved interface in cyclins involved in cell cycle progression.Based on the affinity constants and physicochemical properties of the peptides (helicity and solubility), we selected the stapled peptide P2 and P2short for further characterization of their specificity for cyclin D1 and their inhibitory potential in NSCLC cells. Fluorescence titration of TP2-Rho-labelled P2short revealed very similar affinity for cyclins D1, A and E, but significantly lower affinity for the less conserved cyclin Y, with average Kd values of 12 +/- 6 nM, 25 ± 15 nM, 10 ± 7 nM, and 47 ± 40 nM, respectively Figure ). Control titration of fluorescent mant-ATP with P2short peptide alone did not reveal any significant interaction. These results reveal that P2short can indeed bind cyclin D1 precomplexed to CDK4, and that this enables CDK4 to bind ATP more tightly, suggesting a possible conformational effect on CDK4.Cyclin binding to a CDK induces a rotation of its N-terminal lobe, therefore aligning the ATP binding site with the catalytic cleft Figure A. In contrast, P2short internalized into A549 cells and exhibited very intense cytoplasmic fluorescence emission within 1 h Figure A. Indirect immunofluorescence experiments revealed that P2short partially colocalized with CDK4 and cyclin D1 in the cytoplasm Figure B. Moreover comparison of CDK4 and cyclin D1 localization in untreated cells and cells treated with P2short did not reveal any differences, indicating that P2short did not directly affect the subcellular localization of its targets.The ability of TAMRA-labelled CDK4 C helix and P2short peptides to enter A549 cells was investigated by fluorescence microscopy. As expected for most peptides, the CDK4 C helix peptide was unable to cross the cell membrane Figure ). The inhibitory efficacy of P2short was further assessed in NSCLC proliferation assays, and compared to the ATP-competitive CDK4/CDK6 inhibitor Abemaciclib.We therefore investigated the effect of P2short peptide on CDK4 activity in NSCLC cells, by addressing its ability to inhibit phosphorylation of p107Rb, the main substrate of CDK4. P2short effectively inhibited phosphorylation of p107 Rb in A549 cells, and to a greater extent than the ATP-competitive inhibitor of CDK4 Abemaciclib, after both 24 h and 48 h, and in a dose-dependent fashion . Abemaciclib induced more cytotoxicity than P2shortA . Calculation of IC50 values for P2shortA and Abemaciclib in A549 cells yielded 10.81 ± 0.26 µM and 2.72 ± 2.21 µM, respectively ( and S2E). In contrast the longer P2 stapled peptide had no inhibitory effect whatsoever, nor did the Ctrl stapled peptide Figure B. Interestingly, coadministration of 10 μM P2short with a sub-IC50 concentration of Abemaciclib (500 nM) revealed a synergistic effect between these two compounds Figure C, with more than 80% inhibition of A549 proliferation, in agreement with in vitro studies described above in which ATP analog binding to CDK4/cyclin D1 was increased by P2short . Finally, comparative proliferation inhibition studies performed on several NSCLC cell lines with mutant KRAS (A549 and H358) or wild-type KRAS (PC9) revealed that P2short was more potent than Abemaciclib in all cases Figure D, 5E.To avoid dimerization in solution through disulfide bond formation and any possible inconsistencies that could arise over time in cellular studies, we resorted to use the analog P2shortA. Comparison of P2short and P2shortA in proliferation assays revealed very similar results, inducing a dose-dependent inhibition of A549 cell proliferation with a significant effect at 5-10 μM .Since CDK4 is essentially active during the G). In contrast, P2shortA administered intravenously circulated at 5 h and was observed in the liver and kidneys after 24 h in mice with A549 or H358 subcutaneous tumour, suggesting hepatic and renal elimination of the peptide . Based on these results, intraperitoneal injection was not continued, and the biodistribution of Cy5.5-labeled P2shortA following intravenous administration was compared to intrapulmonary administration using orthotopic mouse models of human NSCLC and healthy mice.The behaviour of Cy5.5-labeled P2shortA was first assessed in mice with human NSCLC tumour following intravenous, intraperitoneal, or intrapulmonary administration. P2shortA administered intraperitoneally was mainly detected in the abdomen of mice with A549 subcutaneous tumour, suggesting the non-specific binding of the peptide in the peritoneal cavity . Thoracic P2shortA fluorescence remained stable over 24 h after intrapulmonary administration and slowly decreased until 72 h . No significant difference was observed between healthy and mice with H358 lung tumours. The fluorescence of the lungs ex vivo was elevated, remained constant over the next 24-48 h, and was similar in mice bearing tumours or not . Moreover P2shortA fluorescence signal co-localized with the bioluminescent signal in lung tumours, inferring that intrapulmonary P2shortA indeed reached the lung tumours . No significant fluorescence signal was observed in organs other than lungs , nor in plasma , thus confirming the strong retention of the P2shortA in the airways.Cy5.5-labeled P2shortA fluorescence was detected in the thoracic region of healthy mice or mice with H358 orthotopic lung tumour, by ). The fluorescence of the lungs ex vivo showed little P2shortA signal 5 h after intravenous injection, ten times lower than after intrapulmonary administration, and no signal at 24 h . In addition, no colocalization was observed between the weak P2shortA fluorescence signal and the bioluminescent signal in the lung tumours . P2shortA fluorescence was detected in several organs 5 h following intravenous injection, indicating that the peptide circulated largely in the different organs without any specific targeting . This was further evidenced by the detection of P2shortA in plasma samples of healthy mice at different times following intravenous injection . P2shortA was also detected in the liver and kidneys at 24 h .In contrast, P2shortA administered intravenously rapidly increased in the thorax at 2 h and then decreased over 24 h , Abemaciclib , P2shortA , or a combination of Abemaciclib and P2shortA (n=10) .Based on the results of the biodistribution and cell proliferation studies, we performed therapeutic studies to address the Figure ). Interestingly, the combination of Abemaciclib and P2shortA significantly inhibited tumour growth as compared to the control or P2shortA-treated groups . In contrast, P2shortA administered intravenously  alone or in combination with Abemaciclib showed the same modest reduction of tumour growth, similar to Abemaciclib alone .The concentration of Abemaciclib was chosen based on a preliminary experiment, so as to have a sub-therapeutic effect on the H358 orthotopic lung tumour model. Bioluminescence follow-up of the tumours indicated that the low concentration of Abemaciclib slightly reduced tumour growth, whereas P2shortA had no effect , and no significant change in serum biochemical values was observed irrespective of treatments . Taken together these results reveal that intrapulmonary P2shortA restrict tumour growth over time when combined with Abemaciclib, whereas intravenous P2shortA failed to have any therapeutic effect probably because it was not retained long enough in the tumour at the concentration used.All treatments were well tolerated. No significant weight loss was observed in the H358 tumour-bearing mice . Tumour sections stained with Ki67 and pRb revealed a significant reduction of both Ki67-positive cells and pRb-positive cells in tumours following intrapulmonary administration of P2shortA combined with Abemaciclib, indicative of a reduced proliferative index and reduced CDK4 activity, respectively . In contrast intrapulmonary administration of P2shortA or Abemaciclib alone did not reduce Ki67 or pRb levels, indicating that, at least in this experimental design, and at the concentrations of treatments chosen, tumour growth remain unaffected . Likewise, intravenous administration of P2shortA either alone, or in combination with Abemaciclib did not promote significant reduction of Ki67-positive cells . In contrast, intravenous administration of P2shortA with Abemaciclib led to a much broader distribution of pRb-positive cells . Taken together these results confirm that intrapulmonary administration of P2shortA inhibits proliferation when it is combined with Abemaciclib in H358 orthotopic lung tumours in vivo by effectively inhibiting Rb phosphorylation, due to dual targeting of CDK4/cyclin D1.Analysis of tumours by Hematoxylin and eosin (HE) staining did not reveal any lung damage following intrapulmonary P2shortA administration, but pulmonary toxicity will have to be formally assessed to confirm the safety of P2shortA , which has previously been reported to be optimal for cellular internalization of stapled peptides 1, as would be expected from a CDK4/cyclin D1 inhibitor, and observed for Abemaciclib. Furthermore fluorescence microscopy and indirect immunofluorescence studies showed that P2short partially colocalized with CDK4 and cyclin D1, but did not induce subcellular relocalization of these proteins. However, P2short indeed induced a decrease in Rb phosphorylation to a similar extent as Abemaciclib, and also inhibited proliferation of NSCLC cell lines. Taken together, our results indicate that P2short indeed affects the ability of CDK4/cyclin D1 to phosphorylate its substrate. However, since P2short binds several cyclins, it might also affect the kinase activities of several CDKs, thereby explaining the overall inhibition of cell proliferation, but the lack of an observable accumulation of cells in a specific cell cycle phase.Although the P2short peptide was derived from CDK4 and interacted with cyclin D1 with high affinity, we also found it bound other cyclins in vitro and in cell extracts. This is not surprising since the amino acids involved in the α5-helix that interacts with the C helix of CDKs are highly conserved. This lack of selectivity for cyclin D1 was also evidenced by the flow cytometry experiments, which did not reveal any accumulation of cells treated with P2shortA in Gin vivo, it did however completely inhibit growth of orthotopic NSCLC tumours in mice. This original combined effect between a therapeutic peptide targeting an essential PPI and a SMKI  has a very strong potential for reducing the doses required to treat lung cancer. Indeed, it has been shown that drug combinations achieve favorable outcomes such as enhanced efficacy, decreased dosage, reduced or delayed development of drug resistance indicative of the beneficial combination of drugs with different targets or mechanisms of action 1, where the availability of monomeric CDK4 and cyclin D1 are much greater than in other phases of the cell cycle, since CDK4 is degraded during mitosis and resynthesized in G11 than during the other phases of the cell cycle where the CDK4/cyclin D1 complex is already assembled. Our in vitro studies have shown that P2short enhances the affinity of recombinant CDK4/cyclin D kinase for ATP, suggesting that P2short might in turn increase Abemaciclib binding and efficacy. The interaction between P2short and cyclin D1 might not preclude CDK4/Cyclin complex formation, since we did not observe either subcellular delocalization of either partner upon treatment with P2short, nor disruption of the recombinant complex in vitro (data not shown), but might instead enact on the kinase complex conformation, thereby promoting increased binding of Abemaciclib in the ATP pocket, in line with our experimental results with the fluorescent ATP analog. Hence by treating cells with an ATP-competitive inhibitor that accumulates cells in G1, this would improve accessibility and targeting of P2short, which would in turn sensitize CDK4 to Abemaciclib.Despite an antiproliferative effect on several NSCLC cell lines, intravenous injection of P2short failed to inhibit lung tumour growth in mice, most likely because the concentration retained at the tumour site was too low. In contrast, direct administration of P2short into the airways was associated with its strong accumulation in the tumours, as well as throughout the healthy parts of the lungs, but not in any other organs. Intrapulmonary administration of antitumoral agents can enhance their concentration at the targeted lung cancer site, and has already been successfully reported for lung cancer treatment in vitro and in vivo, constitutes a promising therapeutic strategy to target the hyperactivity of CDK4 in lung cancer with mutant KRAS. In addition, it is very unlikely that an essential PPI will undergo mutations leading to emergence of resistance, in contrast to the ATP pocket of protein kinases as classically observed in patients following treatment with ATP-competitive drugs such as Erlotinib or Gefinitib. Our stapled P2short peptide thus constitutes an attractive means of sensitizing CDK4 to ATP-competitive therapeutics for the treatment of KRAS-mutant lung cancer patients.In conclusion, the strategy described here consisting in targeting an essential protein/protein interaction, mediated by two α-helices, with a stapled peptide derived from one of these interfaces, for which we have provided a convincing proof-of-concept by successfully targeting CDK4/Cyclin D


In this study, periphytic biofilms (epiphyton and epixylon) were used to bioremediate heavy metals (HMs) and to biodegrade PE under high  methane (CH4) doses. Both periphytic biofilms were actively involved in methane oxidation, HMs accumulation and PE degradation. Epiphyton and epixylon both completely removed Pb2+ and Cd2+ at concentrations of 2 mg L−1 and 50 mg L−1, respectively, but only partially removed these HMs at a relatively higher concentration (100 mg L−1). Treatment containing 12% 13CH4 proved to be most effective for biodegradation of PE. A synergistic effect of HMs and PE drastically changed microbial biota and methanotrophic communities. High-throughput 16S rRNA gene sequencing revealed that Cyanobacteria was the most abundant class, followed by Gammaproteobacteria and Alphaproteobacteria in all high-methane-dose treatments. DNA stable-isotope probing was used to label 13C in a methanotrophic community. A biomarker for methane-oxidizing bacteria, pmoA gene sequence of a 13C-labeled fraction, revealed that Methylobacter was most abundant in all high-methane-dose treatments compared to near atmospheric methane (NAM) treatment, followed by Methylococcus. Methylomonas, Methylocystis, Methylosinus, and Methylocella were also found to be increased by high doses of methane compared to NAM treatment. Overall, Cd+2 had a more determinantal effect on methanotrophic activity than Pb2+. Epiphyton proved to be more effective than epixylon in HMs removal and PE biodegradation. The findings proved that both epiphyton and epixylon can be used to bioremediate HMs and biodegrade PE as an efficient ecofriendly technique under high methane concentrations.Priority pollutants such as polyethylene (PE) microplastic, lead (Pb Periphytic biofilms consist of different kinds of microorganisms including cyanobacteria, algae, diatoms, and protozoans, along with detritus, and are found either attached to submerged surfaces or floating freely in freshwater ecosystems ,2,3. TheGenerally, the microbial consortium of the periphytic biofilm has a relatively impervious nature, which tends to maintain internal processes and resist outer physicochemical variations in the surrounding environment ,12. Due Pollutants removal by periphytic biofilms involve the following three mechanisms: biosorption, bioaccumulation, and biodegradation ,18,19. PMethylobactor and Methylococcus and type II (Alphaproteobacteria) Methylocystic and Methylocella were found to be the most abundant methanotrophic species during plastic degradation [A report on plastic pollution speculated that, in 2050, there will be more plastic in the marine ecosystem than fish . This plradation . Despiteradation .2+ and Cd2+ are also considered toxic and perilous to the environment [2+ and Cd2+ extensively, thus causing aqueous pollution by dumping these metals into the aquatic ecosystem [Methylococcus capsulatus (Bath) efficiently bioremediated chromium (Cr6+) over a wide range of concentrations (1.4−1000 mg L−1) [2+ and Cd2+ by periphytic methanotrophs.HMs are also persistent in the aquatic environment and can bioaccumulate in the human body, causing carcinogenic effects . Similarironment ,29,30. Vcosystem . Periphycosystem , or hypecosystem . In our cosystem . A singl mg L−1) . However2+ and Cd2+) and biodegrade polyethylene (PE) under high methane doses. The specific aims of the current study were (1) to investigate the biodegradation of PE and bioremediation of Cd2+ and Pb2+ by two different kinds of periphytic biofilms, i.e., epiphyton (EPP) and epixylon (EPX); (2) to use the stable isotope probing (SIP) technique to study the impact of high methane dose on degradation and microbial resistance against HMs; (3) to investigate the kind of methanotrophs responsible for metal remediation and PE degradation in lake water; and (4) to assess the microbial community shift of epiphyton and epixylon biofilms as a result of their exposure to PE, Cd2+, and Pb2+.Considering the capability of periphytons to remove HMs and degrade plastic from the aquatic ecosystem and the presence of a methanotrophic community in these periphytic biofilms, we aimed to evaluate the capability of periphytic biofilms to bioremediate HMs  to the microcosm adversely affected the PE biodegradation capability of epiphyton and epixylon, but high-methane-gas amendment stimulated the biodegradation. Previously, different microbial communities were reported to grow and adhere to the surface of micro- and nano-plastic surfaces [2+ showed more degradation of PE compared to Cd2+ for both epiphyton and epixylon biofilms.Epiphytic and epixylic biofilms were found to colonize and grow on the surface of PE pellets compared to the smooth surface of the control pellet. SEM photos of 20-day samples depicted pitted, rough, cracked, and scratchy surfaces . The SEMsurfaces ,35 to erThe Biolog experiment showed that epiphytic and epixylic biofilms produced darker colors by feeding on different substrates revealing their higher microbial activity . Diversi13C, EPP + 13C and treatments with 2 mg L−1 of metals (Pb2+ and Cd2+) for both epiphyton and epixylon biofilms consumed headspace methane within 14 days of injection  treatments showed high bundance . Both ep−1 and 50 mg L−1 of Pb2+ and Cd2+ completely removed HMs, but treatment with EPP + 13C + M1C3 only removed 84% of Pb2+, whereas EPP + 13C + M2C3 showed a 78% Cd2+ removal efficiency after 20 days of incubation  in terms of time duration compared to epixylon. Similarly, epiphyton took less time to completely remove Pb2+ compared to Cd2+. Treatments EPP + 13C + M1C1 and EPP + 13C + M1C2 completely removed Pb2+ within 8 and 14 days, respectively, whereas EPX + 13C + M1C1 and EPX + 13C + M1C2 took 10 and 16 days to remove Pb2+, respectively. According to Faheem et al., high methane oxidation could enhance bioremediation of Pb2+ and Cd2+ [13C + M1C2 + PE and EPP + 13C + M2C2 + PE completely remediated Pb2+ and Cd2+ in 16 and 18 days, respectively, compared to treatments without PE pellets. EPX + 13C + M1C2 + PE and EPX + 13C + M2C2 + PE adsorbed Pb2+ and Cd2+ in 18 and 20 days, respectively. Li et al. [Similarly, EPX + ectively b. Epiphyi et al.  found th2+ and Pb2+) inhibited the biodegradation of PE (~3–5%), whereas Cd2+ addition resulted in lower biodegradation compared to Pb2+. Epiphyton showed better results compared to epixylon. The overall reduction in molecular weight was obvious in the values of polydispersity (Mw/Mn), which also decreased. The decrease in molecular weight is an indication of the depolymerization of long chains found in PE as well as the formation of small fragments by periphytic biofilms [GPC was used to evaluate the impact of metal addition and methane oxidation on the biodegradation of PE after 20 days of treatment. The results of GPC are shown in biofilms .−1 represents the O–H bond of the oxidized methylenic carbons (CH2). The other peaks found in the control at 2914, 2846.90, 1463, and 728 cm−1 represent −CH bond stretches and bends, whereas the peak at 1376 cm−1 represents C–H bending in the polymer structure [The spectra obtained by ATR-FTIR for different PE samples collected after 20 days showed differences in numbers and intensities of peaks . In the −1 and 3184.86 cm−1 disappeared in all the treatments except in EPP + NAM + PE, where 3394 cm−1 shifted to 3389.21 cm−1. In the EPP + 13C + PE treatment  and C–O stretch, respectively. Two new peaks appeared in EPX + 13C + PE , indicating the formation of carbonyl bond (−C = O−). The samples supplied with near atmospheric methane (D) showing the lowest weight loss had a small new peak at 1055.35 cm−1 representing the formation of an alkoxy group (C–O), which indicates the hydrolysis of PE [In the samples inoculated with epiphyton, some new peaks were seen in ATR-FTIR . The peareatment B, two ne13C + PE B at 1564as added A in epixis of PE .13C + PE > EPX + 13C + M1C2 + PE > EPX + 13C + M2C2 + PE > EPX + NAM + PE)  . PrevioupmoA genes, respectively. Addition of HMs and PE alone and in combination significantly inhibited total microbiome and methanotrophic communities. EPP+ 13C treatment showed 1.23-, 2.30-, 1.80-, and 2.90-fold increases in 16S rRNA gene copy numbers compared to EPP + 13C + M1C2, EPP + 13C + M1C3, EPP + 13C + M2C2, and EPP + 13C + M2C3, respectively  compared to control [EPP + 13C) obtained from isopycnic centrifugation of SIP DNA was subjected to QPCR and clearly indicated active methanotrophic cell propagation in all high methane dose treatments, except for NAM treatments, which showed no significant active propagation. A peak shift of relative pmoA abundance of the heavy fraction was clearly visible around a buoyant density of 1.735 g mL−1 in all high methane dose treatments for both epiphyton , respectively. Within epixylon treatments, pmoA gene copy number was also reduced to 9%, 16.50%, and 17.19% in EPX + 13C + PE, EPX + 13C + M1C2 + PE, and EPX + 13C + M2C2 + PE, respectively, compared to the epixylon control (EPX + 13C). From the results of QPCR of pmoA, we further confirmed that copy numbers of 16S rRNA gene were also reduced due to the determinantal effect of HMs and PE. Brennecke et al. [2+ proved to be more toxic to methanotrophic communities than Pb2+. Methane oxidizers presented relatively discrete metal resistance patterns by showing resistance to different metals but proved to be sensitive to cadmium [Epixylon treatments EPX + ectively a. Similae et al.  found th cadmium . Methano cadmium .13C had 1.13%, 8.65%, 13.48%, 2.14%, 7.17%, and 9.52% more OTUs of Cyanobacteria than EPP + 13C + M1C1, EPP + 13C + M1C2, EPP + 13C + M1C3, EPP + 13C + M2C1, EPP + 13C + M2C2, and EPP + 13C + M2C3, respectively  adversely effected Alphaproteobacteria in EPP + 13C + M1C3 and EPP + 13C + M2C3 treatments by reducing its OTUs to 2.85% and 3.19%, respectively, compared to EPP + 13C. Alphaproteobacteria and Gammaproteobacteria were found to rapidly colonize the surface of marine plastics [Unlike Gammaproteobacteria, Alphaproteobacteria was favored by the presence of HMs, with treatments EPP + plastics . Pyroseqplastics .13C had 3.13%, 18.10%, 23.47%, 5.27%, 17.31%, and 13.65% more OTUs of Cyanobacteria than EPX + 13C + M1C1, EPX + 13C + M1C2, EPX + 13C + M1C3, EPX + 13C + M2C1, EPX + 13C + M2C2, and EPX + 13C + M2C3, respectively  were subjected to pmoA gene sequencing for the identification of active methanotrophs responsible for Pb2+, Cd2+ removal, and PE biodegradation.Based on complete removal of HMs and PE biodegradation under high methane doses, heavy fractions of control treatments of both biofilms (containing only 12% of methane), treatments containing 50 mg L13C fraction pmoA genes indicated that Type I methanotrophs outnumbered Type II in all high methane dose treatments. Type I Methylobacter sp. was the most abundant species in all treatments, but in the presence of HMs, methanotrophic abundance reduced by 16.30%, 14.28%, 7.82%, 22.27%, and 22.18% in EPP + 13C + M1C2, EPP + 13C + M2C2, EPP + 13C + PE, EPP + 13C + M1C2 + PE, and EPP + 13C + M2C2 + PE, respectively, compared to EPP + 13C  of metal salts were prepared using deionized water and diluted accordingly. All the chemicals and PE were purchased from Sigma-Aldrich  and were of analytical grade.The PE used in this study was 1–4 mm diameter spheroids in the form of pellets, which were mechanically resized to obtain 1000 µm pellets. The structure and properties of PE are described in Epiphyton (from the base of water lily flower plant) and epixylon (from the base of dock wooden poles) were isolated from Xuan Wu Lake, Nanjing, China, using standard isolation methods. The periphytic biofilms were transported to the laboratory under low temperature (4 °C) and were grown separately using modified woods hole culture (WC) media, as described previously . Briefly−1, ammonia 0.59 mg L−1, total nitrogen (TN) 2.10 mg L−1, total phosphorus (TP) 0.21 mg L−1, and pH 7.74; epixylon: nitrate 0.74 mg L−1, ammonia 0.63 mg L−1, TN 2.14 mg L−1, TP 0.25 mg L−1, and pH 7.79.The sampling sites for both epiphyton and epixylon biofilms were tested for their physicochemical properties, i.e., epiphyton: nitrate 0.69 mg LThe morphological changes in epiphyton and epixylon biofilms were determined before and after treatment with a high methane dose using scanning electron microscopy . Similarly, morphological changes in PE were also observed under SEM before and after treatment with a high methane dose and HMs. The Biolog™ plate technique  with different substrates was employed to assess the microbial activities of biofilms, as previously described . Briefly−1) of HMs  was employed in quintuplicate to these microcosms to assess the bioremediation potential of epiphyton and epixylon biofilms.We studied metals bioremediation and PE biodegradation in microcosms (sterile 120 mL serum bottles) containing 20 mL of suspended material (5 mL of suspended biofilms and 15 mL of modified WC medium), as previously described . A serie−1 each of Pb2+ and Cd2+), biofilms, and PE pellets were prepared. After the preparation of biofilm-treatment mixtures, microcosms were sealed with butyl stoppers and headspace 13CH4 and 12CH4 gases were employed to each microcosm. NAM concentration for low methane dose treatments and 12% methane for high methane dose treatments were used for all prepared treatments. The detailed experimental design is described in 2 and O2 according to atmospheric volumetric ratios.To evaluate the capability of biofilms to biodegrade microplastic, five replicates of each treatment were studied using a microcosm containing 5 mL of each biofilm and 15 mL of modified WC along with a pellet of PE. To determine the effect of HMs on the biodegradation potential and microbial diversity under high methane doses, four separate treatments containing HMs including (50 mg L13CH4 (>99% 13C-atom pure) was obtained from Cambridge Isotope Laboratories . Headspace CH4 concentration was measured using gas chromatography pro-2010  every 48 h. The incubation was stopped when the change in methane concentration was undetectable. PE samples were collected after changes in methane concentration were no longer detectable. Biofilms were collected from each treatment and stored at −20 °C for further investigation. For 13C-atom relative abundance, 1 g of vacuum-freeze-dried sample of each treatment was subjected to a Flash 2000 elemental analyzer  attached to a Delta V™ IRMS Advantage isotope mass ratio spectrometer . Inductively coupled plasma mass spectrometry (ICP-MS) was used to measure the concentrations of metals (Pb2+ and Cd2+). To avoid HMs precipitation, a few drops of concentrated HNO3 were added to avoid precipitation before metals detection.All microcosms were placed in a shaker incubator at 150 rpm with 16 h of light and 8 h of dark at 28 °C and pH was adjusted to 6.8, which is optimum for biofilm growth. The W˳ is the initial weight of the PE pellet and tW is the weight of PE after biodegradation.PE pellets were removed from the microcosms after 20 days of incubation with methane level ≤100 ppm and biodegradation by epiphyton and epixylon. Biofilm constituents were removed from pellets by deionized water and washed with ethanol. The pre-weighed PE pellets were weighed using Mettler Toledo AL54 four-decimal-place analytical lab balance . The percentage weight loss (%) for all treatments was calculated by the following equation (Equation (1)), as described earlier:−1 and a 500–4000 cm−1 range. PE samples from all treatments including the control (non-inoculated sample) were subjected to FTIR analysis.PE polymeric structural change after biodegradation under a high methane dose was recorded by Fourier-transform infrared spectroscopy coupled with attenuated total reflectance  with a scanning power with a resolution of 4 cmPE biodegradation was further confirmed through gel permeation chromatography (GPC) by evaluating the molecular weight changes. A PL-GPC PL50  system coupled with the triple mixed-b columns and an RI detector was used for this purpose.−1 with a column temperature of 160 °C. Agilent chemstation software A.10.02  was used for data analysis and Agilent  polystyrene standards were used for calibration curves.PE was solubilized in 1,2,4-tricholobenzene (TCB) and 0.15 butylated hydroxytoluene (BHT) at 160 °C and the same chemicals were used as eluents in GPC. The eluent flow rate was maintained at 1 mL min12CH4 from 13CH4 fractions of all treatments, as described earlier [The fast DNA spin kit for soil  was used to extract whole DNA from 500 mg biofilm samples from each treatment according to the manufacturer’s instructions. The quantity of extracted DNA was assessed by a NanoDrop ND-1000 UV-visible light spectrophotometer . Density gradient isopycnic centrifugation of total DNA was employed to isolate  earlier . PolyethpmoA genes from total DNA and fractionated DNA (1–15) were quantified using a PCR CFX96 Optical Real-Time Detection System  to ascertain the abundances of methane oxidizing bacteria (MOB) and incorporation of 13C into methanotrophic communities of biofilms. Thermal cycling conditions and the primer pair used are detailed in R2 values of 0.996 to 0.999.Methanotrophic biomarker 13C) CsCl fractions (buoyant density around 1.735 g mL−1) and pmoA gene were subjected to amplicon sequencing [12CH4-treated microcosms with similar buoyant density were also sequenced to compare contextual details.16S rRNA genes from total DNA as well as DNA extracted from the heavy  MultiClassifier was used to obtain taxonomic units at a similarity of 97% (or 3% cutoff) [Quantitative insights into microbial ecology (QIIME) pipeline key-quality control steps were applied to remove low quality sequences as previously described ,60. A to cutoff) .https://www.reproject.org/) for both periphytic biofilms. PE data were subjected to statistical analysis of variance with the p < 0.05 indicating statistical significance .Diversity indices such as operational taxonomic unit (OUT) richness, Chao1, Simpson index, Evenness index, and Shannon index were computed using the Vegan package in R 3.4.3 (2+ and Cd2+ bioremediation along with PE biodegradation potential. After 20 days of incubation under high methane doses and chronic HMs and PE exposure, the individual and combined effects were measured based on HMs adsorption by epiphyton and epixylon. Treatments with 2 mg L−1 and 50 mg L−1 in both periphytic biofilms consumed all headspace methane along with HMs, but 100 mg L−1 only partially removed HMs. We found that the mechanism of HMs bioremediation, methane oxidation, and PE biodegradation is dependent on HM concentrations. The synergistic toxic effect of HMs and PE is more detrimental and enhanced than the discrete effect on methane oxidation and microbial communities. Methane oxidation enhanced PE biodegradation but the effect was reduced by the presence of HMs. In the presence of HMs and PE, the diversity and abundance of microbiota of specific phylotypes changed. Methylobacter sp. was dominant in all treatments, but the relative abundance in the presence of HMs and PE was lower. The relative abundance of Methylococcus increased. All the identified methanotrophic biota were enhanced in high-methane-dose treatments compared to NAM treatments. From the results of the current study, we conclude that epiphyton is more effective than epixylon, and both epiphyton and epixylon biofilms are ecofriendly and can be used for the remediation of multiple pollutants.Epiphyton and epixylon biofilms showed excellent methane oxidation, and Pb


Nairoviridae family, was recently shown to be the target of a protective antibody against CCHFV. Here, we present the crystal structure of GP38, which revealed a novel fold with distant homology to another CCHFV glycoprotein that is suggestive of a gene duplication event. We also demonstrate that antibody 13G8 protects STAT1-knockout mice against heterologous CCHFV challenge using a clinical isolate from regions where CCHFV is endemic. Collectively, these data advance our understanding of GP38 structure and antigenicity and should facilitate future studies investigating its function.Crimean-Congo hemorrhagic fever virus (CCHFV) is a priority pathogen that poses a high risk to public health. Due to the high morbidity and mortality rates associated with CCHFV infection, there is an urgent need to develop medical countermeasures for disease prevention and treatment. CCHFV GP38, a secreted glycoprotein of unknown function unique to the  Crimean-Congo hemorrhagic fever virus (CCHFV) is the causative agent of the most widespread tick-borne viral infection in humans. CCHFV encodes a secreted glycoprotein (GP38) of unknown function that is the target of a protective antibody. Here, we present the crystal structure of GP38 at a resolution of 2.5 Å, which revealed a novel fold primarily consisting of a 3-helix bundle and a β-sandwich. Sequence alignment and homology modeling showed distant homology between GP38 and the ectodomain of Gn , suggestive of a gene duplication event. Analysis of convalescent-phase sera showed high titers of GP38 antibodies indicating immunogenicity in humans during natural CCHFV infection. The only protective antibody for CCHFV in an adult mouse model reported to date, 13G8, bound GP38 with subnanomolar affinity and protected against heterologous CCHFV challenge in a STAT1-knockout mouse model. Our data strongly suggest that GP38 should be evaluated as a vaccine antigen and that its structure provides a foundation to investigate functions of this protein in the viral life cycle.IMPORTANCE Crimean-Congo hemorrhagic fever virus (CCHFV) is a priority pathogen that poses a high risk to public health. Due to the high morbidity and mortality rates associated with CCHFV infection, there is an urgent need to develop medical countermeasures for disease prevention and treatment. CCHFV GP38, a secreted glycoprotein of unknown function unique to the Nairoviridae family, was recently shown to be the target of a protective antibody against CCHFV. Here, we present the crystal structure of GP38, which revealed a novel fold with distant homology to another CCHFV glycoprotein that is suggestive of a gene duplication event. We also demonstrate that antibody 13G8 protects STAT1-knockout mice against heterologous CCHFV challenge using a clinical isolate from regions where CCHFV is endemic. Collectively, these data advance our understanding of GP38 structure and antigenicity and should facilitate future studies investigating its function. Orthonairovirus genus are tick-borne members of the Nairoviridae family in the Bunyavirales order of negative-strand RNA viruses. Orthonairoviruses belong to more than 14 different species and at least 4 of these species contain viruses which can cause disease in humans , medium (M), and large (L) segments . The CCHN-glycosylated in the endoplasmic reticulum (ER) and cleaved by subtilisin kexin isozyme-1/site-1 protease (SKI-1/S1P) in the Golgi complex to liberate it from Gn  (GP38 (GPC residues 248 to 519) does not share substantial sequence homology with other viral or cellular proteins. It is  from Gn . SKI-1/S from Gn . Later, in (MLD) . Mutatinin (MLD) . To datePreviously, Bertolotti-Ciarlet et al. described murine monoclonal antibodies (MAbs) that provided protection against lethal CCHFV challenge in a neonatal mouse model . The neuTo gain insight into the function of GP38, we initiated structural and biophysical studies and obtained a 2.5-Å structure of GP38. GP38 has a novel fold composed primarily of a 3-helix bundle and a β-sandwich. Interestingly, sequence alignment of GP38 showed distant homology with the Gn ectodomain. In addition, the reactivity of convalescent human sera to GP38 indicates it is immunogenic in humans. We also characterized the binding of GP38 to MAb 13G8 and successfully demonstrated the protective efficacy of MAb 13G8 against a heterologous clinical CCHFV isolate strain (Turkey2004) in a STAT1-knockout mouse model. Our data strongly suggest that GP38 should be evaluated as a vaccine antigen.We expressed a construct containing GPC residues 1 to 515 of CCHFV strain IbAr 10200 in FreeStyle 293 cells. The N-terminal MLD (GPC residues 1 to 247) was cleaved at the native furin cleavage site (GPC residues 244 to 247), and secreted GP38 was purified via affinity chromatography followed by size exclusion chromatography (SEC). SEC revealed a sharp symmetrical peak corresponding to a glycosylated GP38 monomer indicating a well-behaved monodispersed protein . SDS-PAGdK) of 0.4 nM, an association rate constant (onk) of 7.8 × 105 M−1s−1, and a dissociation rate constant (offk) of 3.3 × 10−4 s−1 . c13G8 contained the murine variable fragments fused with human constant domains. Biolayer interferometry (BLI) experiments determined that c13G8 bound to GP38 with an equilibrium dissociation constant . The structure of GP38 was initially determined to 2.8 Å using these data sets, and was subsequently extended to 2.5-Å resolution with an additional data set collected at Advanced Photon Source. The final structure has an Rfree of 25.6% and an Rwork of 22.3% , 2% to 4% polyethylene glycol 400 (PEG 400), and 11% to 21% PEG 3350 and belonged to the of 22.3% .N-linked glycosylation sequons at Asn376 and Asn426, and both of these sites showed well-resolved electron density for glycans extending away from the protein surface  at the C terminus of GP38, suggesting a release of GP38 from Gn similar to that observed for CCHFV . The prenfection , 19, whenfection , 21. We nfection . Asn402 Sequence alignment of CCHFV GP38 and the Gn ectodomain displayed less than 20% identity. However, alignment of GP38 and Gn ectodomain sequences from CCHFV, Hazara virus, Tofla virus, NSDV, Ganjam virus, Dugbe virus, and Kupe virus revealed that some sequence and structural elements were conserved in the region corresponding to the C-terminal β-strands of GP38 . This wa−/−) mice  alongside three chimeric human IgG1 variants of m13G8. The first variant c13G8, as described before, contained the murine variable fragments fused with human IgG1 constant domains. The second was an afucosylated variant of c13G8 (c13G8AF-N) that is likely to have significantly increased affinity to FcγR receptors and enhanced Fc effector functions (LALAPG-N) (It was previously shown that the murine MAb 13G8 (m13G8) is a nonneutralizing but protective CCHFV antibody, now known to specifically target GP38 . m13G8 w/−) mice . Howeverhe world . In ordeunctions . The thiALAPG-N) .n = 5/group) and a PBS-treated control group (n = 2). All groups received their respective treatments 30 minutes postinfection. The c13G8 and c13G8AF-N MAbs both completely protected mice challenged with strain Turkey2004; m13G8 was 80% efficacious and the c13G8LALAPG-N MAb showed significant breakthrough, only protecting 60% of the treated animals  was able to protect mice from uniformly lethal CCHFV challenge. This strongly suggests that GP38 plays a role in CCHFV virulence. The studies performed here extend the protective range of MAb 13G8 beyond strain IbAr 10200 to include strain Turkey2004 (85% sequence identity to IbAr 10200). The ability of c13G8 to completely protect mice infected with strain Turkey2004 was unexpected given that m13G8 showed only limited protection in mice challenged with strain Afg09-2990 (91% sequence identity to IbAr 10200) . In addig09-2990 .AF-N, and c13G8LALAPG-N. Both c13G8 and c13G8AF-N provided complete protection, whereas c13G8LALAPG-N protected only 60% of the mice, suggesting that Fc-induced effector functions contributed positively to the protective mechanism of action of 13G8. Moreover, the c13G8LALAPG-N variant, having no ability to recruit complement or induce Fc effector functions, still protected STAT-1−/− mice against lethal CCHFV infection. This suggests that MAb 13G8 may also inhibit yet-to-be-determined functions of GP38 that contribute to the pathogenesis of CCFHV. Unfortunately, the vulnerability of MAb 13G8 to GP38 sequence variation across CCHFV clades may limit its utility as a viable immunotherapeutic candidate. Our work calls for further studies to identify broadly reactive and protective GP38 antibodies and to decipher the mechanism of protection by understanding the function of GP38.Golden et al. also examined the role of Fc function and complement fixation in the protective efficacy of m13G8 using various genetically modified mouse models . Here, w3.A gene fragment encoding residues 1 to 515 of CCHFV strain IbAr 10200 GPC was codon optimized for human cell expression (via GenScript) and cloned into a pαH eukaryotic expression plasmid with a C-terminal HRV3C protease cleavage site, an 8× HisTag, and a Twin-Strep-tag. This plasmid was transiently transfected into FreeStyle 293 cells (Invitrogen) using polyethyleneimine. Transfected FreeStyle 293 cells were treated with 5 μM kifunensine to ensure uniform high-mannose glycosylation. GP38 was expressed in soluble form and secreted out into the medium, which was harvested and purified over Strep-Tactin resin (IBA Lifesciences). A furin cleavage site (residues 244 to 247) separates the N-terminal MLD and GP38 protein. Because not all of the MLD was cleaved from GP38 by endogenous furin, additional MLD was cleaved off by treatment with furin protease during Strep-tag-based affinity purification. After elution of GP38 from Strep-Tactin resin, the affinity tags were removed by HRV3C cleavage. GP38 was further purified by SEC using a HiLoad 16/600 Superdex 200 pg  in 2 mM Tris (pH 8.0), 200 mM NaCl, and 0.02% NaNdK, onk, and offk.A chimeric version of MAb 13G8 (denoted c13G8) was constructed by fusing the variable heavy and light chain fragments from the original murine MAb 13G8 to the constant domains of a human IgG1. The c13G8 MAb was then used for the binding kinetics experiments described herein. Binding kinetics were performed using BLI with an Octet RED96e system . Anti-human IgG Fc Capture (AHC) sensors  were incubated in kinetics buffer  for 15 min. Baseline readings were determined by equilibrating sensors for 60 s in the kinetics buffer. MAb c13G8 was diluted in kinetics buffer to a concentration of 5 nM. The AHC sensors were loaded by dipping them into 5 nM MAb c13G8 for a capture time of 150 s. Postcapture baseline readings were determined. The sensors loaded with MAb 13G8 were then dipped into wells with different concentrations of GP38 (0.63 to 10 nM range prepared in kinetics buffer) for a 5-min association, followed by a 10-min dissociation step in the kinetics buffer. A c13G8-captured AHC sensor was also dipped in a well containing kinetics buffer (no GP38) to allow single-reference subtraction to compensate for the slow dissociation of c13G8 MAb from the sensors. The data from AHC sensors were analyzed using the binding kinetics function of the Octet analysis software and fitted to a 1:1 binding model to determine CCHF convalescent patients were recruited with the help of the Uganda Virus Research Institute, Entebbe, Uganda. All survivors had documented clinical history of CCHF infection in Agago and Nakaseke districts, Uganda, ranging from 2013 to 2017. The study was approved by the Helsinki committees of Uganda Virus Research Institute (UVRI), Entebbe, Uganda (reference number GC/127/13/01/15); Soroka Hospital, Beer Sheva, Israel (protocol number 0263-13-SOR); and the Ugandan National Council for Science and Technology (UNCST) (registration number HS1332). Written informed consent, as well as a personal health questionnaire, was completed for each subject who participated in this study. Study participants were all adults and not related. We confirm that all experiments were performed in accordance with the relevant guidelines and regulations.The GP38 serum reactivity ELISA was performed in a Greiner high-binding half-area plate. Individual wells were coated with 250 ng of purified, recombinant GP38 diluted in phosphate-buffered saline (PBS) (pH 7.4). Wells were washed three times with PBS supplemented with 0.05% Tween 20 (PBST) and then blocked with 2% bovine serum albumin (BSA) in PBS. Five-fold serial dilutions of CCHF patient serum, control serum, or PBS were added to the blocked wells and incubated for 1 h at ambient temperature. Samples were prepared in duplicate. After 3 washes with PBST, bound GP38-specific antibody was detected with an anti-human Fc secondary conjugated to horseradish peroxidase (HRP). KPL SureBlue TMB microwell peroxidase was used as the HRP substrate, and the reaction was stopped with an equal volume of 2 N sulfuric acid. The absorbance at 450 nm was measured with a Perkin Elmer EnVision multimode plate reader.Crystallization trials for GP38 were set up using the sitting-drop vapor diffusion method. The best diffracting crystals were grown in a solution of 0.1 M sodium acetate (pH 5.5), 2% to 4% PEG 400, and 11% to 21% PEG 3350 via hanging-drop vapor diffusion by mixing 1 μl of GP38 (11.2 mg/ml) with 1 μl of reservoir solution. Crystals were looped directly from the crystallization drop and flash frozen in liquid nitrogen. X-ray diffraction data were collected at the 19-ID beamline  and scaled to 2.50 Å. For sulfur-based phasing, long-wavelength diffraction experiments were performed at the long-wavelength MX beamline I23 at Diamond Light Source  at an enRwork/Rfree of 22.3%/25.6% (Diffraction images from the I23 beamline were recorded on a PILATUS 12M device (Dectris) and processed using XDS and XSCALE . The sul3%/25.6% . The str3%/25.6% .Q8JSZ3), Hazara virus (A6XIP3), Tofla virus (A0A0U5AG15), NSDV (A0A0A7H8I1), Ganjam virus (A0A191KWA4), Dugbe virus (Q02004), and Kupe virus (B8PWH4). ESPript was used to display the alignment , Hazara virus (UniProt accession number A6XIP3), NSDV (UniProt accession number D0PRM8), Dugbe virus (UniProt accession number Q02004), Tamdy virus (UniProt accession number A0A1S5NVL7), Dera Ghazi Khan virus (UniProt accession number A0A191KW89), Artashat orthonairovirus (UniProt accession number A0A1S5NQD0), Hughes orthonairovirus (UniProt accession number A0A191KWA8), Kasokero virus (UniProt accession number A0A0M4LB14), Keterrah virus (UniProt accession number A0A0M4KM64), Qalyub orthonairovirus (UniProt accession number A0A1S5NQX5), Sakhalin orthonairovirus (UniProt accession number A0A1S5NQC0), and Thiafora orthonairovirus (UniProt accession number A0A0M5KMK1). PYMOL was used for visualization of surface conservation  Institutional Animal Care and Use Committee (IACUC). Animal research was carried out in compliance with the Animal Welfare Act and other federally regulated stipulations regarding animals and adherences to the Guide for the Care and Use of Laboratory Animals, National Research Council, 2013. The animal facilities where this research was conducted are accredited by the Association for Assessment and Accreditation of Laboratory Animal Care International. Euthanasia criteria was defined as the follows: mouse displays severely hunched posture, inability or reluctance to move, appears weak (staggering when moving around cage), labored breathing, or weight loss of greater than 25% of starting body weight.The Turkey-200406546 strain   was propagated in Vero E6 cells once followed by expansion in SW13 human adenocarcinoma cells. All work with live CCHFV was performed in a biosafety level 4 facility at the Galveston National Laboratory, University of Texas Medical Branch, Galveston, TX. All cell and viral stocks were tested and are free of mycoplasma by using a PCR kit .AF-N, and c13G8LALAPG-N were constructed and purified at Mapp Biopharmaceutical. c13G8 was expressed transiently in expiCHO cells (Thermo Fisher Scientific). c13G8 was purified from expiCHO cell supernatants using a GE MabSelect SuRe LX protein A affinity chromatography column on an AKTA pure fast protein liquid chromatography (FPLC) system. The c13G8 antibody was eluted using a glycine elution buffer at pH 2.2 and neutralized with 2 M Tris base to a pH of ∼7. The c13G8AF-N and c13G8LALAPG-N afucosylated antibodies were expressed in Nicotiana benthamiana plants (tobacco plants). The plant-derived antibodies were purified as described previously  . After an acclimatization period in barrier conditions within environmentally enriched sterile housing, mice were anesthetized by isoflurane and implanted with subdermal transponders, which provide coded identifiers and permitted body temperature monitoring and measurements . Challenge material preparations were diluted in Hanks balanced salts medium with 2% fetal bovine serum (FBS) for a final targeted challenge dose of 100 PFU. All challenge doses were frozen for storage and verified by back-titrations by plaque assay on SW-13-CDC cells  of each antibody per treatment group. Animals were then observed for clinical scoring, temperature, and weight change. Mice were monitored daily and euthanized upon reaching institutionally approved endpoint score criteria or study endpoint (28 days postinfection [p.i.]).The study utilized 4- to 8-week-old female 129S6/SvEv-DC cells . After a6VKF.The atomic coordinates and structure factors for CCHFV GP38 have been deposited in the Protein Data Bank (PDB) under accession number