Valine Residue Research Articles

Backbone distortions in lactam-bridged helical peptides.

Side‐chain‐to‐side‐chain cyclization is frequently used to stabilize the α‐helical conformation of short peptides. In a previous study, we incorporated a lactam bridge between the side chains of Lys‐i and Asp‐i+4 in the nonapeptide 1Y, cyclo‐(2,6)‐(Ac‐VKRLQDLQY‐NH 2 ), an artificial ligand of the inhibitor of DNA binding and cell differentiation (ID) protein with antiproliferative activity on cancer cells. Herein, we show that only the cyclized five‐residue segment adopts a helical turn whereas the C‐terminal residues remain flexible. Moreover, we present nine 1Y analogs arising from different combinations of hydrophobic residues (leucine, isoleucine, norleucine, valine, and tyrosine) at positions 1, 4, 7, and 9. All cyclopeptides except one build a lactam‐bridged helical turn; however, residue‐4 reveals less helix character than the neighboring Arg‐3 and Gln‐5, especially with residue‐4 being isoleucine, valine, and tyrosine. Surprisingly, only two cyclopeptides exhibit helix propagation until the C‐terminus, whereas the others share a remarkable outward tilting of the backbone carbonyl of the lactam‐bridged Asp‐6 (>40° deviation from the orientation parallel to the helix axis), which prevents the formation of the H‐bond between Arg‐3 CO and residue‐7 NH: As a result, the propagation of the helix beyond the lactam‐bridged sequence becomes unfavorable. We conclude that, depending on the amino‐acid sequence, the lactam bridge between Lys‐i and Asp‐i+4 can stabilize a helical turn but deviations from the ideal helix geometry are possible: Indeed, besides the outward tilting of the backbone carbonyls, the residues per turn increased from 3.6 (typical of a regular α‐helix) to 4.2, suggesting a partial helix unwinding.

Introducing the Chiral Constrained α-Trifluoromethylalanine in Aib Foldamers to Control, Quantify and Assign the Helical Screw-Sense.

Oligomers of α-aminoisobutyric acid (Aib) are achiral peptides that adopt 310 helical structures with equal population of left- and right-handed conformers. The screw-sense preference of the helical chain may be controlled by a single chiral residue located at one terminus. 1 H and 19 F NMR, X-ray crystallography and circular dichroism studies on new Aib oligomers show that the incorporation of a chiral quaternary α-trifluoromethylalanine at their N-terminus induces a reversal of the screw-sense preference of the 310 -helix compared to that of a non-fluorinated analogue having an l-α-methyl valine residue. This work demonstrates that, among the many particular properties of introducing a trifluoromethyl group into foldamers, its stereo-electronic properties are of major interest to control the helical screw sense. Its use as an easy-to-handle 19 F NMR probe to reliably determine both the magnitude of the screw-sense preference and its sign assignment is also of remarkable interest.

Multiple binding modes of an N-terminal CCR5-peptide in complex with HIV-1 gp120.

The N-terminal segment of CCR5 contains four tyrosine residues, sulphation of two of which is essential for high-affinity binding to gp120. In the present study, the interactions of gp120YU2 with a 27-residue N-terminal CCR5 peptide sulphated at position Y10 and Y14, i.e. Nt-CCR5, were studied using 13 C-edited-HMQC methyl-NOESY [1 H(13 C)-1 H], combined with transferred NOE NMR spectroscopy. A large number of pairwise interactions were observed between the methyl protons of methionine, threonine, valine and isoleucine residues of gp120, and the aromatic tyrosine-protons of Nt-CCR5. M434, V120 and V200 of gp120 were found to interact with all four tyrosine residues, Y3, sY10, sY14 and Y15. Particularly intriguing was the observation that Y3 and Y15 interact with the same gp120 methyl protons. Such interactions cannot be explained by the single cryo-EM structure of gp120/CD4/CCR5 complex published recently (Nature, 565, 318-323, 2019). Rather, they are consistent with the existence of a dynamic equilibrium involving two or more binding modes of Nt-CCR5 to gp120. These different modes of binding can coexist because the surface of gp120 contains two sites that can optimally interact with a sulphated tyrosine residue and two sites that can interact favorably with a non-sulphated tyrosine residue. Modelling of gp120YU2 complexed with the Nt-CCR5 peptide or with the entire CCR5 receptor provides an explanation for the NMR observations and the existence of these different binding modes of the disordered N-terminus of CCR5. The data presented extend our understanding of the two-step model and suggest a more variable binding mode of Nt-CCR5 with gp120.

Infantile SOD1 deficiency syndrome caused by a homozygous SOD1 variant with absence of enzyme activity.

Pathogenic variants in SOD1, encoding superoxide dismutase 1, are responsible for about 20% of all familial amyotrophic lateral sclerosis cases, through a gain-of-function mechanism. Recently, two reports showed that a specific homozygous SOD1 loss-of-function variant is associated with an infantile progressive motor-neurological syndrome. Exome sequencing followed by molecular studies, including cDNA analysis, SOD1 protein levels and enzymatic activity, and plasma neurofilament light chain levels, were undertaken in an infant with severe global developmental delay, axial hypotonia and limb spasticity. We identified a homozygous 3-bp in-frame deletion in SOD1. cDNA analysis predicted the loss of a single valine residue from a tandem pair (p.Val119/Val120) in the wild-type protein, yet expression levels and splicing were preserved. Analysis of SOD1 activity and protein levels in erythrocyte lysates showed essentially no enzymatic activity and undetectable SOD1 protein in the child, whereas the parents had ∼50% protein expression and activity relative to controls. Neurofilament light chain levels in plasma were elevated, implying ongoing axonal injury and neurodegeneration. Thus, we provide confirmatory evidence of a second biallelic variant in an infant with a severe neurological syndrome and suggest that the in-frame deletion causes instability and subsequent degeneration of SOD1. We highlight the importance of the valine residues at positions V119-120, and suggest possible implications for future therapeutics research.

Exploration of the Activation Mechanism of the Epigenetic Regulator MLL3: A QM/MM Study.

The mixed lineage leukemia 3 or MLL3 is the enzyme in charge of the writing of an epigenetic mark through the methylation of lysine 4 from the N-terminal domain of histone 3 and its deregulation has been related to several cancer lines. An interesting feature of this enzyme comes from its regulation mechanism, which involves its binding to an activating dimer before it can be catalytically functional. Once the trimer is formed, the reaction mechanism proceeds through the deprotonation of the lysine followed by the methyl-transfer reaction. Here we present a detailed exploration of the activation mechanism through a QM/MM approach focusing on both steps of the reaction, aiming to provide new insights into the deprotonation process and the role of the catalytic machinery in the methyl-transfer reaction. Our finding suggests that the source of the activation mechanism comes from conformational restriction mediated by the formation of a network of salt-bridges between MLL3 and one of the activating subunits, which restricts and stabilizes the positioning of several residues relevant for the catalysis. New insights into the deprotonation mechanism of lysine are provided, identifying a valine residue as crucial in the positioning of the water molecule in charge of the process. Finally, a tyrosine residue was found to assist the methyl transfer from SAM to the target lysine.

Effects of Ribosomal Protein S10 Flexible Loop Mutations on Tetracycline and Tigecycline Susceptibility of Escherichia coli.

Tigecycline is a tetracycline derivative that is being used as an antibiotic of last resort. Both tigecycline and tetracycline bind to the small (30S) ribosomal subunit and inhibit translation. Target mutations leading to resistance to these antibiotics have been identified both in the 16S ribosomal RNA and in ribosomal proteins S3 and S10 (encoded by the rpsJ gene). Several different mutations in the S10 flexible loop tip residue valine 57 (V57) have been observed in tigecycline-resistant Escherichia coli isolates. However, the role of these mutations in E. coli has not yet been characterized in a defined genetic background. In this study, we chromosomally integrated 10 different rpsJ mutations into E. coli, resulting in different exchanges or a deletion of S10 V57, and investigated the effects of the mutations on growth and tigecycline/tetracycline resistance. While one exchange, V57K, decreased the minimal inhibitory concentration (MIC) (Etest) to tetracycline to 0.75 μg/ml (compared to 2 μg/ml in the parent strain) and hence resulted in hypersensitivity to tetracycline, most exchanges, including the ones reported previously in resistant isolates (V57L, V57D, and V57I) resulted in slightly increased MICs to tigecycline and tetracycline. The strongest increase was observed for the V57L mutant, with a MIC (Etest) to tigecycline of 0.5 μg/ml (compared to 0.125 μg/ml in the parent strain) and a MIC to tetracycline of 4.0 μg/ml. Nevertheless, none of these exchanges increased the MIC to the extent observed in previously described clinical tigecycline-resistant isolates. We conclude that, next to S10 mutations, additional mutations are necessary in order to reach high-level tigecycline resistance in E. coli. In addition, our data reveal that mutants carrying S10 V57 exchanges or deletion display growth defects and, in most cases, also thermosensitivity. The defects are particularly strong in the V57 deletion mutant, which is additionally cold-sensitive. We hypothesize that the S10 loop tip residue is critical for the correct functioning of S10. Both the S10 flexible loop and tigecycline are in contact with helix h31 of the 16S rRNA. We speculate that exchanges or deletion of V57 alter the positioning of h31, thereby influencing both tigecycline binding and S10 function.

Sickle Cell Anemia Child presented with Bell's palsy: A Rare Case Report

Sickle cell anemia (SCA) is an inherited red blood cell disorder that results from the replacement of a valine residue for glutamic acid at position 6 in the beta-subunit of haemoglobin.This can lead to tissue ischemia, microcirculation obstructions, infarction, and acute stroke while Bell’s palsy is a neuropathy caused by traumatic, inflammatory, infective, or compressive conditions on the facial nerve. In patients with SCA, however, stroke may occur as an acute clinical syndrome presenting with hemiplegia with the unilateral facial nerve. Until now, no case has been reported in the literature with unilateral facial nerve palsy in SCA patients. Here, a rare case of idiopathic unilateral Bell’s palsy is presented in an SCA patient.

A valine residue deletion in ZmSig2A, a sigma factor, accounts for a revertible leaf-color mutation in maize

A nuclear-encoded sigma (σ) factor is essential for the transcriptional regulation of plant chloroplast-encoded genes. Five putative maize σ factors have been identified by database searches, but their functions are unknown. We report a maize leaf color mutant etiolated/albino leaf 1 (eal1) that was derived from space mutation breeding. The eal1 mutant displays etiolated or albino leaves that then gradually turn to normal green at the seedling stage. The changes in eal1 leaf color are associated with changes in photosynthetic pigment content and chloroplast development. Map-based cloning revealed that a single amino-acid deletion changing Val480-Val481-Val482 to Val480-Val481, in the C-terminal domain σ4 of the putative σ factor ZmSig2A, is responsible for the eal1 mutation. In comparison with the expression level of the wild-type (WT) allele ZmSig2A+ in WT plants, much higher expression of the mutant allele ZmSig2AΔV in eal1 plants was detected before the eal1 plants turned to normal green. ZmSig2A shows the highest similarity to rice OsSig2A and Arabidopsis SIG2. Ectopic expression of ZmSig2A+ or ZmSig2AΔV driven by the cauliflower mosaic virus 35S promoter rescued the pale green leaf of the sig2 mutant, but ectopic expression of ZmSig2AΔV driven by the SIG2 promoter did not. We propose that the Val deletion generated a new weak allele of ZmSig2A that cannot completely abolish the ZmSig2A function. Some genes involved in chloroplast development and photosynthesis-associated nuclear genes showed significant expression differences between eal1 and WT plants. We conclude that ZmSig2A encoding a σ factor is essential for maize chloroplast development. The eal1 mutant with a weak allele of ZmSig2A represents a valuable genetic resource for investigating the regulation of ZmSig2A-mediated chloroplast development in maize. The eal1 mutation may be useful as a marker for early identification and elimination of false hybrids or transgene transmission in the application of genetic male sterility to commercial hybrid seed production.

The spectrum of association in HLA region with rheumatoid arthritis in a diverse Asian population: evidence from the MyEIRA case-control study

BackgroundFine-mapping of human leukocyte antigen (HLA) region for rheumatoid arthritis (RA) risk factors has identified several HLA alleles and its corresponding amino acid residues as independent signals (i.e., HLA-A, HLA-B, HLA-DPB1, and HLA-DQA1 genes), in addition to the well-established genetic factor in HLA-DRB1 gene. However, this was mainly performed in the Caucasian and East Asian populations, and data from different Asian regions is less represented. We aimed to evaluate whether there are independent RA risk variants in both anti-citrullinated protein antibody (ACPA)-positive and ACPA-negative RA patients from the multi-ethnic Malaysian population, using the fine-mapping of HLA region strategy.MethodsWe imputed the classical HLA alleles, amino acids, and haplotypes using the Immunochip genotyping data of 1260 RA cases (i.e., 530 Malays, 259 Chinese, 412 Indians, and 59 mixed ethnicities) and 1571 controls (i.e., 981 Malays, 205 Chinese, 297 Indians, and 87 mixed ethnicities) from the Malaysian Epidemiological Investigation of Rheumatoid Arthritis (MyEIRA) population-based case-control study. Stepwise logistic regression was performed to identify the independent genetic risk factors for RA within the HLA region.ResultsWe confirmed that the HLA-DRB1 amino acid at position 11 with valine residue conferred the strongest risk effect for ACPA-positive RA (OR = 4.26, 95% CI = 3.30–5.49, PGWAS = 7.22 × 10−29) in the Malays. Our study also revealed that HLA-DRB1 amino acid at position 96 with histidine residue was negatively associated with the risk of developing ACPA-positive RA in the Indians (OR = 0.48, 95% CI = 0.37–0.62, PGWAS = 2.58 × 10−08). Interestingly, we observed that HLA-DQB1*03:02 allele was inversely related to the risk of developing ACPA-positive RA in the Malays (OR = 0.17, 95% CI = 0.09–0.30, PGWAS = 1.60 × 10−09). No association was observed between the HLA variants and risk of developing ACPA-negative RA in any of the three major ethnic groups in Malaysia.ConclusionsOur results demonstrate that the RA-associated genetic factors in the multi-ethnic Malaysian population are similar to those in the Caucasian population, despite significant differences in the genetic architecture of HLA region across populations. A novel and distinct independent association between the HLA-DQB1*03:02 allele and ACPA-positive RA was observed in the Malays. In common with the Caucasian population, there is little risk from HLA region for ACPA-negative RA.

Development of a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the analysis of tryptic digest of human hemoglobin exposed to sulfur mustard

Sulfur mustard is a highly reactive chemical warfare agent that causes severe damages to the victims exposed by alkylating multiple biomolecules such as proteins. Resulting alkylated products can be used as biomarkers of exposure to this chemical agent. A liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) method was thus developed to detect alkylated peptides after the tryptic digestion of hemoglobin (50 mg.mL−1) incubated with sulfur mustard at different concentrations (0.25, 0.5, 1, 10 and 100 µg.mL−1). Five new alkylation sites were accurately identified on the protein (α-His72, α-His87, α-His89, β-His2 and β-Val98) and fifteen adducted peptides were detected, among which eight of them resulted from the alkylation of four peptides, each presenting two potential sites of adduction that could be discriminated by the method specificity. Similarly, it was possible to discriminate the three potential adduction sites of the peptide α-T9. Moreover, the method allowed the quantification of all the alkylated peptides with a satisfying repeatability, with RSD ranging from 0.5 to 9.3% for an exposure of hemoglobin to sulfur mustard at 100 µg.mL−1. The analysis of hemoglobin incubated with different concentrations of sulfur mustard levels led to a linear response for all the alkylated peptides with the studied concentrations (0.25, 0.5, 1, 10 and 100 µg.mL−1). A variation of the alkylation rate was also observed between the different peptides studied, with a preferential adduction of sulfur mustard on the histidine residues but also on the N-terminal valine residues of both globin chains and on the Val98 residue of globin β. Furthermore, the presented method proved to be sensitive, with a theoretical possibility to detect alkylated peptides resulting from in vitro incubation of hemoglobin in deionized water with sulfur mustard at 2.63 ng.mL−1. After further development, this method could potentially be used for the analysis of blood samples in vivo exposed to sulfur mustard.

The G protein coupled receptor CXCR4 designed by the QTY code becomes more hydrophilic and retains cell signaling activity

G protein-coupled receptors (GPCRs) are vital for diverse biological functions, including vision, smell, and aging. They are involved in a wide range of diseases, and are among the most important targets of medicinal drugs. Tools that facilitate GPCR studies or GPCR-based technologies or therapies are thus critical to develop. Here we report using our QTY (glutamine, threonine, tyrosine) code to systematically replace 29 membrane-facing leucine, isoleucine, valine, and phenylalanine residues in the transmembrane α-helices of the GPCR CXCR4. This variant, CXCR4QTY29, became more hydrophilic, while retaining the ability to bind its ligand CXCL12. When transfected into HEK293 cells, it inserted into the cell membrane, and initiated cellular signaling. This QTY code has the potential to improve GPCR and membrane protein studies by making it possible to design functional hydrophilic receptors. This tool can be applied to diverse α-helical membrane proteins, and may aid in the development of other applications, including clinical therapies.

Site-specific modifications to AAV8 capsid yields enhanced brain transduction in the neonatal MPS IIIB mouse

Mucopolysaccharidosis type IIIB (MPS IIIB) is an autosomal recessive lysosomal disease caused by defective production of the enzyme α-N-acetylglucosaminidase. It is characterized by severe and complex central nervous system degeneration. Effective therapies will likely target early onset disease and overcome the blood–brain barrier. Modifications of adeno-associated viral (AAV) vector capsids that enhance transduction efficiency have been described in the retina. Herein, we describe for the first time, a transduction assessment of two intracranially administered adeno-associated virus serotype 8 variants, in which specific surface-exposed tyrosine (Y) and threonine (T) residues were substituted with phenylalanine (F) and valine (V) residues, respectively. A double-mutant (Y447 + 733F) and a triple-mutant (Y447 + 733F + T494V) AAV8 were evaluated for their efficacy for the potential treatment of MPS IIIB in a neonatal setting. We evaluated biodistribution and transduction profiles of both variants compared to the unmodified parental AAV8, and assessed whether the method of vector administration would modulate their utility. Vectors were administered through four intracranial routes: six sites (IC6), thalamic (T), intracerebroventricular, and ventral tegmental area into neonatal mice. Overall, we conclude that the IC6 method resulted in the widest biodistribution within the brain. Noteworthy, we demonstrate that GFP intensity was significantly more robust with AAV8 (double Y–F + T–V) compared to AAV8 (double Y–F). This provides proof of concept for the enhanced utility of IC6 administration of the capsid modified AAV8 (double Y–F + T–V) as a valid therapeutic approach for the treatment of MPS IIIB, with further implications for other monogenic diseases.

Herpes Simplex Virus-1 infection in human primary corneal epithelial cells is blocked by a stapled peptide that targets processive DNA synthesis

PurposeAcyclovir is most commonly used for treating ocular Herpes Keratitis, a leading cause of infectious blindness. However, emerging resistance to Acyclovir resulting from mutations in the thymidine kinase gene of Herpes Simplex Virus −1 (HSV-1), has prompted the need for new therapeutics directed against a different viral protein. One novel target is the HSV-1 Processivity Factor which is essential for tethering HSV-1 Polymerase to the viral genome to enable long-chain DNA synthesis. MethodsA series of peptides, based on the crystal structure of the C-terminus of HSV-1 Polymerase, were constructed with hydrocarbon staples to retain their alpha-helical conformation. The stapled peptides were tested for blocking both HSV-1 DNA synthesis and infection. The most effective peptide was further optimized by replacing its negative N-terminus with two hydrophobic valine residues. This di-valine stapled peptide was tested for inhibiting HSV-1 infection of human primary corneal epithelial cells. ResultsThe stapled peptides blocked HSV-1 DNA synthesis and HSV-1 infection. The unstapled control peptide had no inhibitory effects. Specificity of the stapled peptides was confirmed by their inabilities to block infection by an unrelated virus. Significantly, the optimized di-valine stapled peptide effectively blocked HSV-1 infection in human primary corneal epithelial cells with selectivity index of 11.6. ConclusionsHydrocarbon stapled peptides that simulate the α-helix from the C-terminus of HSV-1 DNA polymerase can specifically block DNA synthesis and infection of HSV-1 in human primary corneal epithelial cells. These stapled peptides provide a foundation for developing a topical therapeutic for treating human ocular Herpes Keratitis.

An integrated computational investigation to unveil the structural impacts of mutation on the InhA structural gene of Mycobacterium tuberculosis

Growing concern about the difficulty in diagnosis and treatments of drug-resistant tuberculosis falls under the major global health issues. There is an urgent need for finding novel strategies to develop drugs or bioactive molecules against the global threat of Mycobacterium tuberculosis (MTB). Isoniazid (INH) is a front line drug against tuberculosis; it primarily targets the enoyl-acyl carrier protein reductase (InhA), a potent drug target in the mycolic acid pathway of MTB. To gain deeper insight into the impact of INH resistant mutation and its influence on the structural dynamics of InhA, combined conformational dynamics and residue interaction network (RIN) studies were performed. The molecular dynamics investigation provided a hint about the structural changes altering protein activity. The principal component analysis (PCA) based free energy landscape plot highlighted the highest stable part of wild-type (WT) and mutant structures. Intriguingly, the mutation at the 78th position of InhA from its native residue valine to alanine increases the structural stability with higher NADH binding affinity. The MM-PBSA based binding energy calculations confirm that electrostatic interactions played a critical role in the binding of NADH at the binding site of InhA. The calculated binding energy score, as well as potential hydrogen bonds and salt bridge networks, proved the strong binding of mutant InhA as compared to WT. Further, the mutation potentially altered the protein network topology, thereby subsequently affected the landscape of NADH binding. The present study is an attempt to understand the structural and functional impact associated with a drug-resistant mutation (V78A) thus it will be helpful in designing potent inhibitors against drug-resistant tuberculosis.

Unusual complexation behavior between daclatasvir and γ-Cyclodextrin. A multiplatform study.

During a screening of cyclodextrins (CDs) as chiral selectors for the separation of daclatasvir (DCV) and its enantiomer by capillary electrophoresis (CE), an unusual phenomenon for CDs was observed, that is two peaks with a plateau in between using γ-CD as chiral selector. The same result was encountered when enantiopure DCV was injected or when analyzing a sample containing enantiopure DCV and γ-CD in a CD-free background electrolyte. Peak coalescence was observed at 45°C and at a pH above 3.5. Two peaks with a plateau were also observed for DCV stereoisomers as well as a structural analog. However, only a single peak was detected if one or both amino acid moieties of DCV were lacking. Nuclear magnetic resonance (NMR) experiments including Nuclear Overhauser effect-based methods showed that in solution DCV adopted a folded conformation in which the isopropyl side chain of the valine residues pointed toward the aromatic rings of DCV. Moreover, NMR unequivocally demonstrated the simultaneous formation of DCV-γ-CD inclusion complexes with 1:1 and 2:1 stoichiometry, which was corroborated by mass spectrometry. In both complexes, DCV also adopted a folded structure. The RSSR-diastereomer of DCV as well as an analog lacking one of the amino acid moieties also formed 1:1 and 2:1 complexes with γ-CD although a plateau was only observed in the case of the RSSR-diastereomer. As shown by CE-MS, both DCV-γ-CD complexes surprisingly comigrated as the first peak, while the second migrating peak represents non-complexed DCV.

Chromones bearing amino acid residues: Easily accessible and potent inhibitors of the breast cancer resistance protein ABCG2

The Breast Cancer Resistance Protein (BCRP/ABCG2) belongs to the G class of ABC (ATP-Binding Cassette) proteins, which is known as one of the main transporters involved in the multidrug resistance (MDR) phenotype that confer resistance to anticancer drugs. The aim of this study was to design, synthesize and develop new potent and selective inhibitors of BCRP that can be used to abolish MDR and potentialize clinically used anticancer agents. In previous reports, we showed the importance of chromone scaffold and hydrophobicity for the inhibition of ABC transporters. In the present study we report the design and development of chromones linked to one or two amino acids residues that are either hydrophobic or found in the structure of FTC, one of most potent (but highly toxic) inhibitors of BCRP. Herewith, we report the synthesis and evaluation of 13 compounds. The studied molecules were found to be not toxic and showed strong inhibition activity as well as high selectivity toward BCRP. The highest activity was obtained with the chromone bearing a valine residue (9c) which showed an inhibition activity against BCRP of 50 nM. The rationalization of the inhibition potential of the most active derivatives was performed through docking studies. Taken together, the ease of synthesis and the biological profile of these compounds render them as promising candidates for further development in the field of anticancer therapy.

Antibacterial Activity and Mechanism of a Bacteriocin Derived from the Valine-Cecropin A(1–8)-Plantaricin ZJ5(1–18) Hybrid Peptide Against Escherichia coli O104

Peptide fragment hybridization is an effective method to obtain novel hybrid antimicrobial peptides with higher antibacterial activities. The novel peptide, valine-cecropin A(1–8)-plantaricin ZJ5(1–18) (CA-P), was designed by coupling the amphiphilic N-terminal fragment of CA with the N-terminal core helix of P and adding a valine residue to the N-terminus of the hybrid fragment. CA-P showed higher antibacterial activities than the parental peptide P against all indicator strains in the experiment, with no hemolytic activity against sheep red blood cells. Observations by scanning electron microscopy and transmission electron microscopy confirmed that CA-P destroyed the surface structure of the bacteria and caused leakage of the cellular contents. As determined by fluorescence microscopy, the antibacterial mechanism of CA-P is microorganism killing. It was observed that CA-P and Litsea mollis Hemsl. essential oil showed a significant synergistic effect against Salmonella enterica serovar Newport.

Unexpected Enhancement of HDACs Inhibition by MeS Substitution at C-2 Position of Fluoro Largazole.

Given our previous finding that fluorination at the C18 position of largazole showed reasonably good tolerance towards inhibitory activity and selectivity of histone deacetylases (HDACs), further modification on the valine residue in the fluoro-largazole’s macrocyclic moiety with S-Me l-Cysteine or Glycine residue was performed. While the Glycine-modified fluoro analog showed poor activity, the S-Me l-Cysteine-modified analog emerged to be a very potent HDAC inhibitor. Unlike all previously reported C2-modified compounds in the largazole family (including our recent fluoro-largazole analogs) where replacement of the Val residue has failed to provide any potency improvement, the S-Me l-Cysteine-modified analog displayed significantly enhanced (five–nine-fold) inhibition of all the tested HDACs while maintaining the selectivity of HDAC1 over HDAC6, as compared to largazole thiol. A molecular modeling study provided rational explanation and structural evidence for the enhanced inhibitory activity. This new finding will aid the design of novel potent HDAC inhibitors.

Computationally Guided Tuning of Amino Acid Configuration Influences the Chiroptical Properties of Supramolecular Peptide-π-Peptide Nanostructures.

Self-assembled supramolecular materials derived from peptidic macromolecules with π-conjugated building blocks are of enormous interest because of their aqueous solubility and biocompatibility. The design rules to achieve tailored optoelectronic properties from these types of materials can be guided by computation and virtual screening rather than intuition-based experimental trial-and-error. Using machine learning, we reported previously that the supramolecular chirality in self-assembled aggregates from VEVAG-π-GAVEV type peptidic materials was most strongly influenced by hydrogen bonding and hydrophobic packing of the alanine and valine residues. Herein, we build upon this idea to demonstrate through molecular-level experimental characterization and all-atom molecular modeling that varying the stereogenic centers of these residues has a profound impact on the optoelectronic properties of the supramolecular aggregates, whereas the variation of stereogenic centers of other residues has only nominal influence on these properties. This study highlights the synergy between computational and experimental insight relevant to the control of chiroptical or other electronic properties associated with supramolecular materials.

Abstract PR04: Anti peptide-HLA (TCR-like) antibodies specific for the KRAS G12V neoantigen

Abstract The remarkably high prevalence of Ras driver mutations in tumors of diverse origins and stages indicates that Ras neoantigens are poor substrates for HLA-mediated detection by the native immune system. Nevertheless, immunotherapies based upon tumor-infiltrating lymphocytes targeting Ras neoantigens have been reported, indicating that such mutations are subject to HLA display and immune surveillance. Antibodies capable of recognizing specific peptide-HLA complexes at the cell surface have the potential to target intracellular tumor neoantigens for therapeutic intervention. However, such reagents have proved to be challenging to develop. Using a novel antibody discovery platform in combination with a proprietary human germline scFv library, we have developed a rapid method for the isolation of antibodies against peptide-HLA complexes. Applying this technology to KRAS G12V, we have isolated two scFvs that recognize the peptide VVVGAVGVGK in complex with HLA-A*11:01. This peptide-HLA combination forms functional complexes both in vitro and at the cell surface and has been reported to elicit CTL responses in vivo. In contrast, the native peptide is a poor substrate for binding to HLA-A*11:01. Binding affinities of the two scFv binders for the target HLA complex are in the sub-100nM range. Both show highly specific, peptide-dependent recognition of the KRAS-HLA-A*11:01 complex, as demonstrated by the failure to bind complexes containing wild-type KRAS peptides or other control peptides. Alanine scanning mutagenesis reveals multiple residues within the neoepitope peptide that are critical for antibody binding, including the mutated valine residue. The extended binding footprint encompassing the neoepitope and the poor presentation of the unmutated peptide are likely to contribute to the on-target specificity of these antibodies. Incorporation of the scFvs into a bispecific T-cell engager (BiTE) format has demonstrated the simultaneous engagement of both the KRAS HLA-A*11:01 complex and the CD3 surface marker of T cells. Ongoing work is aimed to demonstrate BiTE-dependent CTL-mediated lysis of target cells displaying the KRAS HLA-A*11:01 complex. We are currently developing CAR-T cells carrying peptide-HLA specific scFvs and are screening further mutant Ras peptides in the context of other HLA-A subtypes. This abstract is also being presented as Poster B48. Citation Format: Avisa Maserati, Dahna Makris, Ben Kiefel, Matt Beasley, Brad McColl, Ben Clifton. Anti peptide-HLA (TCR-like) antibodies specific for the KRAS G12V neoantigen [abstract]. In: Proceedings of the AACR Special Conference on Targeting RAS-Driven Cancers; 2018 Dec 9-12; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(5_Suppl):Abstract nr PR04.