Study Of Novel HIV-1Fusion Inhibitor And Six-helix Bundle-based Artificial Enzyme

This study contains two parts. Part I is “Study of Novel HIV-1Fusion Inhibitor”. PartII is “Study of Six-helix Bundle-basedArtificial Enzyme”.Part I Study of Novel HIV-1Fusion InhibitorFusion inhibitors were introduced as a third class of anti-HIV-1drugs, followingreverse transcriptase inhibitors and protease inhibitors. HIV entry is mediated by theenvelope glycoproteins gp120and gp41. The gp41subunit contains several functionaldomains: the N-terminal heptad repeat （NHR） domains fold a triple stranded coiled-coil forming a meta-stable prefusion intermediate. C-terminal heptad repeat （CHR） sub-sequently folds onto the hydrophobic grooves of the NHR coiled-coil to form a stable6-helix bundle, which juxtaposes the viral and cellular membranes for fusion. Inhibitionof the formation of this6-helixbundle prevents fusion of HIV-1and targeted host cellmembranes. Compared to the previous two classes of anti-HIV drugs, fusion inhibitorsact extracellularly prior to invasion of the host cell, interrupting transmission in theinitial infection of HIV-1. Peptide fusion inhibitor T-20was approved in2003. A grow-ing number of fusion inhibitors are under clinical development. The majority issues ofT-20in the clinical are the resistance, low bioavailability, and high price. Therefore,how to solve clinical resistance is becoming the focus of study fusion inhibitors （espe-cially C-peptide inhibitors）Multivalent drug design is a good strategy to improve the affinity of the inhibitorand targets. It is an efficient strategy for inhibitor design based on an additive effect ofG. Many successful multivalent drugs has been reported.In this research, we chose C-peptides as the template sequences to study bivalentinhibitors targeting HIV-1gp41NHR domain. We optimized the crosslink sites andlinkers to improve anti-HIV activities of the bivalent fusion inhibitors. The terminalcysteine was introduced to connect with linker and active peptide. Based on this design,we just need to prepare a single peptide to obtain bivalent molecule after oxidation.Two single peptides of one bivalent molecules are connected with terminal cysteinethiol groups by disulfide bridge. Such preparation is efficient, high yield and clean. We designed the two main categories of bivalent inhibitors, containing T-20or C34-serieslong peptide and C29, C22and SC22EK-series short peptide. It was easy to find thelaw of inhibitors based on T-20or C34-series bivalent peptides, which have been ex-tensively studied. All peptide sequences were designed to the N-terminal or C-terminalcrosslink mode to find the best active compounds. Compared to long peptide, shorterpeptides have advantages in synthesis, bioavailability and cost. Therefore, the short bi-valent peptide molecules are value in clinical more than long peptides.Peptides were synthesized by standard Fmoc solid phase synthesis, purified by pre-parative liquid, and the polypeptide structure were identified by MALDI-TOF, whosepurity was determined by HPLC （the purity of all compounds were more than94%）.We have synthesized41compounds （C34and T-20series20, C29Series13, C22series5and SC22EK series3）. Anti-HIV-1activity was evaluated by cell-cell fusion exper-iment.In this study, we examined the cross-linking sites, linkers and bioactive peptidesequences over cooperative effects. Compared to monovalent molecules, significantcooperative effects in the anti-cell-cell fusion activity were observed in the bivalentinhibitors, at either N-or C-terminal crosslink of both C34and T-20. However, C34and T-20series bivalent compounds cooperate obviously, but the absolute activity donot still surpass template sequences （C34or T-20）. It may caused by one dimensionalsequence design. And C29series compounds, only N-terminus can cause cooperativeeffect. C22series compounds, due to its own low affinity, so its N-and C-terminalcooperative effect was not observed.Linker is crucial for cooperative effect. β-alanine may be the most suitable linkerfor N-terminal crosslink, while no linker is best for C-terminal crosslink. Specially, theanti-HIV IC50of peptide （CβA-C34）₂[（Cys-CONH-CH₂CH₂CO-C34）₂] was improvedfrom43.7nM to6.4nm, indicating the two C-peptide chains had a cooperative effect.The choice of the active sequence is the most import factor for cooperative effect.C34and T-20series compounds, their absolute activity still does not exceed templatesequence, due to binding mobility rate. We further reduce the peptide sequence length,and the peptide sequence was reduced to29amino acids length. The C29bivalent in-hibitors appear a significant activity improvement, and such activity caused by cooper-ative effect between the two-peptide chains. We believe the N-terminal is the suitablecrosslink site for C29-series peptides, because the activity of N-crosslink product ishigher. Especially, the molar IC50of peptide （CAca-C29）₂was improved from49.02 nM （monomer form） to5.71nM, and （CAca-SC22EK）₂was improved to4.92nM closeto the high active peptide fusion inhibitor C34. C29series compounds, cross-linked toform the bivalent inhibitors, and, relative template sequence C29, its activity increasedfrom2.4to35-fold （except C29-AcaC）. Activity increased maximum peptide（CTegβA-C29）[（Cys-CONH（CH₂CH₂O）₄-CONH-CH₂CH₂CO-C29）₂] increased to3.08nM activity has been equal to high activity sequence C34and T-20, and is mean-ingful in clinical. And we continue to selecte a shorter peptide C22to synthesize thebivalent inhibitors. Due its weak helicity, no cooperative effect appeared. Therefore, wechose SC22EK, which were introduced salt bridges to consolidate the helicity based onthe natural sequence C22. The cross-linked （CAca-SC22EK）₂was⁸times more potentthan the monomer SC22EK in anti-HIV activity.The strategy used in this study could be used to design new fusion inhibitors tointerfere similar processes. The activity of short bivalent peptide has been equal to highactivity sequence C34and T-20, and is meaningful in clinical. Shorter peptides haveadvantages in synthesis, bioavailability and cost.Part II Study of Six-helix Bundle-based Artificial EnzymeNatural enzymes lose their activities under extreme environments （i.e. high tem-perature, solvents with denaturants or high-salt concentration et. al.） due to their tertiarystructure dissociation. Here, we try to elucidate a novel strategy to enhance the stabilityand activity of biomolecules under extreme environments by constructing peptidic ar-tificial enzymes with inter-helical covalent bonds.Histidine-based residue reactive sites for the catalysis of hydrolysis reaction of p-nitrophenyl esters have been engineered into a helix in a designed6-helix, and it hasbeen shown to function through cooperative nucleophilic and general-acid catalysis.We construct a covalent bond stablized polypeptide six helix, into which cooperativehistidines are intrduced as a functional group for artificial enzyme research. The six-helix was derived from HIV-1gp41core structure of the fusion state. In the fusion state,the N-and C-heptad repeats（CHR） fold in an antiparallel manner to create a six-helixbundle （6HB）consisting of a trimeric NHR coiled-coil core with three antiparallel CHRthat pack in the grooves formed by the NHR core. We designed single covalent bondcrosslinked six-helix （6HB-1） group, two covalently cross-linked six-helix （6HB-2） andtwo relative non-crosslinked control groups. Peptides were synthesized by standard Fmoc solid phase synthesis, purified by pre-parative liquid, and the polypeptide structure were identified by MALDI-TOF, whosepurity was determined by HPLC （the purity of all compounds were more than97%）.Inter-helical covalent bonds were introduced via an acyl-transfer reaction betweenC-peptides and N-peptides. The choices of crosslink sites were based on our previouswork.6HB-1and6HB-2were employed to test catalytic ability and determination thecontent of helix structure under the conditions of high temperature, high salt concen-tration and high surfactant concentration.It was observed that the introduced inter-helical covalent bonds did not interruptthe assembly.The covalent bond stabilized compounds reflects a distinct advantage in stability.For the saturation kinetics, inter-helical covalent bonds did not affect the catalytic char-acteristics of the enzyme system; as if natural enzyme. Double covalent bonds stabi-lized,6HB structure is more stable under high temperature conditions. Above50℃,covalently stable6HB catalytic ability was stronger than the control group （withoutcovalent bond6HB）.From the point of view of the absolute activity, under the condi-tions of80℃, double covalent bond cross-linking group still maintained a high catalyticactivity. In the condition of4M urea, the two6HBs control group lost all helix structure.In addition,6HB-2, although in8M urea, almost still maintains100%helical. In thecondition of8M urea,6HB-2remains57%of the hydrolysis ability. When the concen-tration of Brji35increased to1%,6HB-1and6HB-2retained⁵0%activities. On thecontrary,6HB-1C and6HB-2C only retained¹0%activities.Covalent bonds are introduced into the six-helix bundles via acyl-transfer reaction.The stabilized artificial enzymes with hydrolysis catalytic ability were tested under con-ditions of high temperature, high salt concentration and high surfactant agents concen-tration. Covalent bonds stabilizing maintain the catalytic energy in extreme condition.Perhaps, this conclusion could give some clues to reveal the mysterious phenomenonof life understand the extreme chemical environment or physical environmental condi-tions.