C-peptides Research Articles

The C34 Peptide Fusion Inhibitor Binds to the Six-Helix Bundle Core Domain of HIV-1 gp41 by Displacement of the C-Terminal Helical Repeat Region.

The conformational transition of the core domain of HIV-1 gp41 from a prehairpin intermediate to a six-helix bundle is responsible for virus-cell fusion. Several inhibitors which target the N-heptad repeat helical coiled-coil trimer that is fully accessible in the prehairpin intermediate have been designed. One such inhibitor is the peptide C34 derived from the C-heptad repeat of gp41 that forms the exterior of the six-helix bundle. Here, using a variety of biophysical techniques, including dye tagging, size-exclusion chromatography combined with multiangle light scattering, double electron-electron resonance EPR spectroscopy, and circular dichroism, we investigate the binding of C34 to two six-helix bundle mimetics comprising N- and C-heptad repeats either without (core(SP)) or with (core(S)) a short spacer connecting the two. In the case of core(SP), C34 directly exchanges with the C-heptad repeat. For core(S), up to two molecules of C34 bind the six-helix bundle via displacement of the C-heptad repeat. These results suggest that fusion inhibitors such as C34 can target a continuum of transitioning conformational states from the prehairpin intermediate to the six-helix bundle prior to the occurrence of irreversible fusion of viral and target cell membranes.

The N-Terminal T-T Motif of a Third-Generation HIV-1 Fusion Inhibitor Is Not Required for Binding Affinity and Antiviral Activity.

The highlighted next-generation HIV-1 fusion inhibitor peptide 1 is capped by two threonines. Here, we generated peptide 2 by deleting the T-T motif and compared their structural and antiviral properties. Significantly, two peptides showed similar helical and oligomeric states in solution, comparable binding affinities to the target, and no significant difference to inhibit HIV-1 fusion and infection. Also, the T-T motif was not associated with peptide 1 resistant mutations and its deletion did not affect peptide 1 against enfuvirtide-resistant HIV-1 mutants. The redundancy of the T-T motif was further verified by the model peptide C34 and short peptide inhibitors that mainly target the gp41 pocket, suggesting that the N-terminal T-T motif of peptide 1 could be removed or modified toward the development of new anti-HIV-1 drugs. Consistently, our data have verified that the M-T hook structure rather than the T-T motif is an efficient strategy for short peptide fusion inhibitors.

Enhanced HIV Fusion Inhibitors Efficacy Requires Membrane Affinity and Exposure of the Pocket Binding Domain of C34 Derivatives

Viral fusion inhibitors block the fusion between the membranes of an enveloped virus and a target cell, therefore preventing the entry of the viral content. It has been demonstrated that the combination of cholesterol-tagging and dimerization of the C34 peptide sequence resulted both in an increase of the antiviral potency and extension of the in vivo half-life of two new HIV fusion inhibitors: HIVP3 (C34-PEG4-cholesterol) and HIVP4 ([C34-PEG4]2-cholesterol). Given the importance of lipophilicity and selective affinity for different lipid domains, the aim was to evaluate the interaction of these HIV fusion inhibitor peptides with biomembranes model systems and human blood cells, in order to clarify where and how they are located. This study allowed the understanding of the mechanism of action of these peptides at the molecular level, and which strategies may be followed to increase their fusion inhibition efficacy. Membrane partition, dipole potential and surface pressure measurements indicated that HIVP3 and HIVP4 interact preferentially with cholesterol-rich liquid ordered membranes. HIVP3 and HIVP4 are able to interact with human erythrocytes and peripheral blood mononuclear cells to a similar degree as previously described for the simpler drug C34-cholesterol. However, the pocket binding domain(PBD) of both HIVP3 and HIVP4 is more exposed to the aqueous environment than in C34-cholesterol. The efficient blocking of HIV entry results from the synergic effect between the membranotropic behavior and the enhanced exposure of the PBD[1]. Maximizing antiviral activity requires finding the proper balance of membrane affinity and exposure of the peptide moiety, through variations in the lipid-binding domain, PEG spacer region, and number of peptide moieties in the construct.[1] Augusto MT et al.(2014)Improvement of the HIV fusion inhibitor C34 efficacy by membrane anchoring and enhanced exposure.J Antimicrob Chemother,69:1286-1297

Engineered FVIII-Specific Human T-Effector Cells: Can the T-Cell Receptor Modulate CD4+ T-Helper Phenotypes or Is It Just a “Switch”?

Engineered T cells are a vital component in the armamentarium of cellular therapies. In this presentation, we examine how human CD4+ T cells, genetically engineered to express a T-cell receptor (TCR) specific for a C2 domain epitope of the coagulation protein cofactor FVIII, can be skewed or polarized to different T-helper subsets. Two TCRs were cloned from Th2 and Th17/Th1 phenotyped CD4+ T cells isolated via a tetramer guided epitope mapping (TGEM) technique from a hemophilia A subject after clinical diagnosis of an inhibitor (neutralizing antibody) to FVIII given as replacement therapy. The two TCRs were cloned using a 5’ RACE with semi-nested PCR and transduced via a retroviral vector into healthy non-hemophilia A human donor CD4+ T cells. Based on proliferation and HLA class II tetramer staining data, engineered CD4+ T cells expressing the different cloned TCRs exhibited different avidities for the same C2 peptide (containing the epitope) over a dose titration curve, despite similar levels of TCR expression on the CD4 T-cell surface. IFN-γ, TNF-α, IL-6, and IL-10 cytokine production levels following stimulation with C2 peptide and DR1 antigen presenting cells, as measured by cytokine bead analysis, were significantly greater for the higher avidity TCR, which was cloned from a “Th2” phenotyped CD4+ T-cell clone. Interestingly, neither the engineered CD4+ T cells expressing the Th2 TCR nor the cells expressing the Th17/Th1 TCR produced cytokines characteristic of their respective original parental clones. Rather, they reflected the cytokine profiles of the donor populations used for transduction. These preliminary data led us to investigate how the different avidities of the two cloned TCRs can modulate the T-helper subset skewing/differentiation potential of engineered CD4+T cells. We hypothesized that the TCR is merely a switch that can activate or direct engineered CD4+ T cells to an antigen-specific response that would be skewed to the T-helper phenotypes of the cells prior to TCR transduction. We further hypothesized that this response could be modulated after TCR transduction according to the apparent tetramer avidity of the engineered cells. We successfully skewed the engineered human T-helper cells to Th1, Th2 and Th17 lineages, based on T-helper signature cytokine expression and the transcription factors T-bet, Gata3 and RORγt. Moreover, we observed that TCR transduction into naïve human CD4+ T cells did not itself affect the T-helper subset skewing of the cells. Preliminary experiments showed a trend toward Th2 skewing for the high avidity Th2 CD4+ T cells having an engineered TCR when they were cultured under either Th1 or Th2 polarizing conditions and stimulated with the C2 peptide, compared to the phenotypes obtained following stimulation of polyclonal CD4 T cells with anti-CD3. These studies will improve our designing of engineered TCRs for CD4+T-cell therapy, especially when concerns of T-helper effector function and plasticity are important to clinical outcomes.Supported by NIH RO1-HL061883 (DWS), funding from Bayer and CSL Behring (KPP) and intramural support from NIAID (EMS). We thank Dr. Arthur Thompson (Puget Sound Blood Center) for enrolling patients and we thank all blood donors. DisclosuresNo relevant conflicts of interest to declare.

783. Cross-Clade Inhibition of HIV on Primary Cells by CXCR4 or CCR5 Fused to the C34 Peptide from gp41 HR2

783. Cross-Clade Inhibition of HIV on Primary Cells by CXCR4 or CCR5 Fused to the C34 Peptide from gp41 HR2

Collagen VI and hyaluronan: the common role in breast cancer.

Collagen VI and hyaluronan are widely distributed extracellular matrix macromolecules that play a crucial role in tissue development and are highly expressed in cancers. Both hyaluronan and collagen VI are upregulated in breast cancer, generating a microenvironment that promotes tumour progression and metastasis. A growing number of studies show that these two molecules are involved in inflammation and angiogenesis by recruiting macrophages and endothelial cells, respectively. Additionally, collagen VI induces epithelial-mesenchymal transition that is correlated to increased synthesis of hyaluronan in mammary cells. Hyaluronan has also a specific role in cellular functions that depends mainly on the size of the polymer, whereas the effect of collagen VI in tumour progression may be the result of the intact molecule or the C5 peptide of α3(VI) chain, known as endotrophin. Collectively, these findings strongly support the parallel role of these molecules in tumour progression and suggest that they may be used as prognostic factors for the breast cancer treatment.

A New Paradigm for Enzymatic Control of α-Cleavage and β-Cleavage of the Prion Protein

The cellular form of the prion protein (PrP(C)) is found in both full-length and several different cleaved forms in vivo. Although the precise functions of the PrP(C) proteolytic products are not known, cleavage between the unstructured N-terminal domain and the structured C-terminal domain at Lys-109↓His-110 (mouse sequence), termed α-cleavage, has been shown to produce the anti-apoptotic N1 and the scrapie-resistant C1 peptide fragments. β-Cleavage, residing adjacent to the octarepeat domain and N-terminal to the α-cleavage site, is thought to arise from the action of reactive oxygen species produced from redox cycling of coordinated copper. We sought to elucidate the role of key members of the ADAM (a disintegrin and metalloproteinase) enzyme family, as well as Cu(2+) redox cycling, in recombinant mouse PrP (MoPrP) cleavage through LC/MS analysis. Our findings show that although Cu(2+) redox-generated reactive oxygen species do produce fragmentation corresponding to β-cleavage, ADAM8 also cleaves MoPrP in the octarepeat domain in a Cu(2+)- and Zn(2+)-dependent manner. Additional cleavage by ADAM8 was observed at the previously proposed location of α-cleavage, Lys-109↓His-110 (MoPrP sequencing); however, upon addition of Cu(2+), the location of α-cleavage shifted by several amino acids toward the C terminus. ADAM10 and ADAM17 have also been implicated in α-cleavage at Lys-109↓His-110; however, we observed that they instead cleaved MoPrP at a novel location, Ala-119↓Val-120, with additional cleavage by ADAM10 at Gly-227↓Arg-228 near the C terminus. Together, our results show that MoPrP cleavage is far more complex than previously thought and suggest a mechanism by which PrP(C) fragmentation responds to Cu(2+) and Zn(2+).

Structure activity relationships of peptidic analogs of MyoD for the development of Id1 inhibitors as antiproliferative agents

Id proteins, inhibitors of DNA binding proteins, have highly conserved dimerization motif known as the helix-loop-helix (HLH) domain that acts as a negative regulator of basic HLH (bHLH) transcription factors. In signaling pathways, Id proteins play an important role in cellular development, proliferation, and differentiation. The mechanism of Id proteins is to antagonize bHLH proteins, thereby preventing them from binding to DNA and inhibiting transcription of cellular differentiation-associated genes in cancer. Recently, we reported an inhibitor of Id1, peptide 3C, which showed good affinity to Id1 protein and exhibited inhibitory effects in cancer cells. In this study, Ala (A)-substituted analogs of peptide 3C were synthesized by SPPS, purified by RP-HPLC, and characterized by MALDI-TOF MS. Binding of each peptide to Id1 or Id1-HLH (the HLH domain of Id1) was monitored by surface plasmon resonance (SPR)-based biosensor. Biological effect of each peptide in MCF-7 breast cancer cells was analyzed by MTT cell viability assay. The secondary structure of substituted analogs of peptide 3C was investigated by circular dichroism (CD) spectroscopy. SPR results revealed that A-substituted analogs of peptide 3C showed weaker binding to Id1 than that of peptide 3C, indicating that the six amino acid residues in the N-terminal of peptide 3C were all essential for binding to Id1 and the importance of amino acid residue was I(2) > Q(6) > Y(1) > G(4) > L(5) > E(3). In addition, substitution of E(3) in peptide 3C with D, Q, and R did not improve the binding potency of peptide 3C. MTT assay demonstrated that neither A-substituted nor position 3-substituted analogs of peptide 3C showed increased antiproliferative effect in MCF-7 cancer cells. CD results indicated that peptide 3C exhibited the highest content of α-helical structure (39.37%), suggesting that the α-helical structure may contribute to its binding potency for Id1 and Id1-HLH. SAR results provided important information for the development of peptidic inhibitors of Id1 as anticancer agents and demonstrated peptide 3C as a promising lead for further modifications.

Multimerized CHR-derived peptides as HIV-1 fusion inhibitors

To date, several HIV-1 fusion inhibitors based on the carboxy-terminal leucine/isoleucine heptad repeat (CHR) region of an HIV-1 envelope protein gp41 have been discovered. We have shown that a synthetic peptide mimetic of a trimer form of the CHR-derived peptide C34 has potent inhibitory activity against the HIV-1 fusion mechanism, compared to a monomer C34 peptide. The present study revealed that a dimeric form of C34 is evidently structurally critical for fusion inhibitors, and that the activity of multimerized CHR-derived peptides in fusion inhibition is affected by the properties of the unit peptides C34, SC34EK, and T20. The fluorescence-based study suggested that the N36-interactive sites of the C34 trimer, including hydrophobic residues, are exposed outside the trimer and that trimerization of C34 caused a remarkable increase in fusion inhibitory activity. The present results could be useful in the design of fusion inhibitors against viral infections which proceed via membrane fusion with host cells.

46. T-Cells Edited To Express the C34 Peptide from gp41 Heptad Repeat-2 Fused to CCR5 or CXCR4 Exhibit Robust Protection from Diverse HIV-1 Isolates

46. T-Cells Edited To Express the C34 Peptide from gp41 Heptad Repeat-2 Fused to CCR5 or CXCR4 Exhibit Robust Protection from Diverse HIV-1 Isolates

Design, Synthesis, and Biological Evaluation of Highly Potent Small Molecule–Peptide Conjugates as New HIV-1 Fusion Inhibitors

The small molecule fusion inhibitors N-(4-carboxy-3-hydroxyphenyl)-2,5-dimethylpyrrole (NB-2) and N-(3-carboxy-4-hydroxyphenyl)-2,5-dimethylpyrrole (A12) target a hydrophobic pocket of HIV-1 gp41 and have moderate anti-HIV-1 activity. In this paper, we report the design, synthesis, and structure-activity relationship of a group of hybrid molecules in which the pocket-binding domain segment of the C34 peptide was replaced with NB-2 and A12 derivatives. In addition, the synergistic effect between the small molecule and peptide moieties was analyzed, and lead compounds with a novel scaffold were discovered. We found that either the nonpeptide or peptide part alone showed weak activity against HIV-1-mediated cell-cell fusion, but the conjugates properly generated a strong synergistic effect. Among them, conjugates Aoc-βAla-P26 and Noc-βAla-P26 exhibited a low nanomolar IC50 in the cell-cell fusion assay and effectively inhibited T20-sensitive and -resistant HIV-1 strains. Furthermore, the new molecules exhibited better stability against proteinase K digestion than T20 and C34.

Crystal structure of human tyrosylprotein sulfotransferase-2 reveals the mechanism of protein tyrosine sulfation reaction

Post-translational protein modification by tyrosine-sulfation plays an important role in extracellular protein-protein interactions. The protein tyrosine sulfation reaction is catalyzed by the Golgi-enzyme called the tyrosylprotein sulfotransferase (TPST). To date, no crystal structure is available for TPST. Detailed mechanism of protein tyrosine sulfation reaction has thus remained unclear. Here we present the first crystal structure of the human TPST isoform 2 (TPST2) complexed with a substrate peptide (C4P5Y3) derived from complement C4 and 3’-phosphoadenosine-5’-phosphate (PAP) at 1.9Å resolution. Structural and complementary mutational analyses revealed the molecular basis for catalysis being an SN2-like in-line displacement mechanism. TPST2 appeared to recognize the C4 peptide in a deep cleft by using a short parallel β-sheet type interaction, and the bound C4P5Y3 forms an L-shaped structure. Surprisingly, the mode of substrate peptide recognition observed in the TPST2 structure resembles that observed for the receptor type tyrosine kinases.

The M-T Hook Structure Is Critical for Design of HIV-1 Fusion Inhibitors

CP621-652 is a potent HIV-1 fusion inhibitor peptide derived from the C-terminal heptad repeat of gp41. We recently identified that its N-terminal residues Met-626 and Thr-627 adopt a unique hook-like structure (termed M-T hook) thus stabilizing the interaction of the inhibitor with the deep pocket on the N-terminal heptad repeat. In this study, we further demonstrated that the M-T hook structure is a key determinant of CP621-652 in terms of its thermostability and anti-HIV activity. To directly define the structure and function of the M-T hook, we generated the peptide MT-C34 by incorporating Met-626 and Thr-627 into the N terminus of the C-terminal heptad repeat-derived peptide C34. The high resolution crystal structure (1.9 Å) of MT-C34 complexed by an N-terminal heptad repeat-derived peptide reveals that the M-T hook conformation is well preserved at the N-terminal extreme of the inhibitor. Strikingly, addition of two hook residues could dramatically enhance the binding affinity and thermostability of 6-helix bundle core. Compared with C34, MT-C34 exhibited significantly increased activity to inhibit HIV-1 envelope-mediated cell fusion (6.6-fold), virus entry (4.5-fold), and replication (6-fold). Mechanistically, MT-C34 had a 10.5-fold higher increase than C34 in blocking 6-helix bundle formation. We further showed that MT-C34 possessed higher potency against T20 (Enfuvirtide, Fuzeon)-resistant HIV-1 variants. Therefore, this study provides convincing data for our proposed concept that the M-T hook structure is critical for designing HIV-1 fusion inhibitors.

The Conserved Residue Arg46 in the N-Terminal Heptad Repeat Domain of HIV-1 gp41 Is Critical for Viral Fusion and Entry

During the process of HIV-1 fusion with the target cell, the N-terminal heptad repeat (NHR) of gp41 interacts with the C-terminal heptad repeat (CHR) to form fusogenic six-helix bundle (6-HB) core. We previously identified a crucial residue for 6-HB formation and virus entry - Lys63 (K63) in the C-terminal region of NHR (aa 54–70), which forms a hydrophobic cavity. It can form an important salt bridge with Asp121 (D121) in gp41 CHR. Here, we found another important conserved residue for virus fusion and entry, Arg46 (R46), in the N-terminal region of NHR (aa 35–53), which forms a hydrogen bond with a polar residue, Asn43 (N43), in NHR, as a part of the hydrogen-bond network. R46 can also form a salt bridge with a negatively charged residue, Glu137 (E137), in gp41 CHR. Substitution of R46 with the hydrophobic residue Ala (R46A) or the negatively charged residue Glu (R46E) resulted in disruption of the hydrogen bond network, breakage of the salt bridge and reduction of 6-HB’s stability, leading to impairment of viral fusion and decreased inhibition of N36, an NHR peptide. Similarly, CHR peptide C34 with substitution of E137 for Ala (E137A) or Arg (E137R) also exhibited reduced inhibitory activity against HIV-1 infection and HIV-1-mediated cell-to-cell fusion. These results suggest that the positively charged residue R46 and its hydrogen bond network, together with the salt bridge between R46 and E137, are important for viral fusion and entry and may therefore serve as a target for designing novel HIV fusion/entry inhibitors.

HIV-1 gp120 Vaccine Induces Affinity Maturation in both New and Persistent Antibody Clonal Lineages

Most antibodies that broadly neutralize HIV-1 are highly somatically mutated in antibody clonal lineages that persist over time. Here, we describe the analysis of human antibodies induced during an HIV-1 vaccine trial (GSK PRO HIV-002) that used the clade B envelope (Env) gp120 of clone W6.1D (gp120(W6.1D)). Using dual-color antigen-specific sorting, we isolated Env-specific human monoclonal antibodies (MAbs) and studied the clonal persistence of antibodies in the setting of HIV-1 Env vaccination. We found evidence of V(H) somatic mutation induced by the vaccine but only to a modest level (3.8% ± 0.5%; range 0 to 8.2%). Analysis of 34 HIV-1-reactive MAbs recovered over four immunizations revealed evidence of both sequential recruitment of naïve B cells and restimulation of previously recruited memory B cells. These recombinant antibodies recapitulated the anti-HIV-1 activity of participant serum including pseudovirus neutralization and antibody-dependent cell-mediated cytotoxicity (ADCC). One antibody (3491) demonstrated a change in specificity following somatic mutation with binding of the inferred unmutated ancestor to a linear C2 peptide while the mutated antibody reacted only with a conformational epitope in gp120 Env. Thus, gp120(W6.1D) was strongly immunogenic but over four immunizations induced levels of affinity maturation below that of broadly neutralizing MAbs. Improved vaccination strategies will be needed to drive persistent stimulation of antibody clonal lineages to induce affinity maturation that results in highly mutated HIV-1 Env-reactive antibodies.

The use of antiendotoxin peptides in obstructive jaundice endotoxemia

Two novel antiendotoxin peptides, P6 and C1, may reduce the inflammatory response. This study aimed to determine the effect of endotoxin on hepatic cytokine response and to assess P6 and C1-related attenuation of the proinflammatory response to endotoxemia, in experimental biliary obstruction. 15 Male Wistar rats were randomized to one of three groups: bile duct ligation (BDL)+P6 (n=5), BDL+C1 (n=5), and BDL+no peptide (n=5). Rats were weighed and underwent BDL surgery on day 1. On day 8, the rats were reweighed and isolated hepatic perfusion was carried out. P6 or C1 peptide (10 μmol/l) was preincubated with 300 ml of endotoxin-containing Krebs perfusate. After perfusion of 10 min with endotoxin-free perfusate, the livers were perfused for another 10 min with 300 ml of perfusate-containing endotoxin on its own or endotoxin plus peptide. This was followed by a further 100 min of perfusion with endotoxin-free perfusate. Effluent perfusate was collected at 20-min intervals for subsequent biochemical and cytokine analyses. Perfusion with endotoxin+P6 or endotoxin+C1 resulted in no significant difference in weight loss, or interleukin-6 response compared with perfusion with endotoxin alone. However, perfusion with endotoxin+P6 or endotoxin+C1 significantly reduced the tumor necrosis factor-α response to portal endotoxemia compared with perfusion with endotoxin alone. This study demonstrates that novel antiendotoxin peptides may attenuate the hepatic inflammatory response in portal endotoxemia. In obstructive jaundice, preoperative peptide administration may reduce endotoxin-related postoperative complications.

Preexposure Prophylaxis with Albumin-Conjugated C34 Peptide HIV-1 Fusion Inhibitor in SCID-hu Thy/Liv Mice

PC-1505 is a C34 peptide derived from the heptad repeat 2 region of HIV-1 gp41 conjugated to human serum albumin for sustained in vivo activity. One single preexposure dose of PC-1505 reduced viral RNA in HIV-1-infected SCID-hu Thy/Liv mice by 3.3 log₁₀ and protected T cells from virus-mediated depletion. In contrast, a single preexposure dose of Truvada reduced viral RNA by only 0.8 log₁₀ and was substantially less effective in preventing T cell depletion.

Effects of Analogues of Hydra Peptide Morphogen on DNA Synthesis in the Myocardium of Newborn Albino Rats

DNA-synthetic activity of myocardial cells was studied by (3)H-thymidine autoradiography in newborn albino rats after intraperitoneal injection of hydra peptide morphogen and its analogues. Administration of hydra peptide morphogen stimulated proliferative activity in the myocardium. Short analogues of hydra peptide morphogen, 6C and 3C peptides, produced a similar effect. Administration of arginine-containing analogue of hydra peptide morphogen significantly reduced the number of DNA-synthesizing nuclei in the ventricular myocardium of newborn albino rats. The role of the structure of the peptide molecule in the realization of the morphogenetic effects of hydra peptide morphogen is discussed.

Heteromeric Assembled Polypeptidic Artificial Hydrolases with a Six‐Helical Bundle Scaffold

Enzyme efficiency results from the cooperation of functional groups in the catalytic site. In order to mimic a natural enzyme, a definite 3D scaffold must be carefully designed so that the functional groups can work cooperatively. During the HIV-1 fusion process, the gp41 N- and C-terminal heptad repeat regions form a coiled-coil six-helical bundle (6HB) that brings the viral and target cell membranes into close proximity for fusion. We used 6HB as the molecular model for a novel scaffold for the design of an artificial enzyme, in which the modified C34 and N36 peptides formed a unique 6HB structure through specific molecular recognition, and the position and orientation of the side-chain groups on this scaffold were predictable. The histidine modified 6HB C34(H13/20)/N36(H15/22) showed enzyme-like hydrolytic activity towards p-nitrophenyl acetate (PNPA; k(cat)/K(M) =3.66 M(-1) s(-1)) through the cooperation of several inter- or intrahelical imidazole groups. Since the catalytic activity of 6HB depends on the C- and N-peptide assembly, either HIV fusion inhibitors that can compete with the formation of catalytic 6HB or denaturants that can destroy the ordered structure were able to modulate its activity. Further engineering of the solvent-exposing face with Glu(-)-Lys(+) salt bridges enhanced the helicity and the stability of 6HB. As a result, the population and stability of cooperative catalytic units increased. In addition, the Glu(-)-Lys(+) -stabilized 6HB SC35(H13/20)/N36(H15/22) had increased catalytic efficiency (k(cat)/K(M) =6.30 M(-1) s(-1)). A unique 6HB system was specifically assembled and provided a scaffold sufficiently stable to mimic the function of enzymes or other biomolecules.

Structure–activity relationships of a peptidic antagonist of Id1 studied by biosensor method, circular dichroism spectroscopy, and bioassay

Id1 proteins, inhibitors of differentiation or DNA binding, act as dominant negative antagonists of the bHLH family of transcription factors, which play an important role in cellular development, proliferation, and differentiation. The mechanism of Id proteins is to antagonize bHLH proteins by forming high-affinity heterodimers with other bHLH proteins, thereby preventing them from binding to DNA and inhibiting transcription of differentiation-associated genes. Our goal is to study the SARs of a peptidic antagonist of Id1, peptide 3C, which exhibits high affinity for Id1 and inhibitory effect on the proliferation of cancer cells. A series of N-terminal- and C-terminal-deleted analogs of peptide 3C were designed, synthesized, and characterized. Affinity of each peptide for Id1 or Id1-HLH domain was determined by SPR-based biosensor. The secondary structure of each peptide was studied by CD spectroscopy. Biological effect of each peptide in breast cancer cell (MCF-7) was analyzed by the MTT cell viability assay. Results demonstrated that peptide 3C and peptide 3C-CtD4 exhibited higher affinity for Id1-HLH and the equilibrium dissociation constants (K(D) ) were 3.16 and 2.77 µM, respectively. CD results indicated that the percentage of α-helix (%) in the secondary structure of deleted peptides were different, ranging from 7.93 to 10.45%. Although MTT results showed that treatment of MCF-7 with these peptides did not cause antiproliferative effects in cancer cells, SPR results demonstrated that the high-affinity peptides 3C and 3C-CtD4 are promising for further modifications to enhance their affinity for Id1-HLH and antiproliferative effects in cancer cells and for the development of peptidic antagonists as anticancer agents.