Qiuyun Dai

Potent HIV fusion inhibitors against Enfuvirtide-resistant HIV-1 strains

T20 (generic name: Enfuvirtide, brand name: Fuzeon) is the only FDA-approved HIV fusion inhibitor that is being used for treatment of HIV/AIDS patients who have failed to respond to current antiretroviral drugs. However, it rapidly induces drug resistance in vitro and in vivo. On the basis of the structural and functional information of anti-HIV peptides from a previous study, we designed an HIV fusion inhibitor named CP32M, a 32-mer synthetic peptide that is highly effective in inhibiting infection by a wide range of primary HIV-1 isolates from multiple genotypes with R5- or dual-tropic (R5X4) phenotype, including a group O virus (BCF02) that is resistant to T20 and C34 (another anti-HIV peptide). Strikingly, CP32M is exceptionally potent (at low picomolar level) against infection by a panel of HIV-1 mutants highly resistant to T20 and C34. These findings suggest that CP32M can be further developed as an antiviral therapeutic against multidrug resistant HIV-1.

Identification of a Critical Motif for the Human Immunodeficiency Virus Type 1 (HIV-1) gp41 Core Structure: Implications for Designing Novel Anti-HIV Fusion Inhibitors

ABSTRACT Human immunodeficiency virus type 1 (HIV-1) entry into the host cell involves a cascade of events and currently represents one of most attractive targets in the search for new antiviral drugs. The fusion-active gp41 core structure is a stable six-helix bundle (6-HB) folded by its trimeric N-terminal heptad repeat (NHR) and C-terminal heptad repeat (CHR). Peptides derived from the CHR region of HIV-1 gp41 are potent fusion inhibitors that target the NHR to block viral and cellular membrane fusion in a dominant negative fashion. However, all CHR peptides reported to date are derived primarily from residues 628 to 673 of gp41; little attention has been paid to the upstream sequence of the pocket binding domain (PBD) in the CHR. Here, we have identified a motif (621QIWNNMT627) located at the upstream region of the gp41 CHR, immediately adjacent to the PBD (628WMEWEREI635). Biophysical characterization demonstrated that this motif is critical for the stabilization of the gp41 6-HB core. The peptide CP621-652, containing the 621QIWNNMT627 motif, was able to interact with T21, a counterpart peptide derived from the NHR, to form a typical 6-HB structure with a high thermostability (thermal unfolding transition [Tm] value of 82°C). In contrast, the 6-HB formed by the peptides N36 and C34, which has been considered to be a core structure of the fusion-active gp41, had a Tm of 64°C. Different from T-20 (brand name Fuseon), which is the first and only HIV-1 fusion inhibitor approved for clinical use, CP621-652 could efficiently block 6-HB formation in a dose-dependent manner. Significantly, CP621-652 had potent inhibitory activity against HIV-1-mediated cell-cell fusion and infection, especially against T-20- and C34-resistant virus. Therefore, our works provide important information for understanding the core structure of the fusion-active gp41 and for designing novel anti-HIV peptides.

Characterization of novel M-superfamily conotoxins with new disulfide linkage

The M‐superfamily with the typical Cys framework (–CC–C–C–CC–) is one of the seven major superfamilies of conotoxins found in the venom of cone snails. Based on the number of residues in the last Cys loop (between C4 and C5), M‐superfamily conotoxins can be provisionally categorized into four branches (M‐1, M‐2, M‐3, M‐4) [Corpuz GP, Jacobsen RB, Jimenez EC, Watkins M, Walker C, Colledge C, Garrett JE, McDougal O, Li W, Gray WR, et al. (2005) Biochemistry44, 8176–8186]. Here we report the purification of seven M‐superfamily conotoxins from Conus marmoreus (five are novel and two are known as mr3a and mr3b) and one known M‐1 toxin tx3a from Conus textile. In addition, six novel cDNA sequences of M‐superfamily conotoxins have been identified from C. marmoreus, Conus leopardus and Conus quercinus. Most of the above novel conotoxins belong to M‐1 and M‐2 and only one to M‐3. The disulfide analyses of two M‐1 conotoxins, mr3e and tx3a, revealed that they possess a new disulfide bond arrangement (C1–C5, C2–C4, C3–C6) which is different from those of the M‐4 branch (C1–C4, C2–C5, C3–C6) and M‐2 branch (C1–C6, C2–C4, C3–C5). This newly characterized disulfide connectivity was confirmed by comparing the HPLC profiles of native mr3e and its two regioselectively folded isoforms. This is the first report of three different patterns of disulfide connectivity in conotoxins with the same cysteine framework.

A highly immunogenic fragment derived from Zaire Ebola virus glycoprotein elicits effective neutralizing antibody.

In order to produce polyvalent vaccines based on single rVSV vector, we investigated the immunogenicity, antibody neutralizing activity, and antigenic determinant domain of Zaire Ebolas fragment MFL (aa 393-556) that contains furin site and internal fusion loop. Both the recombinant protein and the recombinant plasmid of fragment MFL elicited high levels of antibody, similar to those of Zaire Ebola GP (ZGP). The MFL fragment of ZGP also elicited high levels of neutralizing antibody and induced moderate cellular immune response in mice, as revealed by the proliferation and cytokine secretion of splenocytes. Through the analysis of the induction of neutralizing antibody by pVAX1-based recombinant plasmids that expressed truncated fragments of MFL, we found that the domain containing the internal fusion loop and the furin site was the major contributor of fragment MFLs immunogenicity. Furthermore, the rVSV-based bivalent vaccine expressing Sudan Ebola GP (SGP) and MFL fragment elicited efficient cross-immunity against ZGP and SGP with high levels of neutralizing antibody. Our results indicate that fragment MFL is an effective and novel antigen for the production of neutralizing antibody and polyvalent vaccines of Ebola virus.

Diversity and evolution of conotoxins in Conus virgo, Conus eburneus, Conus imperialis and Conus marmoreus from the South China Sea.

The venom peptides of cone snails are encoded by a large gene family, and can selectively bind to voltage-gated ion channels (Na⁺, K⁺ and Ca²⁺ channels) and to membrane receptors (nAChR, 5-HT3R, NMDAR). To identify novel conotoxin genes and analyze the evolution of typical conotoxin superfamily genes from different Conus species, we have constructed cDNA libraries derived from the venom ducts of Conus virgo, Conus eburneus, Conus imperialis and Conus marmoreus, which were collected from the South China Sea. 1312 transcripts from four Conus venom duct cDNA libraries were analyzed and 38.7-49.6% of the transcripts encoded conotoxin sequences. In addition to known conotoxins, 34 novel conotoxins have been identified and can be classified into eleven superfamilies, some of which showed unique patterns of cysteines or different signal peptide sequences. The evolutionary trees of T- and A-superfamily conotoxins were analyzed. Likelihood approaches revealed that T-superfamily conotoxins from the four Conus species undergo positive selection, mostly located in the mature toxin region. These findings contribute to a better understanding of the diversity and evolution of conotoxins from the South China Sea, and some novel conotoxins are valuable for further functional investigations.

Subtype-selective antagonism of N-methyl-D-aspartate receptor ion channels by synthetic conantokin peptides

Conantokin-G (con-G), conantokin-T (con-T), a truncated conantokin-R (con-R[1-17]), that functions the same as wild-type con-R, and variant sequences of con-T, were chemically synthesized and employed to investigate their selectivities as antagonists of glutamate/glycine-evoked ion currents in human embryonic kidney-293 cells expressing various combinations of NMDA receptor (NMDAR) subunits (NR), viz., NR1a/2A, NR1a/2B, NR1b/2A and NR1b/2B. Con-G did not substantially affect ion flow into NR1a,b/NR2A-transfected cells, but potently inhibited cells expressing NR1a,b/NR2B, showing high NR2B selectivity. Con-T and con-R served as non-selective antagonists of all of four NMDAR subunit combinations. C-terminal truncation variants of the 21-residue con-T were synthesized and examined in this regard. While NMDAR ion channel antagonist activity, and the ability to adopt the Ca(2+)-induced alpha-helical conformation, diminished as a function of shortening the COOH-terminus of con-T, NMDAR subtype selectivity was enhanced in the con-T[1-11], con-T[1-9], and con-T[1-8] variants toward NR2A, NR1b, and NR1b/2A, respectively. Receptor subtype selectivity was also obtained with Met-8 sequence variants of con-T. Con-T[M8A] and con-T[M8Q] displayed selectivity with NR2B-containing subunits, while con-T[M8E] showed enhanced activity toward NR1b-containing NMDAR subtypes. Of those studied, the most highly selective variant was con-T[M8I], which showed maximal NMDAR ion channel antagonism activity toward the NR1a/2A subtype. These studies demonstrate that it is possible to engineer NMDAR subtype antagonist specificity into con-T. Since the subunit composition of the NMDAR varies temporally and spatially in developing brain and in various disease states, conantokins with high subtype selectivities are potentially valuable drugs that may be used at specific stages of disease and in selected regions of the brain.

NR2B-selective conantokin peptide inhibitors of the NMDA receptor display enhanced antinociceptive properties compared to non-selective conantokins

NR2B-selective inhibitors show lower side-effects in preclinical pain models than non-selective NMDA receptor (NMDAR) antagonists, but it is unclear whether the improved safety of NR2B-selective inhibitors is due to their subtype selectivity or to a unique mode of inhibition of the receptor. In this study, the analgesic effects of intracerebral bolus injections of conantokin peptides with different NMDAR subunit selectivity were determined in mice by the standard hot-plate test, and following stimuli with acetic acid, formalin and complete Freunds adjuvant (CFA). In the standard hot-plate model, con-G[S16Y], a NR2B-selective inhibitor, showed the highest analgesic activity among conantokin peptides tested. In the acetic acid- and CFA-induced pain models, con-G[S16Y] and, to a lesser extent, con-G exhibited higher analgesic activity compared to non-selective inhibitors, such as con-R[1-17]. In the formalin test, while all conantokin peptides could partially suppress the first phase response, only con-G[S16Y] and con-G significantly inhibited the second phase response and suppressed paw edema. Our results suggest that the antinociceptive action of the conantokins may be related to their NR2B-selectivity and that these peptides may be useful as both neurobiological tools for probing mechanisms of nociception and as therapeutic agents for pain relief.

Helix-Helix Interactions between Homo- and Heterodimeric γ-Carboxyglutamate-containing Conantokin Peptides and Their Derivatives

The conantokins are a family of small, naturally occurring γ-carboxyglutamate (Gla)-rich peptides that specifically antagonize the N-methyl-d-aspartate (NMDA) subtype of ionotropic glutamate receptor. One member of this family, conantokin-G (con-G), undergoes Ca2+-mediated self-assembly to form an antiparallel helical dimer. Subunit interactions in this complex are incumbent upon intermolecular Ca2+ bridging of Gla residues spaced at i, i + 4, i + 7, i + 11 intervals within the monomer. Herein, we further probe the molecular determinants governing such helix-helix interactions. Select variants were synthesized to evaluate the contributions of non-Gla residues to conantokin self-association. Con-G dimerization was shown to be exothermic and accompanied by positive heat capacity changes. Using positional Gla variants of conantokin-R (con-R), a non-dimerizing conantokin, i, i + 4, i + 7, i + 11 Gla spacing alone was shown to be insufficient for self-assembly. The Ca2+-dependent antiparallel heterodimerization of con-G and con-T(K7γ), two peptides that harbor optimal Gla spacing, was established. Last, the effects of covalently constrained con-G dipeptides on NMDA-evoked current in HEK293 cells expressing combinations of NR1a, NR1b, NR2A, and NR2B subunits of the NMDA receptor were investigated. The antiparallel dipeptide was unique in its ability to potentiate current at NR1a/2A receptors and, like monomeric con-G, was inhibitory at NR1a/2B and NR1b/2B combinations. In contrast, the parallel species was completely inactive at all subunit combinations tested. These results suggest that, under physiological Ca2+ concentrations, equilibrium levels of con-G dimer most likely exist in an antiparallel orientation and exert effects on NMDA receptor activity that differ from the monomer.

Conantokins and variants derived from cone snail venom inhibit naloxone-induced withdrawal jumping in morphine-dependent mice

The biochemical processes underlying opiate addiction are complex, but n-methyl-d-aspartate receptor (NMDAR) dysfunction appears to be one contributing factor. NMDAR antagonists, including MK-801 and memantine, have previously been shown to assuage symptoms stemming from opiate withdrawal. The conantokins are a small family of naturally occurring peptide toxins known to specifically antagonize the NMDAR. In the present study, the effects of wild-type and variant conantokins on the suppression of naloxone-induced jumping in morphine-dependent mice were evaluated. Our results demonstrate that NR2B-selective conantokins, viz., con-G, con-G[S(16)Y] and con-G[gamma(7)K], are potent inhibitors of naloxone-induced jumping at low doses (2-15 nmol/kg) compared with con-T, con-R[1-17], and small-molecule antagonists of the NMDAR, including the NR2B-selective agent, ifenprodil. We conclude that the NR2B-selective conantokins may find utility as neuropharmacological tools for probing NMDAR-related mechanisms of opiate dependence.

Design, synthesis, and biological activity of novel 1,4-disubstituted piperidine/piperazine derivatives as CCR5 antagonist-based HIV-1 entry inhibitors.

A series of novel 1,4-disubstituted piperidine/piperazine derivatives were designed, synthesized and evaluated for their in vitro activities against HIV-1 Bal (R5) infection in CEMX174 5.25M7 cells. A majority of these compounds showed potent anti-HIV-1 activities with IC(50) at nanomolar levels. N-(4-Fluoro-benzyl)piperazine analog B07 hydrochloride exhibited potency against HIV-1 activity similar to that of TAK-220 hydrochloride, but it had much better water solubility (25 mg/ml in phosphate sodium buffer at 25 °C) and oral bioavailability (56%) than TAK-220 hydrochloride (a solubility of 2 mg/ml and oral bioavailability of 1.4%). These results suggest that B07 hydrochloride may serve as a better lead for the development of new anti-HIV-1 therapies or microbicides for treatment and prevent of HIV-1 infection.