In an effort to identify broadly active inhibitors of HIV-1 entry into host cells, we had previously reported a family of dodecamer triazole-peptide conjugates with nanomolar affinity for viral ichaiken@drexelmed NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript surface protein gp120. This class of peptides exhibits potent antiviral activity and the capacity to simultaneously inhibit interaction of viral envelope protein with both CD4 and co-receptor. In the current investigation, we used minimization of structural complexity of the lead triazole inhibitor HNG-156 (peptide 1) in order to explore the limits of the pharmacophore that enables dual antagonism and to improve opportunities for peptidomimetic design. Truncations of both carboxyl-and amino-terminal residues of the initial 12 amino acid residues of peptide 1 were found to have minimal effect on both affinity and antiviral activity. In contrast, the central triazole Pro-Trp cluster at residues 6 and 7 with ferrocenyl-triazole-Pro (Ftp) was found to be critical for bioactivity. Amino terminal residues distal to the central triazole Pro-Trp sequence tolerated decreasing degrees of side chain variation upon approaching the central cluster. A peptide fragment containing residues 3-7 (Asn-Asn-Ile-Ftp-Trp) exhibited substantial direct binding affinity, antiviral potency, dual receptor site antagonism and induction of gp120 structuring, all properties defining the functional signature of the parent compound 1. This active core contains a stereochemically specific hydrophobic triazole-Pro-Trp cluster, with a short N-terminal peptide extension providing groups for potential main chain and side chain hydrogen binding. The results of this work argue that the pharmacophore for dual antagonism is structurally limited, enhancing the potential to develop minimized peptidomimetic HIV-1 entry inhibitors that simultaneously suppress binding of envelope protein to both of its host cell receptors. The results also argue that the target epitope on gp120 is relatively small, pointing to a localized allosteric inhibition site in the HIV-1 envelope that could be targeted for small-molecule inhibitor discovery.

The recombinant product of the hemoglobin gene of the cyanobacterium Synechocystis sp. PCC 6803 forms spontaneously a covalent bond linking one of the heme vinyl groups to a histidine located in the C-terminal helix (His117, or H16). The present report describes the (1)H, (15)N, and (13)C NMR spectroscopy experiments demonstrating that the recombinant hemoglobin from the cyanobacterium Synechococcus sp. PCC 7002, a protein sharing 59% identity with Synechocystis hemoglobin, undergoes the same facile heme adduct formation. The observation that the extraordinary linkage is not unique to Synechocystis hemoglobin suggests that it constitutes a noteworthy feature of hemoglobin in non-N(2)-fixing cyanobacteria, along with the previously documented bis-histidine coordination of the heme iron. A qualitative analysis of the hyperfine chemical shifts of the ferric proteins indicated that the cross-link had modest repercussions on axial histidine ligation and heme electronic structure. In Synechocystis hemoglobin, the unreacted His117 imidazole had a normal p K(a) whereas the protonation of the modified residue took place at lower pH. Optical experiments revealed that the cross-link stabilized the protein with respect to thermal and acid denaturation. Replacement of His117 with an alanine yielded a species inert to adduct formation, but inspection of the heme chemical shifts and ligand binding properties of the variant identified position 117 as important in seating the cofactor in its site and modifying the dynamic properties of the protein. A role for bis-histidine coordination and covalent adduct formation in heme retention is proposed.

The AIDS epidemic continues to spread at an alarming rate worldwide, especially in developing countries. One approach to solving this problem is the generation of anti-human immunodeficiency virus (HIV) compounds with inhibition spectra broad enough to include globally prevailing forms of the virus. We have examined the HIV type 1 (HIV-1) envelope specificity of a recently identified entry inhibitor candidate, HNG-105, using surface plasmon resonance spectroscopy and pseudovirus inhibition assays. The combined results suggest that the HNG-105 molecule may be effective across the HIV-1 subtypes, and they highlight its potential as a lead for developing therapeutic and microbicidal agents to help combat the spread of AIDS.

The cyanobacterium Synechocystis sp. PCC 6803 (S6803) expresses a two-on-two globin in which His46 (distal side) and His70 (proximal) function as heme iron axial ligands. His46 can be displaced by O 2 , CO, and CN − , among others, whereas His70 is not labile under native conditions. The residue preceding the proximal histidine has been implicated in controlling globin axial ligand reactivity; the details of the mechanism, however, are not well understood, and little information exists for bis-histidyl hexacoordinate proteins. In many vertebrate hemoglobins and in the Synechocystis protein, the position is occupied by an alanine whereas, in myoglobins, it is a serine involved in an intricate hydrogen bond network. We examined the role of Ala69 in S6803 hemoglobin through the effects of an Ala → Ser replacement. The substitution resulted in minor structural perturbations, but the holoprotein's response to temperature-, urea-, and acid-induced denaturation was measurably affected. Enhanced three-state behavior was manifested in the decoupling of heme binding and secondary structure formation. Urea-gradient gel experiments revealed that the stability of the apoprotein was unchanged by the replacement and that a slight alteration of the folding kinetics occurred in the holoproteins. Cyanide-binding experiments were performed to assess trans effects. The apparent rate constant for association decreased two-fold upon Ala69Ser replacement. This deceleration was attributed to a change in the lifetime of a state containing a decoordinated His46. The results demonstrated that, as in vertebrate globins and leghemoglobin, proximal influences operate to determine fundamental dynamic and thermodynamic properties of the protein.Keywords truncated hemoglobin; 2-on-2 globin; hexacoordinate hemoglobin; urea-gradient gel; heme binding; thermodynamic stabilityIron protoporphyrin IX (Fe-PPIX) 1 serves as a cofactor in a large number of essential proteins. In the absence of covalent attachment to the protein matrix via protoporphyrin substituents, the contact between Fe(II)-PPIX (or b heme) and the protein is limited to ligation bond(s) to the iron, hydrogen bonding, van der Waals and electrostatic interactions, and hydrophobic † This study was supported by National Science Foundation grants MCB-091182 and MCB-0349409, National Institutes of Health grant GM-054217, and NASA grant NNG04GN33H (DAV).* To whom correspondence should be addressed. Tel: (814) NIH Public Access Author ManuscriptBiochemistry. Author manuscript; available in PMC 2008 September 11. forces. The affinity for the heme group and its chemical properties, such as redox potential, ability to bind various ligands, and propensity for generating reactive oxygen species, are strictly controlled by the protein environment.In many b hemoproteins the iron is endogenously hexacoordinated, with the axial positions of its octahedral geometry filled by two histidine side chains (1). A well-known example is cytochrome b 5 , an electron-transfer protein that does not bind...

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