Relation between flexibility and positively selected HIV‐1 protease mutants against inhibitors

The antiretroviral chemotherapy helps to reduce the mortality of HIVs infected patients. However, RNA dependant virus replication has a high mutation rate. Human immunodeficiency virus Type 1 protease plays an essential role in viral replication cycle. This protein is an important target for therapy with viral protein inhibitors. There are few works using normal mode analysis to investigate this problem from the structural changes viewpoint. The investigation of protein flexibility may be important for the study of processes associated with conformational changes and state transitions. The normal mode analysis allowed us to investigate structural changes in the protease (such as flexibility) in a straightforward way and try to associate these changes with the increase of fitness for each positively selected HIV‐1 mutant protease of patients treated with several protease inhibitors (saquinavir, indinavir, ritonavir, nelfinavir, lopinavir, fosamprenavir, atazanavir, darunavir, and tripanavir) in combination or separately. These positively selected mutations introduce significant flexibility in important regions such as the active site cavity and flaps. These mutations were also able to cause changes in accessible solvent area. This study showed that the majority of HIV‐1 protease mutants can be grouped into two main classes of protein flexibility behavior. We presented a new approach to study structural changes caused by positively selected mutations in a pathogen protein, for instance the HIV‐1 protease and their relationship with their resistance mechanism against known inhibitors. The method can be applied to any pharmaceutically relevant pathogen proteins and could be very useful to understand the effects of positively selected mutations in the context of structural changes. Proteins 2012; © 2012 Wiley Periodicals, Inc.     

Optimization of peptidic HIV‐1 fusion inhibitor T20 by phage display

The HIV fusion inhibitor T20 has been approved to treat those living with HIV/AIDS, but treatment gives rise to resistant viruses. Using combinatorial phage‐displayed libraries, we applied a saturation scan approach to dissect the entire T20 sequence for binding to a prefusogenic five‐helix bundle (5HB) mimetic of HIV‐1 gp41. Our data set compares all possible amino acid substitutions at all positions, and affords a complete view of the complex molecular interactions governing the binding of T20 to 5HB. The scan of T20 revealed that 12 of its 36 positions were conserved for 5HB binding, which cluster into three epitopes: hydrophobic epitopes at the ends and a central dyad of hydrophilic residues. The scan also revealed that the T20 sequence was highly adaptable to mutations at most positions, demonstrating a striking structural plasticity that allows multiple amino acid substitutions at contact points to adapt to conformational changes, and also at noncontact points to fine‐tune the interface. Based on the scan result and structural knowledge of the gp41 fusion intermediate, a library was designed with tailored diversity at particular positions of T20 and was used to derive a variant (T20v1) that was found to be a highly effective inhibitor of infection by multiple HIV‐1 variants, including a common T20‐escape mutant. These findings show that the plasticity of the T20 functional sequence space can be exploited to develop variants that overcome resistance of HIV‐1 variants to T20 itself, and demonstrate the utility of saturation scanning for rapid epitope mapping and protein engineering.     

Allosteric HIV‐1 integrase inhibitors promote aberrant protein multimerization by directly mediating inter‐subunit interactions: Structural and thermodynamic modeling studies

Allosteric HIV‐1 integrase (IN) inhibitors (ALLINIs) bind at the dimer interface of the IN catalytic core domain (CCD), and potently inhibit HIV‐1 by promoting aberrant, higher‐order IN multimerization. Little is known about the structural organization of the inhibitor‐induced IN multimers and important questions regarding how ALLINIs promote aberrant IN multimerization remain to be answered. On the basis of physical chemistry principles and from our analysis of experimental information, we propose that inhibitor‐induced multimerization is mediated by ALLINIs directly promoting inter‐subunit interactions between the CCD dimer and a C‐terminal domain (CTD) of another IN dimer. Guided by this hypothesis, we have built atomic models of inter‐subunit interfaces in IN multimers by incorporating information from hydrogen‐deuterium exchange (HDX) measurements to drive protein‐protein docking. We have also developed a novel free energy simulation method to estimate the effects of ALLINI binding on the association of the CCD and CTD. Using this structural and thermodynamic modeling approach, we show that multimer inter‐subunit interface models can account for several experimental observations about ALLINI‐induced multimerization, including large differences in the potencies of various ALLINIs, the mechanisms of resistance mutations, and the crucial role of solvent exposed R‐groups in the high potency of certain ALLINIs. Our study predicts that CTD residues Tyr226, Trp235 and Lys266 are involved in the aberrant multimer interfaces. The key finding of the study is that it suggests the possibility of ALLINIs facilitating inter‐subunit interactions between an external CTD and the CCD‐CCD dimer interface.     

Enantioselective Surface Plasmon Resonance Sensor Based on C60 Fullerene‐Glutathione Self‐Assembled Monolayer (SAM)

A novel enantioselective surface plasmon resonance (SPR) sensor based on a self‐assembled monolayer of C₆₀ fullerene as the chiral selector is proposed. A binding assay, apparent affinity constant, and apparent dissociation binding constant were used to analyze and study the enantioselectivity of C₆₀ fullerene‐glutathione film for L‐histidine, which was chosen as the model analyte. The apparent affinity constant for the complex formed by L‐histidine with C₆₀ fullerene‐glutathione film was 5.2 x 10⁹ M^‐1. The proposed SPR sensor can be used for the assay of L‐histidine in the 10^‐10 – 10^‐7 mol/L concentration range. Chirality 26:129–131, 2014. © 2014 Wiley Periodicals, Inc.     

Rational optimization of a HIV‐1 Tat inhibitor: Rapid progress on combinatorial lead structures

Lead molecules identified by combinatorial chemistry approaches are preferred starting points for straightforward improvements of compound profiles. Structure‐guided rationales can be supported and complemented by systematic variations based on the modular nature of the molecules. A peptoidic compound (CGP 64222), previously identified from a sequential unrandomization process, was shown to specifically inhibit the interaction between the HIV‐1 trans‐activator Tat and its RNA response element TAR. To improve the compound's pharmaceutical attractiveness an approach to reduce both, size and number of charges was pursued. Because this resulted in activity decrease, parallel synthesis with variations on one rationally defined position aimed at the identification of structural determinants was undertaken to regain in vitro activity in biochemical and cellular Tat‐TAR interaction assays. As a result CGP74026 was identified, a drastically simplified but highly active Tat antagonist, which is able to block HIV‐1 replication even in primary human cells. © 1999 John Wiley & Sons, Inc. Biotechnol Bioeng (Comb Chem) 61:155–168, 1998/1999.     

Solution structure of LC5, the CCR5‐ derived peptide for HIV‐1 inhibition

The synthetic peptide fragment (LC5: LRCRNEKKRHRAVRLIFTI) inhibits human immunodeficiency virus type 1 (HIV‐1) infection of MT‐4 cells. In this study, the solution structure of LC5 in SDS micelles was elucidated by using the standard ¹H two‐dimensional NMR spectroscopic method along with circular dichroism and fluorescence quenching. The peptide adopts a helical structure in the C‐terminal region (residues 13–16), whereas the N‐terminal part remains unstructured. The importance of Phe17 in maintaining the structure of LC5 was demonstrated by replacing Phe17 with Ala, which resulted in the dramatic conformational change of LC5. The solution structure of LC5 elucidated in the present work provides a basis for further study of the mechanism of the inhibition of HIV‐1 infection. Copyright © 2010 European Peptide Society and John Wiley & Sons, Ltd.     

Pharmacokinetic stability of macrocyclic peptide triazole HIV‐1 inactivators alone and in liposomes

Previously, we reported the discovery of macrocyclic peptide triazoles (cPTs) that bind to HIV‐1 Env gp120, inhibit virus cell infection with nanomolar potencies, and cause irreversible virion inactivation. Given the appealing virus‐killing activity of cPTs and resistance to protease cleavage observed in vitro, we here investigated in vivo pharmacokinetics of the cPT AAR029b. AAR029b was investigated both alone and encapsulated in a PEGylated liposome formulation that was designed to slowly release inhibitor. Pharmacokinetic analysis in rats showed that the half‐life of FITC‐AAR029b was substantial both alone and liposome‐encapsulated, 2.92 and 8.87 hours, respectively. Importantly, liposome‐encapsulated FITC‐AAR029b exhibited a 15‐fold reduced clearance rate from serum compared with the free FITC‐cPT. This work thus demonstrated both the in vivo stability of cPT alone and the extent of pharmacokinetic enhancement via liposome encapsulation. The results obtained open the way to further develop cPTs as long‐acting HIV‐1 inactivators against HIV‐1 infection.     

Assessing the correlation of microscopy‐based and volumetry‐based measurements for resin swelling in a range of potential greener solvents for SPPS

The degree of resin swelling in a particular solvent system is one of the critical parameters for solid‐phase peptide synthesis (SPPS) and for solid‐phase synthesis in general. Methods used for measuring the degree of resin swelling include microscopy‐based and volumetry‐based methods. This study describes and compares the use of both methods for a number of commercially available resins commonly used in SPPS, with a range of solvents, which have been identified in the literature as ‘greener’ than DCM, DMF and NMP. The results were analysed by statistical methods, and a significant correlation between the two distinct methods has been demonstrated for the first time. The results will likely be used, in conjunction with other literature methods, to help in choosing both the resin and solvent system for greener SPPS, as well as for continuous flow SPPS, which is of growing importance.     

Synthesis and application of N‐[1‐(4‐(4‐fluorophenyl)‐2,6‐dioxocyclohexylidene)ethyl] (Fde)‐protected amino acids for optimization of solid‐phase peptide synthesis using gel‐phase 19F NMR spectroscopy

N‐[1‐(4‐(4‐fluorophenyl)‐2,6‐dioxocyclohexylidene)ethyl] (Fde) protected amino acids have been prepared and applied in solid‐phase peptide synthesis monitored by gel‐phase ¹⁹F NMR spectroscopy. The Fde protective group could be cleaved with 2% hydrazine or 5% hydroxylamine solution in DMF as determined with gel‐phase ¹⁹F NMR spectroscopy. The dipeptide Ac‐L ‐Val‐L ‐Val‐NH₂ 12 was constructed using Fde‐L ‐Val‐OH and no noticeable racemization took place during the amino acid coupling with N,N′‐diisopropylcarbodiimide and 1‐hydroxy‐7‐azabenzotriazole or Fde deblocking. To extend the scope of Fde protection, the hydrophobic nonapeptide LLLLTVLTV from the signal sequence of mucin MUC1 was successfully prepared using Fde‐L ‐Leu‐OH at diagnostic positions. Copyright © 2009 European Peptide Society and John Wiley & Sons, Ltd.     

A convenient microwave‐enhanced solid‐phase synthesis of short chain N‐methyl‐rich peptides

Structural modification of the peptide backbone via N‐methylation is a powerful tool to modulate the pharmacokinetic profile and biological activity of peptides. Here we describe a rapid and highly efficient microwave(MW)‐assisted Fmoc/tBu solid‐phase method to prepare short chain N‐methyl‐rich peptides, using Rink amide p‐methylbenzhydrylamine (MBHA) resin as solid‐phase support. This method produces peptides in high yield and purity, and reduces the time required for Fmoc‐N‐methyl amino acid coupling. Copyright © 2010 European Peptide Society and John Wiley & Sons, Ltd.     

Tetrapeptides,as small‐sized peptidic inhibitors; synthesis and their inhibitory activity against BACE1

β‐Site amyloid precursor protein cleaving enzyme 1 (BACE1) is known to be involved in the production of amyloid β‐peptide in Alzheimer's disease and is a major target for current drug design. We previously reported substrate‐based peptidomimetics, KMI‐compounds as potent BACE1 inhibitors. In this study, we designed and synthesized tetrapeptides as low molecular‐sized inhibitors. These exhibited high potency against recombinant BACE1, with the highest IC₅₀ value of 34.6 nM from KMI‐927. Copyright © 2010 European Peptide Society and John Wiley & Sons, Ltd.     

Solid‐phase synthesis and screening of a library of C‐terminal arginine peptide aldehydes against Murray Valley encephalitis virus protease

Murray Valley encephalitis virus is a member of the flavivirus group, a large family of single‐stranded RNA viruses, which cause serious disease in all regions of the world. Unfortunately, no suitable antivirals are available, and there are commercial vaccines for only three flaviviruses. The solid‐phase synthesis of a library of 400 C‐terminal arginine peptide aldehydes and their screening against Murray Valley encephalitis virus protease are demonstrated. The library was utilised to elucidate several tripeptide sequences that can be used as inhibitors in further SAR studies. Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd.     

Snapin promotes HIV‐1 transmission from dendritic cells by dampening TLR8 signaling

HIV‐1 traffics through dendritic cells (DCs) en route to establishing a productive infection in T lymphocytes but fails to induce an innate immune response. Within DC endosomes, HIV‐1 somehow evades detection by the pattern‐recognition receptor (PRR) Toll‐like receptor 8 (TLR8). Using a phosphoproteomic approach, we identified a robust and diverse signaling cascade triggered by HIV‐1 upon entry into human DCs. A secondary siRNA screen of the identified signaling factors revealed several new mediators of HIV‐1 trans‐infection of CD4⁺ T cells in DCs, including the dynein motor protein Snapin. Inhibition of Snapin enhanced localization of HIV‐1 with TLR8⁺ early endosomes, triggered a pro‐inflammatory response, and inhibited trans‐infection of CD4⁺ T cells. Snapin inhibited TLR8 signaling in the absence of HIV‐1 and is a general regulator of endosomal maturation. Thus, we identify a new mechanism of innate immune sensing by TLR8 in DCs, which is exploited by HIV‐1 to promote transmission.     

Synthesis of ‘head‐to‐tail’ cyclized peptides on solid support using a chelating amide as new orthogonal protecting group

The synthesis of ‘head‐to‐tail’ cyclized peptides requires orthogonal protecting groups. Herein, we report on the introduction of bis(2‐pyridylmethyl)amine (Bpa) as a new protecting group for carboxylic functions in SPPS. The synthesis of the Bpa‐protected aspartic acid was straightforward, and its utility was investigated under standard peptide synthesis conditions. The new protecting group was cleaved in a very mild way using Cu(OAc)₂ and 2‐(trimethylsilyl)ethanol as nucleophile in a microwave oven without affecting other groups. Hence, the new group is ideally suited for the synthesis of ‘head‐to‐tail’ cyclic peptides, as demonstrated for a cyclic pentapeptide and cyclic hexapeptides. Copyright © 2015 European Peptide Society and John Wiley & Sons, Ltd.