Effect of flap mutations on structure of HIV-1 protease and inhibition by saquinavir and darunavir

HIV-1 (human immunodeficiency virus type 1) protease (PR) and its mutants are important antiviral drug targets. The PR flap region is critical for binding substrates or inhibitors and catalytic activity. Hence, mutations of flap residues frequently contribute to reduced susceptibility to PR inhibitors in drug-resistant HIV. Structural and kinetic analyses were used to investigate the role of flap residues Gly48, Ile50, and Ile54 in the development of drug resistance. The crystal structures of flap mutants PR_I50V (PR with I50V mutation), PR_I54V (PR with I54V mutation), and PR_I54M (PR with I54M mutation) complexed with saquinavir (SQV) as well as PR_G48V (PR with G48V mutation), PR_I54V, and PR_I54M complexed with darunavir (DRV) were determined at resolutions of 1.05-1.40 Å. The PR mutants showed changes in flap conformation, interactions with adjacent residues, inhibitor binding, and the conformation of the 80s loop relative to the wild-type PR. The PR contacts with DRV were closer in PR_G48V-DRV than in the wild-type PR-DRV, whereas they were longer in PR_I54M-DRV. The relative inhibition of PR_I54V and that of PR_I54M were similar for SQV and DRV. PR_G48V was about twofold less susceptible to SQV than to DRV, whereas the opposite was observed for PR_I50V. The observed inhibition was in agreement with the association of G48V and I50V with clinical resistance to SQV and DRV, respectively. This analysis of structural and kinetic effects of the mutants will assist in the development of more effective inhibitors for drug-resistant HIV.     

The L76V drug resistance mutation decreases the dimer stability and rate of autoprocessing of HIV-1 protease by reducing internal hydrophobic contacts

The mature HIV-1 protease (PR) bearing the L76V drug resistance mutation (PR(L76V)) is significantly less stable, with a >7-fold higher dimer dissociation constant (K(d)) of 71 ± 24 nM and twice the sensitivity to urea denaturation (UC(50) = 0.85 M) relative to those of PR. Differential scanning calorimetry showed decreases in T(m) of 12 °C for PR(L76V) in the absence of inhibitors and 5-7 °C in the presence of inhibitors darunavir (DRV), saquinavir (SQV), and lopinavir (LPV), relative to that of PR. Isothermal titration calorimetry gave a ligand dissociation constant of 0.8 nM for DRV, ～160-fold higher than that of PR, consistent with DRV resistance. Crystal structures of PR(L76V) in complexes with DRV and SQV were determined at resolutions of 1.45-1.46 ?. Compared to the corresponding PR complexes, the mutated Val76 lacks hydrophobic interactions with Asp30, Lys45, Ile47, and Thr74 and exhibits closer interactions with Val32 and Val56. The bound DRV lacks one hydrogen bond with the main chain of Asp30 in PR(L76V) relative to PR, possibly accounting for the resistance to DRV. SQV shows slightly improved polar interactions with PR(L76V) compared to those with PR. Although the L76V mutation significantly slows the N-terminal autoprocessing of the precursor TFR-PR(L76V) to give rise to the mature PR(L76V), the coselected M46I mutation counteracts the effect by enhancing this rate but renders the TFR-PR(M46I/L76V) precursor less responsive to inhibition by 6 μM LPV while preserving inhibition by SQV and DRV. The correlation of lowered stability, higher K(d), and impaired autoprocessing with reduced internal hydrophobic contacts suggests a novel molecular mechanism for drug resistance.     

Structural and kinetic analyses of the protease from an amprenavir-resistant human immunodeficiency virus type 1 mutant rendered resistant to saquinavir and resensitized to amprenavir

Recent drug regimens have had much success in the treatment of human immunodeficiency virus (HIV)-infected individuals; however, the incidence of resistance to such drugs has become a problem that is likely to increase in importance with long-term therapy of this chronic illness. An analysis and understanding of the molecular interactions between the drug(s) and the mutated viral target(s) is crucial for further progress in the field of AIDS therapy. The protease inhibitor amprenavir (APV) generates a signature set of HIV type 1 (HIV-1) protease mutations associated with in vitro resistance (M46I/L, I47V, and I50V [triple mutant]). Passage of the triple-mutant APV-resistant HIV-1 strain in MT4 cells, in the presence of increasing concentrations of saquinavir (SQV), gave rise to a new variant containing M46I, G48V, I50V, and I84L mutations in the protease and a resulting phenotype that was resistant to SQV and, unexpectedly, resensitized to APV. This phenotype was consistent with a subsequent kinetic analysis of the mutant protease, together with X-ray crystallographic analysis and computational modeling which elucidated the structural basis of these observations. The switch in protease inhibitor sensitivities resulted from (i) the I50V mutation, which reduced the area of contact with APV and SQV; (ii) the compensating I84L mutation, which improved hydrophobic packing with APV; and (iii) the G-to-V mutation at residue 48, which introduced steric repulsion with the P3 group of SQV. This analysis establishes the fine detail necessary for understanding the loss of protease binding for SQV in the quadruple mutant and gain in binding for APV, demonstrating the powerful combination of virology, molecular biology, enzymology, and protein structural and modeling studies in the elucidation and understanding of viral drug resistance.     

Autocatalytic maturation, physical/chemical properties, and crystal structure of group N HIV-1 protease: Relevance to drug resistance

The mature protease from Group N human immunodeficiency virus Type 1 (HIV‐1) (PR1_N) differs in 20 amino acids from the extensively studied Group M protease (PR1_M) at positions corresponding to minor drug‐resistance mutations (DRMs). The first crystal structure (1.09 Å resolution) of PR1_N with the clinical inhibitor darunavir (DRV) reveals the same overall structure as PR1_M, but with a slightly larger inhibitor‐binding cavity. Changes in the 10s loop and the flap hinge propagate to shift one flap away from the inhibitor, whereas L89F and substitutions in the 60s loop perturb inhibitor‐binding residues 29–32. However, kinetic parameters of PR1_N closely resemble those of PR1_M, and calorimetric results are consistent with similar binding affinities for DRV and two other clinical PIs, suggesting that minor DRMs coevolve to compensate for the detrimental effects of drug‐specific major DRMs. A miniprecursor (TFR 1 - 54 ‐PR1_N) comprising the transframe region (TFR) fused to the N‐terminus of PR1_N undergoes autocatalytic cleavage at the TFR/PR1_N site concomitant with the appearance of catalytic activity characteristic of the dimeric, mature enzyme. This cleavage is inhibited at an equimolar ratio of precursor to DRV (～6 μM), which partially stabilizes the precursor dimer from a monomer. However, cleavage at L34/W35 within the TFR, which precedes the TFR 1 - 54 /PR1_N cleavage at pH ≤ 5, is only partially inhibited. Favorable properties of PR1_N relative to PR1_M include its suitability for column fractionation by size under native conditions and >10‐fold higher dimer dissociation constant (150 nM). Exploiting these properties may facilitate testing of potential dimerization inhibitors that perturb early precursor processing steps.     

Structural and kinetic analysis of pyrrolidine-based inhibitors of the drug-resistant Ile84Val mutant of HIV-1 protease

Human immunodeficiency virus (HIV) protease is a well-established drug target in HIV chemotherapy. However, continuously increasing resistance towards approved drugs inevitably requires the development of new inhibitors preferably showing no susceptibility against resistant HIV protease strains. Recently, symmetric pyrrolidine-3,4-bis-N-benzyl-sulfonamides have been developed as a new class of HIV-1 protease inhibitors. The most promising candidate exhibited a K_i of 74 nM towards a wild-type protease. Herein, we report the influence of the active-site mutations Ile50Val and Ile84Val on these inhibitors by structural and kinetic analysis. Although the Ile50Val mutation leads to a significant decrease in affinity for all compounds in this series, they retain or even show increased affinity towards the important Ile84Val mutation. By detailed analysis of the crystal structures of two representatives in complex with wild-type and mutant proteases, we were able to elucidate the structural basis of this phenomenon.     

Exploring the drug resistance mechanism of active site,non-active site mutations and their cooperative effects in CRF01_AE HIV-1 protease: molecular dynamics simulations and free energy calculations

Human immunodeficiency virus type 1 protease is essential for virus replication and maturation and has been considered as one of the important drug target for the antiretroviral treatment of HIV infection. The majority of HIV infections are caused due to non-B subtypes in developing countries. Subtype AE is spreading rapidly and infecting huge population worldwide. Understanding the interdependence of active and non-active site mutations in conferring drug resistance is crucial for the development effective inhibitors in subtype AE protease. In this work, we have investigated the mechanism of resistance against indinavir (IDV) due to therapy selected active site mutation V82F, non-active site mutations PF82V and their cooperative effects PV82F in subtype AE-protease using molecular dynamics simulations and binding free energy calculations. The simulations suggested all the three complexes lead to decrease in binding affinity of IDV, whereas the PF82V complex resulted in an enhanced binding affinity compared to V82F and PV82F complexes. Large positional deviation of IDV was observed in V82F complex. The preservation of hydrogen bonds of IDV with active site Asp25/Asp25′ and flap residue Ile50/50′ via a water molecule is crucial for effective binding. Owing to the close contact of 80s loop with Ile50′ and Asp25, the alteration between residues Thr80 and Val82, further induces conformational change thereby resulting in loss of interactions between IDV and the residues in the active site cavity, leading to drug resistance. Our present study shed light on the effect of active, non-active site mutations and their cooperative effects in AE protease.

Communicated by Ramaswamy H. Sarma     

Interactions of different inhibitors with active-site aspartyl residues of HIV-1 protease and possible relevance to pepsin

The importance of the active site region aspartyl residues 25 and 29 of the mature HIV-1 protease (PR) for the binding of five clinical and three experimental protease inhibitors [symmetric cyclic urea inhibitor DMP323, nonhydrolyzable substrate analog (RPB) and the generic aspartic protease inhibitor acetyl-pepstatin (Ac-PEP)] was assessed by differential scanning calorimetry. DeltaT(m) values, defined as the difference in T(m) for a given protein in the presence and absence of inhibitor, for PR with DRV, ATV, SQV, RTV, APV, DMP323, RPB, and Ac-PEP are 22.4, 20.8, 19.3, 15.6, 14.3, 14.7, 8.7, and 6.5 degrees C, respectively. Binding of APV and Ac-PEP is most sensitive to the D25N mutation, as shown by DeltaT(m) ratios [DeltaT(m)(PR)/DeltaT(m)(PR(D25N))] of 35.8 and 16.3, respectively, whereas binding of DMP323 and RPB (DeltaT(m) ratios of 1-2) is least affected. Binding of the substrate-like inhibitors RPB and Ac-PEP is nearly abolished (DeltaT(m)(PR)/DeltaT(m)(PR(D29N)) > or = 44) by the D29N mutation, whereas this mutation only moderately affects binding of the smaller inhibitors (DeltaT(m) ratios of 1.4-2.2). Of the nine FDA-approved clinical HIV-1 protease inhibitors screened, APV, RTV, and DRV competitively inhibit porcine pepsin with K(i) values of 0.3, 0.6, and 2.14 microM, respectively. DSC results were consistent with this relatively weak binding of APV (DeltaT(m) 2.7 degrees C) compared with the tight binding of Ac-PEP (DeltaT(m) > or = 17 degrees C). Comparison of superimposed structures of the PR/APV complex with those of PR/Ac-PEP and pepsin/pepstatin A complexes suggests a role for Asp215, Asp32, and Ser219 in pepsin, equivalent to Asp25, Asp25', and Asp29 in PR in the binding and stabilization of the pepsin/APV complex.     

HIV-1 protease with 20 mutations exhibits extreme resistance to clinical inhibitors through coordinated structural rearrangements

The escape mutant of HIV-1 protease (PR) containing 20 mutations (PR20) undergoes efficient polyprotein processing even in the presence of clinical protease inhibitors (PIs). PR20 shows >3 orders of magnitude decreased affinity for PIs darunavir (DRV) and saquinavir (SQV) relative to PR. Crystal structures of PR20 crystallized with yttrium, substrate analogue p2-NC, DRV, and SQV reveal three distinct conformations of the flexible flaps and diminished interactions with inhibitors through the combination of multiple mutations. PR20 with yttrium at the active site exhibits widely separated flaps lacking the usual intersubunit contacts seen in other inhibitor-free dimers. Mutations of residues 35-37 in the hinge loop eliminate interactions and perturb the flap conformation. Crystals of PR20/p2-NC contain one uninhibited dimer with one very open flap and one closed flap and a second inhibitor-bound dimer in the closed form showing six fewer hydrogen bonds with the substrate analogue relative to wild-type PR. PR20 complexes with PIs exhibit expanded S2/S2' pockets and fewer PI interactions arising from coordinated effects of mutations throughout the structure, in agreement with the strikingly reduced affinity. In particular, insertion of the large aromatic side chains of L10F and L33F alters intersubunit interactions and widens the PI binding site through a network of hydrophobic contacts. The two very open conformations of PR20 as well as the expanded binding site of the inhibitor-bound closed form suggest possible approaches for modifying inhibitors to target extreme drug-resistant HIV.     

Loss of protease dimerization inhibition activity of darunavir is associated with the acquisition of resistance to darunavir by HIV-1

Dimerization of HIV protease is essential for the acquisition of protease's proteolytic activity. We previously identified a group of HIV protease dimerization inhibitors, including darunavir (DRV). In the present work, we examine whether loss of DRV's protease dimerization inhibition activity is associated with HIV development of DRV resistance. Single amino acid substitutions, including I3A, L5A, R8A/Q, L24A, T26A, D29N, R87K, T96A, L97A, and F99A, disrupted protease dimerization, as examined using an intermolecular fluorescence resonance energy transfer (FRET)-based HIV expression assay. All recombinant HIV(NL4-3)-based clones with such a protease dimerization-disrupting substitution failed to replicate. A highly DRV-resistant in vitro-selected HIV variant and clinical HIV strains isolated from AIDS patients failing to respond to DRV-containing antiviral regimens typically had the V32I, L33F, I54M, and I84V substitutions in common in protease. None of up to 3 of the 4 substitutions affected DRV's protease dimerization inhibition, which was significantly compromised by the four combined substitutions. Recombinant infectious clones containing up to 3 of the 4 substitutions remained sensitive to DRV, while a clonal HIV variant with all 4 substitutions proved highly resistant to DRV with a 205-fold 50% effective concentration (EC(50)) difference compared to HIV(NL4-3). The present data suggest that the loss of DRV activity to inhibit protease dimerization represents a novel mechanism contributing to HIV resistance to DRV. The finding that 4 substitutions in PR are required for significant loss of DRV's protease dimerization inhibition should at least partially explain the reason DRV has a high genetic barrier against HIV's acquisition of DRV resistance.     

Ascidiacyclamides containing oxazoline and thiazole motifs assume square conformations and show high cytotoxicity

Four cyclic octapeptides were designed from ascidiacyclamide [cyclo(–Ile–Oxz–D ‐Val– Thz–)₂] (ASC, 1 ) to investigate the effects of oxazoline (Oxz) and thiazole (Thz) rings on the structures and cytotoxicities of the peptides. cyclo(–Ile–Thz–D ‐Val–Oxz–)₂ ( 2 ) had the same number of Oxz and Thz rings as ASC, but the ring positions were switched. cyclo(–Ile–Oxz–D ‐Val–Thz–Ile–Thz–D ‐Val–Thz–) ( 3 ) and cyclo(–Ile–Thz–D ‐Val–Oxz–Ile–Thz–D ‐Val–Thz–) ( 4 ) contained one Oxz and three Thz rings within the molecule. All Oxz rings were substituted with Thz in cyclo(–Ile–Thz–D ‐Val–Thz–)₂ ( 5 ). These analogues had new Oxz and Thz blocks forming the 24‐membered ring. Based on CD spectra and X‐ray diffraction analyses, the structures of all four analogues were classified as square ASC forms. But the structures of 2 and 5 differed from the original square form of 1 , and they showed no cytotoxicity. The structure of 3 was very similar to that of 1 , and 3 showed 10 times greater cytotoxicity than 1 . Although no definite structure of 4 was obtained, it showed three times greater cytotoxicity than 1 . It appears that the position and number of Oxz residues are essential determinants in the structure‐cytotoxicity relationship of ASC analogues.     

Revealing the dimer dissociation and existence of a folded monomer of the mature HIV‐2 protease

Purification and in vitro protein‐folding schemes were developed to produce monodisperse samples of the mature wild‐type HIV‐2 protease (PR2), enabling a comprehensive set of biochemical and biophysical studies to assess the dissociation of the dimeric protease. An E37K substitution in PR2 significantly retards autoproteolytic cleavage during expression. Furthermore, it permits convenient measurement of the dimer dissociation of PR2_E37K (elevated K_d ～20 nM) by enzyme kinetics. Differential scanning calorimetry reveals a T_m of 60.5 for PR2 as compared with 65.7°C for HIV‐1 protease (PR1). Consistent with weaker binding of the clinical inhibitor darunavir (DRV) to PR2, the T_m of PR2 increases by 14.8°C in the presence of DRV as compared with 22.4°C for PR1. Dimer interface mutations, such as a T26A substitution in the active site (PR2_T26A) or a deletion of the C‐terminal residues 96–99 (PR2_1–95), drastically increase the K_d (>10⁵‐fold). PR2_T26A and PR2_1–95 consist predominantly of folded monomers, as determined by nuclear magnetic resonance (NMR) and size‐exclusion chromatography coupled with multiangle light scattering and refractive index measurements (SMR), whereas wild‐type PR2 and its active‐site mutant PR2_D25N are folded dimers. Addition of twofold excess active‐site inhibitor promotes dimerization of PR2_T26A but not of PR2_1–95, indicating that subunit interactions involving the C‐terminal residues are crucial for dimer formation. Use of SMR and NMR with PR2 facilitates probing for potential inhibitors that restrict protein folding and/or dimerization and, thus, may provide insights for the future design of inhibitors to circumvent drug resistance.     

Selection of Drug-Resistant Feline Immunodeficiency Virus (FIV) Encoding FIV/HIV Chimeric Protease in the Presence of HIV-Specific Protease Inhibitors

An infectious chimeric feline immunodeficiency virus (FIV)/HIV strain carrying six HIV-like protease (PR) mutations (I37V/N55M/V59I/I98S/Q99V/P100N) was subjected to selection in culture against the PR inhibitor lopinavir (LPV), darunavir (DRV), or TL-3. LPV selection resulted in the sequential emergence of V99A (strain S-1X), I59V (strain S-2X), and I108V (strain S-3X) mutations, followed by V37I (strain S-4X). Mutant PRs were analyzed in vitro, and an isogenic virus producing each mutant PR was analyzed in culture for LPV sensitivity, yielding results consistent with the original selection. The 50% inhibitory concentrations (IC₅₀s) for S-1X, S-2X, S-3X, and S-4X were 95, 643, 627, and 1,543 nM, respectively. The primary resistance mutations, V99⁸²A, I59⁵⁰V, and V37³²I, are consistent with the resistance pattern developed by HIV-1 under similar selection conditions. While resistance to LPV emerged readily, similar PR mutations causing resistance to either DRV or TL-3 failed to emerge after passage for more than a year. However, a G37D mutation in the nucleocapsid (NC) was observed in both selections and an isogenic G37D mutant replicated in the presence of 100 nM DRV or TL-3, whereas parental chimeric FIV could not. An additional mutation, L92V, near the PR active site in the folded structure recently emerged during TL-3 selection. The L92V mutant PR exhibited an IC₅₀ of 50 nM, compared to 35 nM for 6s-98S PR, and processed the NC-p2 junction more efficiently, consistent with increased viral fitness. These findings emphasize the role of mutations outside the active site of PR in increasing viral resistance to active-site inhibitors and suggest additional targets for inhibitor development.     

Highly conserved glycine 86 and arginine 87 residues contribute differently to the structure and activity of the mature HIV‐1 protease

The structural and functional role of conserved residue G86 in HIV‐1 protease (PR) was investigated by NMR and crystallographic analyses of substitution mutations of glycine to alanine and serine (PR_G86A and PR_G86S). While PR_G86S had undetectable catalytic activity, PR_G86A exhibited ～6000‐fold lower catalytic activity than PR. ¹H‐¹⁵N NMR correlation spectra revealed that PR_G86A and PR_G86S are dimeric, exhibiting dimer dissociation constants (K_d) of ～0.5 and ～3.2 μM, respectively, which are significantly lower than that seen for PR with R87K mutation (K_d > 1 mM). Thus, the G86 mutants, despite being partially dimeric under the assay conditions, are defective in catalyzing substrate hydrolysis. NMR spectra revealed no changes in the chemical shifts even in the presence of excess substrate, indicating very poor binding of the substrate. Both NMR chemical shift data and crystal structures of PR_G86A and PR_G86S in the presence of active‐site inhibitors indicated high structural similarity to previously described PR/inhibitor complexes, except for specific perturbations within the active site loop and around the mutation site. The crystal structures in the presence of the inhibitor showed that the region around residue 86 was connected to the active site by a conserved network of hydrogen bonds, and the two regions moved further apart in the mutants. Overall, in contrast to the role of R87 in contributing significantly to the dimer stability of PR, G86 is likely to play an important role in maintaining the correct geometry of the active site loop in the PR dimer for substrate binding and hydrolysis. Proteins 2010. © 2009 Wiley‐Liss, Inc.     

Loss of the Protease Dimerization Inhibition Activity of Tipranavir (TPV) and Its Association with the Acquisition of Resistance to TPV by HIV-1

Tipranavir (TPV), a protease inhibitor (PI) inhibiting the enzymatic activity and dimerization of HIV-1 protease, exerts potent activity against multi-PI-resistant HIV-1 isolates. When a mixture of 11 multi-PI-resistant (but TPV-sensitive) clinical isolates (HIV_11MIX), which included HIV_B and HIV_C, was selected against TPV, HIV_11MIX rapidly (by 10 passages [HIV_11MIX^P10]) acquired high-level TPV resistance and replicated at high concentrations of TPV. HIV_11MIX^P10 contained various amino acid substitutions, including I54V and V82T. The intermolecular FRET-based HIV-1 expression assay revealed that TPV''s dimerization inhibition activity against cloned HIV_B (cHIV_B) was substantially compromised. The introduction of I54V/V82T into wild-type cHIV_NL4-3 (cHIV_{NL4-3^I54V/V82T}) did not block TPV''s dimerization inhibition or confer TPV resistance. However, the introduction of I54V/V82T into cHIV_B (cHIV_B^I54V/V82T) compromised TPV''s dimerization inhibition and cHIV_B^I54V/V82T proved to be significantly TPV resistant. L24M was responsible for TPV resistance with the cHIV_C genetic background. The introduction of L24M into cHIV_NL4-3 (cHIV_NL4-3^L24M) interfered with TPV''s dimerization inhibition, while L24M increased HIV-1''s susceptibility to TPV with the HIV_NL4-3 genetic background. When selected with TPV, cHIV_{NL4-3^I54V/V82T} most readily developed TPV resistance and acquired E34D, which compromised TPV''s dimerization inhibition with the HIV_NL4-3 genetic background. The present data demonstrate that certain amino acid substitutions compromise TPV''s dimerization inhibition and confer TPV resistance, although the loss of TPV''s dimerization inhibition is not always associated with significantly increased TPV resistance. The findings that TPV''s dimerization inhibition is compromised with one or two amino acid substitutions may explain at least in part why the genetic barrier of TPV against HIV-1''s development of TPV resistance is relatively low compared to that of darunavir.     

The covalent structure of pig kidney fructose 1,6-bisphosphatase: sequence of the 60-residue NH2-terminal peptide produced by digestion with subtilisin

Digestion of the native pig kidney fructose 1,6-bisphosphatase tetramer with subtilisin cleaves each of the 35,000-molecular-weight subunits to yield two major fragments: the S-subunit (M_{r ca.} 29,000), and the S-peptide (M_r 6,500). The following amino acid sequence has been determined for the S peptide: AcThrAspGlnAlaAlaPheAspThrAsnIle Val ThrLeuThrArgPheValMetGluGlnGlyArgLysAla ArgGlyThrGlyGlu MetThrGlnLeuLeuAsnSerLeuCysThrAlaValLys AlaIleSerThrAla z.sbnd;ValArgLysAlaGlyIleAlaHisLeuTyrGlyIleAla. Comparison of this sequence with that of the NH₂-terminal 60 residues of the enzyme from rabbit liver (El-Dorry et al., 1977, Arch. Biochem. Biophys.182, 763) reveals strong homology with 52 identical positions and absolute identity in sequence from residues 26 to 60.Although subtilisin cleavage of fructose 1,6-bisphosphatase results in diminished sensitivity of the enzyme to AMP inhibition, we have found no AMP inhibition-related amino acid residues in the sequenced S-peptide. The loss of AMP sensitivity that occurs upon pyridoxal-P modification of the enzyme does not result in the modification of lysyl residues in the S-peptide. Neither photoaffinity labeling of fructose 1,6-bisphosphatase with 8-azido-AMP nor modification of the cysteinyl residue proximal to the AMP allosteric site resulted in the modification of residues located in the NH₂-terminal 60-amino acid peptide.     

Epitope analysis of the multiphosphorylated peptide αs1‐casein(59–79)

The multiphosphorylated tryptic peptide α_s1‐casein(59–79) has been shown to be antigenic with anti‐casein antibodies. In an approach to determine the amino acyl residues critical for antibody binding we undertook an epitope analysis of the peptide using overlapping synthetic peptides. With α_s1‐casein(59–79) as the adsorbed antigen in a competitive ELISA only two of five overlapping synthetic peptides at 1 mM significantly inhibited binding of the anti‐casein antibodies. Peptides Glu‐Ser(P)‐Ile‐Ser(P)‐Ser(P)‐Ser(P)‐Glu‐Glu and Ile‐Val‐Pro‐Asn‐Ser(P)‐Val‐Glu‐Glu inhibited antibody binding by 20.0±3.6% and 60.3±7.9%, respectively. The epitope of Glu⁶³‐Ser(P)‐Ile‐Ser(P)‐Ser(P)‐Ser(P)‐Glu‐Glu⁷⁰ was further localised to the phosphoseryl cluster as the peptide Ser(P)‐Ser(P)‐Ser(P) significantly inhibited binding of the anti‐casein antibodies to α_s1‐casein(59–79) by 29.5±7.4%. Substitution of Ser(P)⁷⁵ with Ser⁷⁵ in the second inhibitory peptide Ile‐Val‐Pro‐Asn‐Ser(P)⁷⁵‐Val‐Glu‐Glu also abolished inhibition of antibody binding to α_s1‐casein (59–79) demonstrating that Ser(P)⁷⁵ is also a critical residue for recognition by the antibodies. These data show that the phosphorylated residues in the cluster sequence ‐Ser(P)⁶⁶‐Ser(P)‐Ser(P)⁶⁸ and in the sequence ‐Pro⁷³‐Asn‐Ser(P)‐Val‐Glu⁷⁷‐ are critical for antibody binding to α_s1‐casein(59–79) and further demonstrate that a highly phosphorylated segment of a protein can be antigenic. Copyright © 1999 European Peptide Society and John Wiley & Sons, Ltd.     

Angiotensin II restricted analogs with biological activity in the erythrocytic cycle of Plasmodium falciparum

The anti‐plasmodial activity of conformationally restricted analogs of angiotensin II against Plasmodium gallinaceum has been described. To observe activity against another Plasmodium species, invasion of red blood cells by Plasmodium falciparum was analyzed. Analogs restricted with lactam or disulfide bridges were synthesized to determine their effects and constraints in the peptide–parasite interaction. The analogs were synthesized using tert‐butoxycarbonyl and fluoromethoxycarbonyl solid phase methods, purified by liquid chromatography, and characterized by mass spectrometry. Results indicated that the lactam bridge restricted analogs 1 (Glu‐Asp‐Arg‐Orn ‐Val‐Tyr‐Ile‐His‐Pro‐Phe) and 3 (Asp‐Glu‐Arg‐Val‐Orn ‐Tyr‐Ile‐His‐Pro‐Phe) showed activity toward inhibition of ring formation stage of P. falciparum erythrocytic cycle, preventing invasion in about 40% of the erythrocytes. The disulfide‐bridged analog 10 (Cys‐Asp‐Arg‐Cys ‐Val‐Tyr‐Ile‐His‐Pro‐Phe) was less effective yet significant, showing a 25% decrease in infection of new erythrocytes. In all cases, the peptides presented no pressor activity, and hydrophobic interactions between the aromatic and alkyl amino acid side chains were preserved, a factor proven important in efficacy against P. gallinaceum. In contrast, hydrophilic interactions between the Asp¹ carboxyl and Arg² guanidyl groups proved not to be as important as they were in the case of P. gallinaceum, while interactions between the Arg² guanidyl and Tyr⁴ hydroxyl groups were not important in either case. The β‐turn conformation was predominant in all of the active peptides, proving importance in anti‐plasmodial activity. This approach provides insight for understanding the importance of each amino acid residue on the native angiotensin II structure and a new direction for the design of potential chemotherapeutic agents. Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.     

Modulating the structure of phenylalanine‐incorporated ascidiacyclamide through fluorination

We designed four fluorinated Phe‐incorporated ascidiacyclamide ([Phe]ASC) analogs, (cyclo(?Xxx1‐oxazoline2‐d ‐Val3‐thiazole4‐Ile5‐oxazoline6‐d ‐Val7‐thiazole8‐)), [(4‐F)Phe]ASC (Xxx1: 4‐fluorophenylalanine), [(3,5‐F₂)Phe]ASC (Xxx1: 3,5‐difluorophenylalanine), [(3,4,5‐F₃)Phe]ASC (Xxx1: 3,4,5‐trifluorophenylalanine) and [(F₅)Phe]ASC (Xxx1: pentafluorophenylalanine), to modulate the π‐electron density of the aromatic ring of the Phe residue. X‐ray diffraction analysis, ¹H NMR and CD spectra all suggested that the interactions between the benzene ring of the Xxx1 residue and the alkyl groups of oxazoline2 contribute to the stability of the folded structure of these analogs. Substituting fluorines for the hydrogens progressively weakened those interactions through reducing the π‐electron density, thereby mediating transformation from the folded to square structure. As a result, [(F₅)Phe]ASC preferred the square form more than the other analogs did. Also contributing to the preference for the square form may be the hindrance of the rotation around the Cα–Cβ bond by the two ortho‐fluoro substituents of [(F₅)Phe]ASC. These findings demonstrate that the structure of ASC can be modulated by using fluorine as an electron‐withdrawing group. Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.     

Incorporation of β‐amino acids into ascidiacyclamides: Effects on conformation,cytotoxicity and interaction with copper (II) ion

Seven ascidiacyclamide [cyclo(–Ile–oxazoline–d ‐Val–thiazole–)₂] (ASC) analogues incorporating the β‐amino acids βIle, βoxazoline, and/or d ‐βVal were synthesized. We then investigated the effects of the position and number of incorporated β‐amino acids on the structure, cytotoxicity, and copper binding by these seven analogues. The structural analyses revealed that both βIle and d ‐βVal favor a gauche‐type θ torsion angles, while βoxazoline favors a trans‐type θ torsion angle. Expansion of the macrocycle by incorporation of βIle or d ‐βVal readily induced molecular folding. On the other hand, the incorporation of two βoxazoline residues strongly extended the peptide conformation, and the incorporation of one was sufficient for the moderate restriction important for conformational equilibrium and cytotoxicity. Despite expansion of the macrocycles, the structure‐cytotoxicity relationships were largely maintained. In studies of complexation of the analogues with Cu (II) ion, the position and number of incorporated β‐amino acids had a large impact on the structure of the metal complex and may contribute to its stabilization.