Polymer Cancerostatics Targeted by Recombinant Antibody Fragments to GD2-Positive Tumor Cells.

A water-soluble polymer cancerostatic actively targeted against cancer cells expressing a disialoganglioside antigen GD2 was designed, synthesized and characterized. A polymer conjugate of an antitumor drug doxorubicin with a N-(2-hydroxypropyl)methacrylamide-based copolymer was specifically targeted against GD2 antigen-positive tumor cells using a recombinant single chain fragment (scFv) of an anti-GD2 monoclonal antibody. The targeting protein ligand was attached to the polymer-drug conjugate either via a covalent bond between the amino groups of the protein using a traditional nonspecific aminolytic reaction with a reactive polymer precursor or via a noncovalent but highly specific interaction between bungarotoxin covalently linked to the polymer and the recombinant scFv modified with a C-terminal bungarotoxin-binding peptide. The GD2 antigen binding activity and GD2-specific cytotoxicity of the targeted noncovalent polymer-scFv complex proved to be superior to the covalent polymer-scFv conjugate.

Optimization of the crystallizability of a single-chain antibody fragment.

Single-chain variable antibody fragments (scFvs) are molecules with immense therapeutic and diagnostic potential. Knowledge of their three-dimensional structure is important for understanding their antigen-binding mode as well as for protein-engineering approaches such as antibody humanization. A major obstacle to the crystallization of single-chain variable antibody fragments is their relatively poor homogeneity caused by spontaneous oligomerization. A new approach to optimization of the crystallizability of single-chain variable antibody fragments is demonstrated using a representative single-chain variable fragment derived from the anti-CD3 antibody MEM-57. A Thermofluor-based assay was utilized to screen for optimal conditions for antibody-fragment stability and homogeneity. Such an optimization of the protein storage buffer led to a significantly improved ability of the scFv MEM-57 to yield crystals.

Design of HIV protease inhibitors based on inorganic polyhedral metallacarboranes.

HIV protease (HIV PR) is a primary target for anti-HIV drug design. We have previously identified and characterized substituted metallacarboranes as a new class of HIV protease inhibitors. In a structure-guided drug design effort, we connected the two cobalt bis(dicarbollide) clusters with a linker to substituted ammonium group and obtained a set of compounds based on a lead formula [H(2)N-(8-(C(2)H(4)O)(2)-1,2-C(2)B(9)H(10))(1',2'-C(2)B(9)H(11))-3,3'-Co)(2)]Na. We explored inhibition properties of these compounds with various substitutions, determined the HIV PR:inhibitor crystal structure, and computationally explored the conformational space of the linker. Our results prove the capacity of linker-substituted dual-cage cobalt bis(dicarbollides) as lead compounds for design of more potent inhibitors of HIV PR.

Potent inhibition of drug-resistant HIV protease variants by monoclonal antibodies.

The monoclonal antibodies 1696 and F11.2.32 strongly inhibit the activity of wild-type HIV-1 protease (PR) by binding to epitopes at the enzyme N-terminus (residues 1-6) and flap residues 36-46, respectively. Here we demonstrate that these antibodies are also potent inhibitors of PR variants resistant to active-site inhibitors used as anti-AIDS drugs. Our in vitro experiments revealed that the inhibitory potency of single-chain fragments (scFv) of these antibodies is not significantly affected by the presence of mutations in PR; inhibition constants for drug-resistant protease variants are 5-11 nM and 13-169 nM for scFv1696 and for scFvF11.2.32, respectively. Tethered dimer of HIV-1 PR variant proved to be a model protease variant resistant to dissociative inhibition by 1696, and, strikingly, it also displayed resistance to inhibition by F11.2.32 suggesting that dimer dissociation also plays a role in the inhibitory action of F11.2.32.

Stabilization of antibody structure upon association to a human carbonic anhydrase IX epitope studied by X-ray crystallography, microcalorimetry, and molecular dynamics simulations.

Specific antibodies interfere with the function of human tumor-associated carbonic anhydrase IX (CA IX), and show potential as tools for anticancer interventions. In this work, a correlation between structural elements and thermodynamic parameters of the association of antibody fragment Fab M75 to a peptide corresponding to its epitope in the proteoglycan-like domain of CA IX, is presented. Comparisons of the crystal structures of free Fab M75 and its complex with the epitope peptide reveal major readjustments of CDR-H1 and CDR-H3. In contrast, the overall conformations and positions of CDR-H2 and CDR-L2 remain unaltered, and their positively charged residues may thus present a fixed frame for epitope recognition. Adoption of the altered CDR-H3 conformation in the structure of the complex is accompanied by an apparent local stabilization. Analysis of domain mobility with translation-libration-screw (TLS) method shows that librations of the entire heavy chain variable domain (V(H)) decrease and reorient in the complex, which correlates well with participation of the heavy chain in ligand binding. Isothermal titration microcalorimetry (ITC) experiments revealed a highly unfavorable entropy term, which can be attributed mainly to the decrease in the degrees of freedom of the system, the loss of conformational freedom of peptide and partially to a local stabilization of CDR-H3. Moreover, it was observed that one proton is transferred from the environment to the protein-ligand complex upon binding. Molecular dynamics simulations followed by molecular mechanics/generalized Born surface area (MM-GBSA) calculations of the ligand (epitope peptide) binding energy yielded energy values that were in agreement with the ITC measurements and indicated that the charged residues play crucial role in the epitope binding. Theoretical arguments presented in this work indicate that two adjacent arginine residues (ArgH50 and ArgH52) are responsible for the observed proton transfer.

On the role of the R configuration of the reaction-intermediate isostere in HIV-1 protease-inhibitor binding: X-ray structure at 2.0 A resolution.

Peptidomimetic inhibitors of human immunodeficiency virus-1 protease are successful lead substances for the development of virostatic drugs against HIV as the causative agent of acquired immunodeficiency syndrome (AIDS). The hydroxyethylamine isostere of the proteolytic cleavage intermediate provides a suitable replacement for the peptide bond. A series of acyclic pseudopeptide inhibitors with the hydroxyethylamine isostere varying in chiral carbon configuration and P'2 residue type were structurally analysed by single-crystal X-ray crystallography. The compounds inhibit HIV protease with subnanomolar inhibition constants and block viral replication in tissue cultures. Here, the structure of such a complex with the R configuration of the isosteric group (PDB code 1zsf) is presented together with newly available synchrotron data for a complex with the S stereoisomer of the inhibitor (PDB code 1zsr). Comparison of the structure and binding with other complexes of HIV-1 protease and similar inhibitors contributes to the understanding of how these molecules bind to the wild-type form of this enzyme. The hydroxy group of the R stereoisomer interacts with one of the catalytic aspartic acids by a short hydrogen bond with rather extreme geometry. The change of configuration of the chiral carbon bearing the hydroxyl from S to R does not influence the inhibition efficiency in this case.

From nonpeptide toward noncarbon protease inhibitors: metallacarboranes as specific and potent inhibitors of HIV protease.

HIV protease (PR) represents a prime target for rational drug design, and protease inhibitors (PI) are powerful antiviral drugs. Most of the current PIs are pseudopeptide compounds with limited bioavailability and stability, and their use is compromised by high costs, side effects, and development of resistant strains. In our search for novel PI structures, we have identified a group of inorganic compounds, icosahedral metallacarboranes, as candidates for a novel class of nonpeptidic PIs. Here, we report the potent, specific, and selective competitive inhibition of HIV PR by substituted metallacarboranes. The most active compound, sodium hydrogen butylimino bis-8,8-[5-(3-oxa-pentoxy)-3-cobalt bis(1,2-dicarbollide)]di-ate, exhibited a K(i) value of 2.2 nM and a submicromolar EC(50) in antiviral tests, showed no toxicity in tissue culture, weakly inhibited human cathepsin D and pepsin, and was inactive against trypsin, papain, and amylase. The structure of the parent cobalt bis(1,2-dicarbollide) in complex with HIV PR was determined at 2.15 A resolution by protein crystallography and represents the first carborane-protein complex structure determined. It shows the following mode of PR inhibition: two molecules of the parent compound bind to the hydrophobic pockets in the flap-proximal region of the S3 and S3' subsites of PR. We suggest, therefore, that these compounds block flap closure in addition to filling the corresponding binding pockets as conventional PIs. This type of binding and inhibition, chemical and biological stability, low toxicity, and the possibility to introduce various modifications make boron clusters attractive pharmacophores for potent and specific enzyme inhibition.

Regular arrangement of periodates bound to lysozyme.

The structure of tetragonal hen egg-white lysozyme soaked in a periodate solution has been determined to a resolution of 1.8 A. Four high-occupancy periodate positions have been identified on the basis of the anomalous signal of the I atoms. The four periodates exhibit a regular rectangular arrangement on the surface of the lysozyme molecule. No similar regular arrangement was found either in lysozyme crystals soaked in other heavy-atom anions or in other structures from the Protein Data Bank. Depending on their position on the surface of the protein, the periodate ions deviate to a varying extent from ideal octahedral geometry.

Crystal structure of a cross-reaction complex between an anti-HIV-1 protease antibody and an HIV-2 protease peptide.

The monoclonal antibody 1696, elicited by HIV-1 protease, inhibits the activity of both HIV-1 and HIV-2 proteases with inhibition constants in the low nanomolar range. The antibody cross-reacts with peptides derived from the N-terminal region of both proteases. The crystal structure of the recombinant single-chain Fv fragment of 1696 complexed with an N-terminal peptide from the HIV-2 protease has been determined at 1.88A resolution. Interactions of the peptide with scFv1696 are compared with the previously reported structure of scFv1696 in complex with the corresponding peptide from HIV-1 protease. The origin of cross-reactivity of mAb1696 with HIV proteases is discussed.

Role of hydroxyl group and R/S configuration of isostere in binding properties of HIV-1 protease inhibitors.

The crystal structure of the complex between human immunodeficiency virus type 1 (HIV-1) protease and a peptidomimetic inhibitor of ethyleneamine type has been refined to R factor of 0.178 with diffraction limit 2.5 A. The peptidomimetic inhibitor Boc-Phe-Psi[CH2CH2NH]-Phe-Glu-Phe-NH2 (denoted here as OE) contains the ethyleneamine replacement of the scissile peptide bond. The inhibitor lacks the hydroxyl group which is believed to mimic tetrahedral transition state of proteolytic reaction and thus is suspected to be necessary for good properties of peptidomimetic HIV-1 protease inhibitors. Despite the missing hydroxyl group the inhibition constant of OE is 1.53 nm and it remains in the nanomolar range also towards several available mutants of HIV-1 protease. The inhibitor was found in the active site of protease in an extended conformation with a unique hydrogen bond pattern different from hydroxyethylene and hydroxyethylamine inhibitors. The isostere nitrogen forms a hydrogen bond to one catalytic aspartate only. The other aspartate forms two weak hydrogen bridges to the ethylene group of the isostere. A comparison with other inhibitors of this series containing isostere hydroxyl group in R or S configuration shows different ways of accommodation of inhibitor in the active site. Special attention is devoted to intermolecular contacts between neighbouring dimers responsible for mutual protein adhesion and for a special conformation of Met46 and Phe53 side chains not expected for free protein in water solution.

Inhibitor binding at the protein interface in crystals of a HIV-1 protease complex.

Depending on the excess of ligand used for complex formation, the HIV-1 protease complexed with a novel phenylnorstatine inhibitor forms crystals of either hexagonal (P6(1)) or orthorhombic (P2(1)2(1)2(1)) symmetry. The orthorhombic form shows an unusual complexity of crystal packing: in addition to one inhibitor molecule that is bound to the enzyme active site, the second inhibitor molecule is bound as an outer ligand at the protein interface. Binding of the outer ligand apparently increases the crystal-quality parameters so that the diffraction data allow solution of the structure of the complex at 1.03 A, the best resolution reported to date. The outer ligand interacts with all four surrounding HIV-1 protease molecules and has a bent conformation owing to its accommodation in the intermolecular space. The parameters of the solved structures of the orthorhombic and hexagonal forms are compared.

A phenylnorstatine inhibitor binding to HIV-1 protease: geometry, protonation, and subsite-pocket interactions analyzed at atomic resolution.

The X-ray structure of a complex of HIV-1 protease (PR) with a phenylnorstatine inhibitor Z-Pns-Phe-Glu-Glu-NH(2) has been determined at 1.03 A, the highest resolution so far reported for any HIV PR complex. The inhibitor shows subnanomolar K(i) values for both the wild-type PR and the variant representing one of the most common mutations linked to resistance development. The structure comprising the phenylnorstatine moiety of (2R,3S)-chirality displays a unique pattern of hydrogen bonding to the two catalytic aspartate residues. This high resolution makes it possible to assess the donor and acceptor relations of this hydrogen bonding and to indicate a proton shared by the two catalytic residues. A structural mechanism for the unimpaired inhibition of the protease Val82Ala mutant is also suggested, based on energy calculations and analyses.

Structure of a single-chain Fv fragment of an antibody that inhibits the HIV-1 and HIV-2 proteases.

The monoclonal antibody 1696, which was raised against the HIV-1 protease, inhibits the catalytic activity of the enzyme from both the HIV-1 and HIV-2 strains. The antibody cross-reacts with peptides containing the N-terminus of the enzyme, which is highly conserved between these strains. The crystal structure of a single-chain Fv fragment of 1696 (scFv-1696) in the non-complexed form, solved at 1.7 A resolution, is compared with the previously reported non-complexed Fab-1696 and antigen-bound scFv-1696 structures. Large conformational changes in the third hypervariable region of the heavy chain and differences in relative orientation of the variable domains are observed between the different structures.

Hydroxyethylamine isostere of an HIV-1 protease inhibitor prefers its amine to the hydroxy group in binding to catalytic aspartates. A synchrotron study of HIV-1 protease in complex with a peptidomimetic inhibitor.

A complex structure of HIV-1 protease with a hydroxyethylamine-containing inhibitor Boc-Phe-Psi[(S)-CH(OH)CH2NH]-Phe-Gln-Phe-NH2 has been determined by X-ray diffraction to 1.8 A resolution. The inhibitor is bound in the active site of the protease dimer with its hydroxyethylamine isostere participating in hydrogen bonds to the catalytic aspartates 25 and 25' and glycine 27' of the active site triads via five hydrogen bonds. The isostere amine interactions with the catalytic aspartates result in a displacement of the isostere hydroxy group in comparison with the common position known for analogous hydroxyethylamine containing inhibitors. A comparison with another inhibitor of this series shows that the change of one atom of the P2' side chain (Glu/Gln) leads to an altered ability of creating hydrogen bonds to the active site and within the inhibitor molecule. The diffraction data collected at a synchrotron radiation source enabled a detailed analysis of the complex solvation and of alternative conformations of protein side chains.