Leonid Kirnarsky

Development of response-selective agonists of human C5a anaphylatoxin: Conformational, biological, and therapeutic considerations

Numerous studies on the relationship between the structure and function of peptide agonists derived from the biologically active, C-terminal region of human C5a anaphylatoxin have been reported over the past decade. These studies have been performed with the objective of parlaying this structure-function information into the design of peptide/peptidomimetic modulators of C5a receptor (C5aR)-mediated function. In this review, we describe a rational approach for the development of conformationally biased, decapeptide agonists of C5a and described how these stabilized and specific conformational features relate to the expression of specific C5a-like activities in vitro and in vivo. The therapeutic potential of such response-selective C5a agonists is discussed and underscored by the results of one such response-selective C5a agonist that was used in vivo as an effective molecular adjuvant capable of generating antigen-specific humoral and cellular immune responses. Finally, we describe the synthesis of a new generation of highly response-selective, conformationally biased C5a agonist and discuss the in vitro and in vivo biologic results that so indicate this biologic selectivity.

Differential activities of decapeptide agonists of human C5a: the conformational effects of backbone N-methylation

Analogues of the potent, conformationally biased, decapeptide agonist of human C5a anaphylatoxin, C5a(65-74)Y65,F67,P69,P71,D-Ala73 (YSFKPMPLaR, peptide 54), were synthesized with methyl groups occupying specific amide nitrogen atoms along the peptide backbone. This N-methylation induced crucial extended backbone conformations in a manner similar to the two Pro residues, but without eliminating the contributions made by the side-chain of the residue for which Pro was substituted. The presence of backbone N-methyl groups on peptide 54 analogues had pronounced detrimental effects on the ability to bind and activate C5aRs expressed on human PMNs, but not on the ability to contract smooth muscle of human umbilical artery. Several N-methylated analogues of peptide 54 (peptides 56, 67, 124, 125, and 137) were significantly more selective for smooth muscle contraction, which is mediated by tissue resident macrophages, than for enzyme release from PMNs. Indeed, peptide 67, YSFKDMP(MeL)aR was almost 3000-fold more selective for smooth muscle contraction than for PMN enzyme release. Consistent with these differential activities was the observation that peptide 67 expressed a significantly greater binding affinity to C5aRs expressed on rat macrophages than on rat PMNs. This differential activity was also observed in vivo in the rat where peptide 67 induced a hypotensive response similar to peptide 54 and rhuC5a, but without accompanying neutropenia.

Improving the efficiency of protein structure determination from NMR

Abstract Comprehensive computational experiments were performed to evaluate efficiency of the newly proposed combine procedure on protein structure calculations from NMR data. This procedure is intended to combine merits of the previously developed FiSiNOE method with the diana program, widely used for NMR structure calculations. The new version of the FiSiNOE program, FiSiNOE-3 , was developed to determine local conformations of proteins consistent with short-range NMR data (intraresidue and sequential distance constraints and coupling constants). For each residue, the program determines the allowed ranges of φ , ψ and χ 1 torsion angles consistent with the NMR data. The benchmark calculations were carried out on three proteins: bovine pancreatic trypsin inhibitor, crambin and avian pancreatic polypeptide. The results of the calculations obtained by the combine protocol were compared with the results obtained by the standard run of the diana program. The combine procedure allowed one to significantly narrow ranges of the dihedral angle constraints before the structure calculations that, in turn, resulted in more stereospecific assignments. The numbers of βCH 2 groups unambiguously assigned using the combine procedure were significantly greater in comparison with those assigned by the standard protocol. For all three proteins, the use of the combine procedure almost doubled the numbers of unambiguously assigned βCH 2 groups in comparison with standard . The computational experiments clearly showed that the use of allowed ranges for torsion angles obtained by the combine procedure as input data for the diana program provides a higher precision and accuracy of 3D protein structures reproduced from NMR constraints. The combine procedure may be incorporated into any protocol using as input data the allowed ranges of torsion angles consistent with a given set of NMR constraints. Since the combine procedure proved to be effective, reliable and robust, it may be recommended for general use in 3D structure determination of proteins and peptides from NMR data.

Improvement in accuracy of protein local structure determination from NMR data

Abstract A method for determining the most probable conformations of amino acid residues from semiquantitatively estimated nuclear Overhauser effects (NOEs) and coupling constants was developed and coded in the FiSiNOE-2 program. This program is a new version of the FiSiNOE program, utilizing NMR data with complementary knowledge-based information on protein structures. In FiSiNOE-2 this information is conformational clusters of the dihedral angles (φ, ψ, gc 1 ) derived from the Protein Data Bank. The FiSiNOE-2 method determines mathematical expectations and standard deviations for the angles φ, ψ, and χ 1 , and provides direct determination of the local structure of proteins from NMR data before building and refining their spatial structure. The results of the FiSiNOE-2 program in combination with the results of the habas program may be used to provide stereospecific assignments of a pair of β-methylene protons and to determine precisely allowed ranges of the φ, ψ, and χ 1 dihedral angles consistent with a given set of NMR data. To do this, a new procedure, combine , was developed. Computational experiments with the NMR data simulated from X-ray coordinates of the BPTI showed that use of the combine procedure, in comparison with results obtained when habas was used alone, increases by more than 30% the number of correct assignments for βCH 2 groups and reduces the total lengths of the combined angular intervals for φ, ψ, and χ 1 angles to 1.9, 2.4, and 1.8 times, respectively. In contrast to the redundant dihedral angle constraints (REDAC) strategy, that derives REDAC from preliminary calculations of the complete structure, the combine procedure reduces the length of the angular intervals before using the variable target function algorithm to determine spatial structures of proteins. This feature of the combine strategy may be especially beneficial in the cases when there is lack of long-range NOEs.