Converting Insulin-like Growth Factors 1 and 2 into High-Affinity Ligands for Insulin Receptor Isoform A by the Introduction of an Evolutionarily Divergent Mutation.

Insulin-like growth factors 1 and 2 (IGF-1 and -2, respectively) are protein hormones involved not only in normal growth and development but also in life span regulation and cancer. They exert their functions mainly through the IGF-1R or by binding to isoform A of the insulin receptor (IR-A). The development of IGF-1 and IGF-2 antagonists is of great clinical interest.

Insulin-like Growth Factor 1 Analogs Clicked in the C Domain: Chemical Synthesis and Biological Activities.

Human insulin-like growth factor 1 (IGF-1) is a 70 amino acid protein hormone, with key impact on growth, development, and lifespan. The physiological and clinical importance of IGF-1 prompted challenging chemical and biological trials toward the development of its analogs as molecular tools for the IGF-1 receptor (IGF1-R) studies and as new therapeutics. Here, we report a new method for the total chemical synthesis of IGF-1 analogs, which entails the solid-phase synthesis of two IGF-1 precursor chains that is followed by the Cu-catalyzed azide-alkyne cycloaddition ligation and by biomimetic formation of a native pattern of disulfides.

Optimized syntheses of Fmoc azido amino acids for the preparation of azidopeptides.

The rise of CuI-catalyzed click chemistry has initiated an increased demand for azido and alkyne derivatives of amino acid as precursors for the synthesis of clicked peptides. However, the use of azido and alkyne amino acids in peptide chemistry is complicated by their high cost. For this reason, we investigated the possibility of the in-house preparation of a set of five Fmoc azido amino acids: β-azido l-alanine and d-alanine, γ-azido l-homoalanine, δ-azido l-ornithine and ω-azido l-lysine.

Synthesis and Evaluation of a Library of Trifunctional Scaffold-Derived Compounds as Modulators of the Insulin Receptor.

We designed a combinatorial library of trifunctional scaffold-derived compounds, which were derivatized with 30 different in-house-made azides. The compounds were proposed to mimic insulin receptor (IR)-binding epitopes in the insulin molecule and bind to and activate this receptor. This work has enabled us to test our synthetic and biological methodology and to prove its robustness and reliability for the solid-phase synthesis and testing of combinatorial libraries of the trifunctional scaffold-derived compounds.

Probing Receptor Specificity by Sampling the Conformational Space of the Insulin-like Growth Factor II C-domain.

Insulin and insulin-like growth factors I and II are closely related protein hormones. Their distinct evolution has resulted in different yet overlapping biological functions with insulin becoming a key regulator of metabolism, whereas insulin-like growth factors (IGF)-I/II are major growth factors. Insulin and IGFs cross-bind with different affinities to closely related insulin receptor isoforms A and B (IR-A and IR-B) and insulin-like growth factor type I receptor (IGF-1R).

Insulin-Insulin-like Growth Factors Hybrids as Molecular Probes of Hormone:Receptor Binding Specificity.

Insulin, insulin-like growth factors 1 and 2 (IGF-1 and -2, respectively), and their receptors (IR and IGF-1R) are the key elements of a complex hormonal system that is essential for the development and functioning of humans. The C and D domains of IGFs (absent in insulin) likely play important roles in the differential binding of IGF-1 and -2 to IGF-1R and to the isoforms of IR (IR-A and IR-B) and specific activation of these receptors. Here, we attempted to probe the impact of IGF-1 and IGF-2 D domains (DI and DII, respectively) and the IGF-2 C domain (CII) on the receptor specificity of these hormones.

Rational steering of insulin binding specificity by intra-chain chemical crosslinking.

Insulin is a key hormone of human metabolism with major therapeutic importance for both types of diabetes. New insulin analogues with more physiological profiles and better glycemic control are needed, especially analogues that preferentially bind to the metabolic B-isoform of insulin receptor (IR-B). Here, we aimed to stabilize and modulate the receptor-compatible conformation of insulin by covalent intra-chain crosslinking within its B22-B30 segment, using the Cu(I)-catalyzed Huisgen 1,3-dipolar cycloaddition reaction of azides and alkynes.

Human insulin analogues modified at the B26 site reveal a hormone conformation that is undetected in the receptor complex.

The structural characterization of the insulin-insulin receptor (IR) interaction still lacks the conformation of the crucial B21-B30 insulin region, which must be different from that in its storage forms to ensure effective receptor binding. Here, it is shown that insulin analogues modified by natural amino acids at the TyrB26 site can represent an active form of this hormone. In particular, [AsnB26]-insulin and [GlyB26]-insulin attain a B26-turn-like conformation that differs from that in all known structures of the native hormone.

Insight into the structural and biological relevance of the T/R transition of the N-terminus of the B-chain in human insulin.

The N-terminus of the B-chain of insulin may adopt two alternative conformations designated as the T- and R-states. Despite the recent structural insight into insulin-insulin receptor (IR) complexes, the physiological relevance of the T/R transition is still unclear. Hence, this study focused on the rational design, synthesis, and characterization of human insulin analogues structurally locked in expected R- or T-states.

Determination of pK(a) values of diastereomers of phosphinic pseudopeptides by CZE.

A CE method was used for the determination of acidity constants (pK(a)) of a series of ten phosphinic pseudopeptides, which varied in number and type of ionogenic groups. Effective electrophoretic mobilities were measured in the 1.8-12.

S-alkylated homocysteine derivatives: new inhibitors of human betaine-homocysteine S-methyltransferase.

A series of S-alkylated derivatives of homocysteine were synthesized and characterized as inhibitors of human recombinant betaine-homocysteine S-methyltransferase (BHMT). Some of these compounds inhibit BHMT with IC50 values in the nanomolar range. BHMT is very sensitive to the structure of substituents on the sulfur atom of homocysteine.

Activation of murine RNase L by isopolar 2'-phosphonate analogues of 2',5' oligoadenylates.

To determine the influence of methylene group insertion in the internucleotide linkage on the binding process of 2',5'-oligoadenylates to RNase L, a series of 2'-phosphonate-modified trimers and tetramers were synthesized from appropriate monomeric units and evaluated for their ability to bind to murine RNase L. Tetramers pAAXA modified by ribo-, arabino-, or xylo-2'-phosphonate unit X in the third position were capable of binding to RNase L in nanomolar concentrations. The replacement of the first residue (pXAAA), or both the first and the third residues (pXAXA), was also tolerated by the enzyme.

Inhibition of betaine-homocysteine S-methyltransferase causes hyperhomocysteinemia in mice.

Inhibitors and methyl donor substrates for betaine-homocysteine S-methyltransferase (BHMT) were used to study the role of this enzyme in the regulation of plasma total homocysteine (tHcy). Mice were administered an i.p.

Dissecting the catalytic mechanism of betaine-homocysteine S-methyltransferase by use of intrinsic tryptophan fluorescence and site-directed mutagenesis.

Betaine-homocysteine S-methyltransferase (BHMT) is a zinc-dependent enzyme that catalyzes the transfer of a methyl group from glycine betaine (Bet) to homocysteine (Hcy) to form dimethylglycine (DMG) and methionine (Met). Previous studies in other laboratories have indicated that catalysis proceeds through the formation of a ternary complex, with a transition state mimicked by the inhibitor S-(delta-carboxybutyl)-l-homocysteine (CBHcy). Using changes in intrinsic tryptophan fluorescence to determine the affinity of human BHMT for substrates, products, or CBHcy, we now demonstrate that the enzyme-substrate complex reaches its transition state through an ordered bi-bi mechanism in which Hcy is the first substrate to bind and Met is the last product released.

Physicochemical characterization of phosphinic pseudopeptides by capillary zone electrophoresis in highly acidic background electrolytes.

Phosphinic pseudopeptides (i.e., peptide isosteres with one peptide bond replaced by a phosphinic acid moiety) were analyzed and physicochemically characterized by capillary zone electrophoresis in the pH range of 1.

Combining combinatorial chemistry and affinity chromatography: highly selective inhibitors of human betaine: homocysteine S-methyltransferase.

A new method to find novel protein targets for ligands of interest is proposed. The principle of this approach is based on affinity chromatography and combinatorial chemistry. The proteins within a crude rat liver homogenate were allowed to interact with a combinatorial library of phosphinic pseudopeptides immobilized on affinity columns.

Determination of dissociation constant of phosphinate group in phosphinic pseudopeptides by capillary zone electrophoresis.

Capillary zone electrophoresis (CZE) was used for determination of dissociation constant of phosphinate group in phosphinic pseudopeptides, i.e. peptides where one peptide bond is substituted by phosphinic acid moiety -PO2--CH2-.

Analysis and characterization of phosphinic pseudopeptides by capillary zone electrophoresis.

Capillary zone electrophoresis (CZE) was applied to analysis and characterization of phosphinic pseudopeptides with the general structure N-Ac-Val-Ala(psi)(PO2(-)-CH(2)) Leu-Xaa-NH(2), where Xaa represents one of 20 proteinogenic amino acid residues. Pseudopeptides containing neutral or acidic amino acid residues in position Xaa were analyzed as anions in weakly alkaline (pH 8.1) Tris-Tricine background electrolyte (BGE), pseudopeptides with basic amino acid residues in position Xaa were analyzed as cations in acid BGEs (Tris-phosphate buffers).