Whyte, Michael P.; McAlister, William H.; and et al, ,"Enzyme-replacement therapy in life-threatening hypophosphatasia." The New England Journal of Medicine.366,10. 904-913. (2012).

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Side-chain lactam bridges linking amino acid residues that are spaced several residues apart in the linear sequence offer a convenient and flexible method for introducing conformational constraints into a peptide structure. The availability of a variety of selectively cleavable protecting groups for amines and carboxylic acids allows for several approaches to the synthesis of monocyclic, dicyclic, and bicyclic lactam-bridged peptides by solid-phase methods. Multicyclic structures are also accessible, but segment-condensation syntheses with solution-phase cyclizations are most likely to provide the best synthetic approach to these more complex constrained peptides. Lactam bridges linking (i, i + 3)-, (i, i + 4), and (i, i + 7)-spaced residue pairs have all proven useful for stabilization of alpha helices, and (i, i + 3)-linked residues have also been demonstrated to stabilize beta-turns. These structures are finding an increasing number of applications in protein biology, including studies of protein folding, protein aggregation, peptide ligand-receptor recognition, and the development of more potent peptide therapeutics. Defining the functional roles of the amphiphilic alpha-helices in medium-sized peptide hormones, and studying helix propagation from rigid, alpha-helix initiating bicyclic peptides are among the most exciting developments currently underway in this field.

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The conformational and pharmacological effects of the introduction of conformational constraints in the form of i-(i + 4) lactam-bridges in the potential amphiphilic alpha-helical region (8-21) of human calcitonin (hCT) were studied. The following three cyclic hCT analogues were synthesized: cyclo17,21-[Lys17,Asp21]hCT (1), cyclo17,21- [Asp17,Lys21]hCT (2) and cyclo10,14-[Lys10,-Asp14]hCT (3). For their syntheses, solid-phase methodology was used in combination with either direct side chain to side chain cyclization on the solid support or a segment-condensation strategy. Circular dichroism studies in aqueous buffer, pH 7.0, indicated that the conformational effects were different for each lactam bridge introduced. Significant induction of alpha-helical structure was observed only for peptide 3. In contrast, peptide 1 and hCT had similar CD spectra, indicative of mixed disordered and beta-sheet conformations, and peptide 2 had a weaker spectrum consistent with the formation of a more ordered but nonhelical structure. In rat brain receptor binding assays, peptide 2 showed a nearly 80-fold higher potency than hCT or peptides 1 and 3. All three analogues stimulated adenylyl cyclase in the rat kidney membrane at 5-fold lower concentrations than hCT and with similar maximal effects. In vivo hypocalcemic assays, performed in mice by analysis of serum calcium levels 1 h after sc injection, indicated that peptide 2 had similar maximal effects to hCT and was 10-20 times more potent than hCT at doses giving half-maximal effects. In contrast, peptides 1 and 3 were not significantly more potent than hCT. Our findings indicate compatibility of all three lactam bridges and, most probably, also the amphiphilic alpha-helix, with the pharmacological activities of hCT. However, the properties of peptide 2 also suggest that another conformation, possibly a type I beta-turn involving residues 17-20, may play an important role. A multistep mechanism of receptor recognition by hCT that might account for these results is discussed.

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In efforts to develop AIDS vaccine components, we generated combinatorial libraries of recombinant human rhinoviruses that display the well-conserved ELDKWA epitope of the membrane-proximal external region of human immunodeficiency virus type 1 (HIV-1) gp41. The broadly neutralizing human monoclonal antibody 2F5 was used to select for viruses whose ELDKWA conformations resemble those of HIV. Immunization of guinea pigs with different chimeras, some boosted with ELDKWA-based peptides, elicited antibodies capable of neutralizing HIV-1 pseudoviruses of diverse subtypes and coreceptor usages. These recombinant immunogens are the first reported that elicit broad, albeit modest, neutralization of HIV-1 using an ELDKWA-based epitope and are among the few reported that elicit broad neutralization directed against any recombinant HIV epitope, providing a critical advance in developing effective AIDS vaccine components.

Background: Our Phase 2, open-label study of 11 infants and young children with lifethreatening perinatal or infantile hypophosphatasia (HPP) demonstrated 1-year safety and efficacy of asfotase alfa, an enzyme replacement therapy. We report outcomes over ~7 years. Methods: Patients received asfotase alfa (1 mg/kg thrice weekly subcutaneously; adjusted to 3 mg/kg thrice weekly if required). HPP skeletal manifestations were evaluated on the Radiographic Global Impression of Change (RGI-C) scale (−3=severe worsening; +3=complete/near complete healing). Respiratory support, growth, and cognitive and motor function were also evaluated. Findings: Ten patients completed a 6-month treatment period and entered an extension; nine received asfotase alfa for ≥6 years and completed the study, with four treated >7 years. Skeletal healing was sustained over 7 years of treatment; all evaluable patients had RGI-C scores ≥+2 at Years 6 and 7. No patient who completed the study required respiratory support after Year 4. Weight Z-scores improved to within normal range from Year 3 to study end; length/height Z-scores improved but remained below normal. Age-equivalent scores on Gross Motor, Fine Motor, and Cognitive subscales of the Bayley Scales of Infant and Toddler Development also improved. Treatment was generally well tolerated; adverse events were similar to those previously published. Interpretation: Patients with perinatal or infantile HPP treated with asfotase alfa for up to 7 years showed early, sustained improvements in skeletal mineralization. Respiratory function, growth, and cognitive and motor function also improved. Asfotase alfa is safe and effective in perinatal/infantile HPP.