Kazuhide Miyamoto

Synthesis and evaluation of pseudopeptide analogues of a specific CXCR4 inhibitor, T140: The insertion of an (E)-alkene dipeptide isostere into the βII'-turn moiety

Abstract A 14-residue peptide, T140, strongly inhibits the T-cell line-tropic HIV-1 (X4-HIV-1) infection, since this peptide functions as a specific antagonist against a chemokine receptor, CXCR4. T140 takes an antiparallel β-sheet structure with a type II′ β-turn. In the present paper, we have designed and synthesized several T140 analogues, in which an (E)-alkene dipeptide isostere was inserted into the type II′ β-turn moiety, as a bridging study to develop nonpeptidic CXCR4 inhibitors. It has been proven that the turn region of T140 can be replaced by the above surrogate with the maintenance of strong anti-HIV activity.

Solution structure of the cytoplasmic linker between domain III‐S6 and domain IV‐S1 (III–IV linker) of the rat brain sodium channel in SDS micelles

The solution structure of the 36-mer peptide MP-5A in SDS micelles was investigated by CD and (1)H-NMR spectroscopies. The MP-5A was dissected from the cytoplasmic linker (K1482-A1517) connecting domain III-segment 6 (IIIS6) and domain IV-segment 1 (IVS1; III-IV linker) of the rat brain type IIA sodium channel. The molecular energy calculations including nuclear Overhauser effect and dihedral angle restraints gave a well-converged set of the structures of MP-5A for the region between I1488 and S1506. It was found that a large hydrophobic cluster is formed by I1488-F1489-M1490 (IFM motif), Y1497-Y1498, and M1501, which may be related to the fast inactivation process of the sodium channel. The solvent-accessible surface area of the IFM motif (195 A(2)), which is known to work essentially as an inactivation gate particle to occlude the ion permeation pore, gave the free energy (DeltaG) of stabilization of -3.9 kcal mol(-1) as a result of the hydrophobic interactions with its receptor. This value agreed well with the free energy of binding (inactivation) of -4.1 kcal mol(-1) calculated for the equilibrium between the open and the inactivated states of the sodium channels. It is concluded that the fast inactivation of the sodium channel is achieved by the environmental polarity-dependent conformational switching at the IFM motif, in response to the voltage-dependent activation and the movement of the S4 segments of the sodium channel.

1H-NMR and Circular Dichroism Spectroscopic Studies on Changes in Secondary Structures of the Sodium Channel Inactivation Gate Peptides as Caused by the Pentapeptide KIFMK

The pentapeptide KIFMK, which contains three clustered hydrophobic amino acid residues of isoleucine, phenylalanine, and methionine (IFM) in the sodium channel inactivation gate on the cytoplasmic linker between domains III and IV (III-IV linker), is known to restore fast inactivation to the mutant sodium channels having a defective inactivation gate or to accelerate the inactivation of the wild-type sodium channels. To investigate the docking site of KIFMK and to clarify the mechanisms for restoring the fast inactivation, we have studied the interactions between KIFMK and the fragment peptide in the III-IV linker GGQDIFMTEEQK (MP-1A; G1484-K1495 in rat brain IIA) by one- and two-dimensional (1)H-NMR and circular dichroism (CD) spectroscopies. KIFMK was found to increase the helical content of MP-1A in 80% trifluoroethanol (TFE) solution by approximately 11%. A pentapeptide, KIFMT, which can restore inactivation but less effectively than KIFMK, also increased the helical content of MP-1A, but to a lesser extent ( approximately 6%) than did KIFMK. In contrast, KDIFMTK, which is ineffective in restoring inactivation, decreased the helical content ( approximately -4%). Furthermore, we studied the interactions between KIFMK and modified peptides from MP-1A, that is, MP-1NA (D1487N), MP-1QEA (E1492Q), or MP-1EQA (E1493Q). The KIFMK was found to increase the helical content of MP-1EQA to an extent nearly identical to that of MP-1A, whereas it was found to decrease those of MP-1NA and MP-1QEA. These findings mean that KIFMK, by allowing each of the Lys residues to interact with D1487 and E1492, respectively, stabilized the helical structure of the III-IV linker around the IFM residues. This helix-stabilizing effect of KIFMK on the III-IV linker may restore and/or accelerate fast inactivation to the sodium channels having a defective inactivation gate or to wild-type sodium channels.