Jane V. Aldrich

Chloroplast DNA evolution among legumes: Loss of a large inverted repeat occurred prior to other sequence rearrangements

SummaryWe have compared the sequence organization of four previously uncharacterized legume chloroplast DNAs - from alfalfa, lupine, wisteria and subclover — to that of legume chloroplast DNAs that either retain a large, ribosomal RNA-encoding inverted repeat (mung bean) or have deleted one half of this repeat (broad bean). The circular, 126 kilobase pair (kb) alfalfa chloroplast genome, like those of broad bean and pea, lacks any detectable repeated sequences and contains only a single set of ribosomal RNA genes. However, in contrast to broad bean and pea, alfalfa chloroplast DNA is unrearranged (except for the deletion of one segment of the inverted repeat) relative to chloroplast DNA from mung bean. Together with other findings reported here, these results allow us to determine which of the four possible inverted repeat configurations was deleted in the alfalfa-pea-broad bean lineage, and to show how the present-day broad bean genome may have been derived from an alfalfa-like ancestral genome by two major sequence inversions. The 147 kb lupine chloroplast genome contains a 22 kb inverted repeat and has essentially complete colinearity with the mung bean genome. In contrast, the 130 kb wisteria genome has deleted one half of the inverted repeat and appears colinear with the alfalfa genome. The 140 kb subclover genome has been extensively rearranged and contains a family of at least five dispersed repetitive sequence elements, each several hundred by in size; this is the first report of dispersed repeats of this size in a land plant chloroplast genome. We conclude that the inverted repeat has been lost only once among legumes and that this loss occurred prior to all the other rearrangements observed in subclover, broad bean and pea. Of those lineages that lack the inverted repeat, some are stable and unrearranged, other have undergone a moderate amount of rearrangement, while still others have sustained a complex series of rearrangement either with or without major sequence duplications and transpositions.

Zyklophin, a systemically active selective kappa opioid receptor peptide antagonist with short duration of action

The cyclic peptide zyklophin {[N-benzylTyr1,cyclo(D-Asp5,Dap8)-dynorphin A-(1–11)NH2, Patkar KA, et al. (2005) J Med Chem 48: 4500–4503} is a selective peptide kappa opioid receptor (KOR) antagonist that shows activity following systemic administration. Systemic (1–3 mg/kg s.c.) as well as central (0.3–3 nmol intracerebroventricular, i.c.v.) administration of this peptide dose-dependently antagonizes the antinociception induced by the selective KOR agonist U50,488 in C57BL/6J mice tested in the 55 °C warm water tail withdrawal assay. Zyklophin administration had no effect on morphine- or SNC-80-mediated antinociception, suggesting that zyklophin selectively antagonizes KOR in vivo. Additionally, the antagonism of antinociception induced by centrally (i.c.v.) administered U50,488 following peripheral administration of zyklophin strongly suggests that the peptide crosses the blood-brain barrier to antagonize KOR in the CNS. Most importantly, the antagonist activity of zyklophin (3 mg/kg s.c.) lasts less than 12 h, which contrasts sharply with the exceptionally long duration of antagonism reported for the established small-molecule selective KOR antagonists such as nor-binaltorphimine (nor-BNI) that last weeks after a single administration. Systemically administered zyklophin (3 mg/kg s.c.) also prevented stress-induced reinstatement of cocaine-seeking behavior in a conditioned place preference assay. In conclusion, the peptide zyklophin is a KOR-selective antagonist that exhibits the desired shorter duration of action, and represents a significant advance in the development of KOR-selective antagonists.

The Macrocyclic Peptide Natural Product CJ-15,208 Is Orally Active and Prevents Reinstatement of Extinguished Cocaine-Seeking Behavior

The macrocyclic tetrapeptide natural product CJ-15,208 (cyclo[Phe-d-Pro-Phe-Trp]) exhibited both dose-dependent antinociception and kappa opioid receptor (KOR) antagonist activity after oral administration. CJ-15,208 antagonized a centrally administered KOR selective agonist, providing strong evidence it crosses the blood-brain barrier to reach KOR in the CNS. Orally administered CJ-15,208 also prevented both cocaine- and stress-induced reinstatement of extinguished cocaine-seeking behavior in the conditioned place preference assay in a time- and dose-dependent manner. Thus, CJ-15,208 is a promising lead compound with a unique activity profile for potential development, particularly as a therapeutic to prevent relapse to drug-seeking behavior in abstinent subjects.

High affinity conformationally constrained nociceptin/orphanin FQ(1-13) amide analogues.

A series of cyclic analogues with a lactam linkage were prepared by solid phase peptide synthesis to explore possible biologically active conformation(s) of nociceptin/orphanin FQ (N/OFQ). cyclo[D-Asp(7),Lys(10)]- and cyclo[Asp (6),Lys(10)]N/OFQ(1-13)NH2 exhibit high affinity (Ki = 0.27 and 0.34 nM, respectively) and high potency in the GTPgammaS assay (EC 50 = 1.6 and 4.1 nM, respectively) at human nociceptin/orphanin FQ peptide (NOP) receptors. These analogues exhibit 2- to 3-fold higher affinity and 2- to 5-fold higher potency than the parent peptide.

Synthesis and opioid activity of conformationally constrained dynorphin A analogues

Several cyclic lactam analogues of Dyn A-(1-13)NH2 were prepared in order to reduce the conformational flexibility in different regions of the native linear peptide. Cyclo[D-Asp(i),Dap(i+3)]Dyn A-(1-13)NH2 (Dap = alpha,beta-diaminopropionic acid) analogues were designed on the basis of molecular modeling using AMBER, which suggested that this constraint may be compatible with an alpha-helix. The cyclic portion of these constrained analogues spanned from residues 3 to 9, a region proposed by Schwyzer (Biochemistry 1986, 25, 4281) to adopt a helical conformation at kappa receptor sites. Analogues containing Dab (alpha,gamma-diaminobutyric acid) or Orn in position i + 3 were also synthesized to examine the effects of larger ring size. The cyclic peptides exhibited marked differences in binding affinities for kappa, mu, and delta receptors and in opioid activity in the guinea pig ileum (GPI). Cyclo[D-Asp6,Dap9]Dyn A-(1-13)NH2 showed both high kappa receptor affinity and potent agonist activity in the GPI, while cyclo[D-Asp3,Dap6]Dyn A-(1-13)NH2 exhibited very weak binding affinity at all opioid receptors as well as very weak opioid activity in the GPI. Cyclo[D-Asp5,Dap8]Dyn A-(1-13)NH2 showed moderate binding affinity for kappa receptors and was the most kappa selective ligand in this study, but this peptide exhibited very weak agonist activity in the GPI assay. Compared to the corresponding linear peptides, all of the cyclic peptides exhibited decreased mu receptor affinity, while kappa receptor affinity was retained or improved. Therefore the corresponding linear peptides were generally mu selective while the cyclic constrained peptides demonstrated slight selectivity for kappa vs mu receptors or were nonselective. Increasing the ring size by incorporating Dab or Orn in positions 6, 8, or 9 did not significantly affect the binding affinity for the three opioid receptor types nor the opioid activity observed in the GPI. Circular dichroism spectra of the cyclo[D-Asp(i),Dap(i+3)] derivatives in 80% trifluoroethanol at 25 and 5 degrees C suggested differences in the stability of a helical structure when the constraint was incorporated near the N-terminus vs in the middle of the peptide.

Synthesis of enkephalin-based affinity labels for delta opioid receptors

Abstract Affinity labels (irreversible ligands) are useful pharmacological tools to study receptor structure and function. As part of a program to prepare affinity labels based on opioid peptides, we developed a general synthetic strategy to introduce different electrophilic labeling moieties (i.e. isothiocyanate and bromoacetamide) via a p-amino functionality on a phenylalanine residue. The affinity labelling groups were incorporated into the Phe 4 position of the delta opioid receptor antagonists ICI-174,864 (1) and N,N-dibenzyl leucine enkephalin (2). Using an orthogonal Boc/Fmoc protection strategy, the common peptide precursors were assembled in solution, the affinity labels introduced and the peptides deprotected. In cloned δ receptors expressed in CHO cells (3), only the isothiocyanate derivative of N,N-dibenzyl leucine enkephalin exhibited wash-resistant inhibition of [ 3 H]DPDPE binding.

Optimized Method to Generate Synthetically Challenging Macrocyclic Tetrapeptides that do not have a Turn Inducer

Natural and synthetic cyclic peptides represent an important class of bioactive molecules that exhibit a broad range of biological activities [1] and are considered as promising lead compounds in the search for new drugs. In particular, macrocyclic tetrapeptides (MTPs) have potential for development into therapeutic agents because of their low molecular weight, small organic molecule-like properties, membrane permeability, selectivity, expected metabolic stability to proteases, and improved pharmacokinetic properties in vivo [1,2]. However, the small 12-membered ring size can make their synthesis difficult, often resulting in low yields of the desired MTP with resulting dimers and oliogomers as the major products [3-5]. Generally MTPs in which the amino acids are all the same chirality are more difficult to synthesize because the conformations of the linear precursor tetrapeptides limit the close approach of the Nand C-termini needed for macrocyclization to occur [5]. Also to date, almost all reported synthesized MTPs contain at least one turn inducing residue such as Pro, an N-alkyl amino acid, a D-amino acid or an unusual amino acid that favors macrocyclization [1,6]. As part of our long standing interest in peptide ligands for kappa opioid receptors (KOR) we are synthesizing analogs of the natural product macrocyclic tetrapeptide CJ-15,208 (Figure 1) which exhibits modest affinity for KOR [4,7]. We use an improved synthetic protocol to prepare larger quantities of the macrocyclic tetrapeptide CJ-15,208, [D-Trp]CJ-15,208 and their analogs for detailed pharmacological evaluation in vivo [8-10]. To demonstrate the broad applicability of this methodology, here we describe the challenging syntheses of two all L-amino acid macrocyclic tetrapeptides (all L-AA MTPs, Figure 2) based on the CJ-15,208 scaffold that do not contain a turn inducer to promote macrocyclization.