Jozef Vanden Broeck

Drosophila molting neurohormone bursicon is a heterodimer and the natural agonist of the orphan receptor DLGR2.

Bursicon is a neurohumoral agent responsible for tanning and hardening of the cuticle and expansion of the wings during the final phase of insect metamorphosis. Although the hormonal activity was described more than 40 years ago, the molecular nature of bursicon has remained elusive. We identify here Drosophila bioactive bursicon as a heterodimer made of two cystine knot polypeptides. This conclusion was reached in part from the unexpected observation that in the genome of the honey bee, the orthologs of the two Drosophila proteins are predicted to be fused in a single open reading frame. The heterodimeric Drosophila protein displays bursicon bioactivity in freshly eclosed neck‐ligated flies and is the natural agonist of the orphan G protein‐coupled receptor DLGR2.

Comparison of active conformations of the insectatachykinin/tachykinin and insect kinin/Tyr-W-MIF-1 neuropeptide family pairs.

Abstract: A comparison of solution conformations of active, restricted‐conformation analogues of two sequence‐similar insect/vertebrate neuropeptide family pairs shed light on the potential existence of molecular evolutionary relationships. Analogues of the locustatachykinins and the mammalian tachykinin substance P, containing a sterically hindered Aib‐NMePhe/Tyr residue block, share similar low‐energy turn conformations incorporating a cis peptide bond. Conversely, restricted conformation analogues of the insect kinins and the mammalian opiate peptide Tyr‐W‐MIF‐1, with near identical C‐terminal tetrapeptide sequences, adopt different conformations. The insect kinins adopt a cisPro 1‐4 β‐turn, in which the Phe1 is critical for bioactivity. Tyr‐W‐MIF‐1 prefers a transPro 2‐5 turn, and an additional N‐terminal Phe severely inhibits μ‐opiate receptor binding. Comparisons of the chemical/conformational requirements for receptor interaction are consistent with a distant evolutionary relationship between the insectatachykinins and tachykinins, but not between the insect kinins and Tyr‐W‐MIF‐1. Therefore, analogues of the insect kinins with pest control potential can be readily designed to avoid mammalian interactions.

Recombinant aequorin as a reporter for receptor-mediated changes of intracellular Ca2+ -levels in Drosophila S2 cells

Abstract. The bioluminescent Ca2+-sensitive reporter protein, aequorin, was employed to develop an insect cell-based functional assay system for monitoring receptor-mediated changes of intracellular Ca2+-concentrations. Drosophila Schneiderxa02 (S2) cells were genetically engineered to stably express both apoaequorin and the insect tachykinin-related peptide receptor, STKR. Lom-TKxa0III, an STKR agonist, was shown to elicit concentration-dependent bioluminescent responses in these S2-STKR-Aeq cells. The EC50 value for the calcium effect detected by means of aequorin appeared to be nearly identical to the one that was measured by means of Fura-2, a fluorescent Ca2+-indicator. In addition, this aequorin-based method was also utilised to study receptor antagonists. Experimental analysis of the effects exerted by spantidexa0I, II and III, three potent substancexa0P antagonists, on Lom-TKxa0III-stimulated S2-STKR-Aeq cells showed that these compounds antagonise STKR-mediated responses in a concentration-dependent manner. The rank order of inhibitory potencies was spantidexa0III > spantidexa0II > spantidexa0I.

Functional comparison of two evolutionary conserved insect neurokinin-like receptors

Tachykinins are multifunctional neuropeptides that have been identified in vertebrates as well as invertebrates. The C-terminal FXGXRa-motif constitutes the consensus active core region of invertebrate tachykinins. In Drosophila, two putative G protein-coupled tachykinin receptors have been cloned: DTKR and NKD. This study focuses on the functional characterization of DTKR, the Drosophila ortholog of the stable flys tachykinin receptor (STKR). Tachykinins containing an alanine residue instead of the highly conserved glycine (FXAXRa) display partial agonism on STKR-mediated Ca(2+)-responses, but not on cAMP-responses. STKR therefore seems to differentiate between a number of tachykinins. Gly- and Ala-containing tachykinins are both encoded in the Drosophila tachykinin precursor, thus raising the question of whether DTKR can also distinguish between these two tachykinin types. DTKR was activated by all Drosophila tachykinins and inhibited by tachykinin antagonists. Ala-containing analogs did not produce the remarkable activation behavior previously observed with STKR, suggesting different mechanisms of discerning ligands and/or activating effector pathways for STKR and DTKR.

Synthesis and Biological Activity of Endomorphin‐2 Analogs Incorporating Piperidine‐2‐, 3‐ or 4‐Carboxylic Acids Instead of Proline in Position 2

Novel endomorphin‐2 (EM‐2) analogs have been synthesized, incorporating unnatural amino acids with six‐membered heterocyclic rings, such as piperidine‐2‐, 3‐ and 4‐carboxylic acids (Pip, Nip and Inp, respectively) instead of Pro in position 2. [(R)‐Nip2]EM‐2 displayed an extremely high affinity for the μ‐opioid receptor with IC50u2003=u20030.04u2003± 0.01u2003nm in comparison with IC50u2003=u20030.69u2003±u20030.03u2003nm for EM‐2. This analog was also very potent in the aequorin luminescence‐based functional calcium assay and showed significantly enhanced stability in rat brain homogenate.

Novel highly potent μ-opioid receptor antagonist based on endomorphin-2 structure

The mu-opioid agonists endomorphin-1 (Tyr-Pro-Trp-Phe-NH(2)) and endomorphin-2 (Tyr-Pro-Phe-Phe-NH(2)) exhibit an extremely high selectivity for the mu-opioid receptor and thus represent a potential framework for modification into mu-antagonists. Here we report on the synthesis and biological evaluation of novel [d-2-Nal(4)]endomorphin-2 analogs, [Sar(2),d-2-Nal(4)]endomorphin-2 and [Dmt(1),Sar(2),d-2-Nal(4)]endomorphin-2 (Dmt=26-dimethyltyrosine; Sar=N-methylglycine, sarcosine; d-2-Nal=3-(2-naphthyl)-d-alanine). [Dmt(1),Sar(2),d-2-Nal(4)]endomorphin-2 possessed very high affinity for the mu-opioid receptor (IC(50)=0.01+/-0.001 nM) and turned out to be a potent and extremely selective mu-opioid receptor antagonist, as judged by the in vitro aequorin luminescence-based calcium assay (pA(2)=9.19). However, in the in vivo hot plate test in mice this analog was less potent than our earlier mu-opioid receptor antagonist, [Dmt(1),d-2-Nal(4)]endomorphin-2 (antanal-2). The exceptional mu-opioid receptor in vitro activity and selectivity of [Dmt(1), Sar(2),d-2-Nal(4)]endomorphin-2 makes this analog a valuable pharmacological tool, but further modifications are needed to improve its in vivo profile.

Insect Basic Leucine Zipper Proteins and Their Role in Cyclic AMP-Dependent Regulation of Gene Expression

The cAMP-protein kinase A (PKA) pathway is an important intracellular signal transduction cascade that can be activated by a large variety of stimuli. Activation or inhibition of this pathway will ultimately affect the transcriptional regulation of various genes through distinct responsive sites. In vertebrates, the best- characterized nuclear targets of PKA are the cyclic AMP response element-binding (CREB) proteins. It is now well established that CREB is not only regulated by PKA, but many other kinases can exert an effect as well. Since CREB-like proteins were also discovered in invertebrates, several studies unraveling their physiological functions in this category of metazoans have been performed. This review will mainly focus on the presence and regulation of CREB proteins in insects. Differences in transcriptional responses to the PKA pathway and other CREB-regulating stimuli between cells, tissues, and even organisms can be partially attributed to the presence of different CREB isoforms. In addition, the regulation of CREB appears to show some important differences between insects and vertebrates. Since CREB is a basic leucine zipper (bZip) protein, other insect members of this important family of transcriptional regulators will be briefly discussed as well.

Comparison of antagonist activity of spantide family at human neurokinin receptors measured by aequorin luminescence-based functional calcium assay.

Neurokinin receptors (NK1, NK2, NK3) are G-protein-coupled receptors, which upon activation by a peptide agonist induce a transient increase in the concentration of intracellular calcium. The functional assay based on aequorin-derived luminescence triggered by receptor-mediated changes in Ca2+ levels was used to compare the effect of spantides I-III on SP-, NKA- and NKB-stimulated NK1, NK2 and NK3 receptors, respectively. Recombinant cell lines expressing neurokinin receptors and apoaequorin were used in the study. The obtained results indicate that all three spantides acted as competitive antagonists at the NK1 and NK2 receptors and inhibited agonist-induced calcium responses. The rank order of antagonism at the NK1 receptor was spantide II>spantide III>spantide I and at the NK2 receptor was spantide III>spantide II>spantide I. All three spantides failed to antagonize NKB-induced calcium responses at the NK3 receptor.

Design, Synthesis and Pharmacological Characterization of Endomorphin Analogues with Non-Cyclic Amino Acid Residues in Position 2

A series of endomorphin-1 (EM-1) and endomorphin-2 (EM-2) analogues, containing non-cyclic amino acids (Ala, D-Ala, beta-Ala, NMeAla, D-NMeAla or Sar) instead of Pro in position 2 was synthesized, where NMeAla = N-methylalanine and Sar = N-methylglycine, sarcosine. The opioid activity profiles of these peptides were determined in mu and delta opioid receptor (MOR and DOR)-representative binding assays and bioassays in vitro, as well as in the mouse hot-plate test in vivo. Finally, the degradation rates of all analogues in the presence of either rat brain homogenate or selected proteolytic enzymes were determined. Analogues of EM-2 were generally more potent than the respective analogues of EM-1. EM-2 analogues with D-Ala or D-NMeAla were about twofold more potent than the parent peptide and were least prone to degradation by brain homogenate, dipeptydyl peptidase IV and aminopeptidase M. In the in vivo test, [D-Ala(2)]EM-2 and [D-NMeAla(2)]EM-2 showed much higher analgesic potency than EM-2 which confirmed the usefulness of structural modifications in obtaining new leads for pain-relief therapeutics.

CHD1 assumes a central role during follicle development.

During Bombyx mori follicle development, fine-tuning of chorion gene expression is under the control of bidirectional promoters. In this work, we show that the silkmoth chromo-helicase/ATPase-DNA binding protein 1 (CHD1) ortholog is responsible for repositioning of nucleosomes on chorion promoters, where the factor binds specifically. Chorion genes, occupying a single chromosomal locus, rely on an almost identical set of cis elements for their differential expression. As a direct consequence of remodeling, interaction of C/EBP and TFIID with promoter elements is facilitated and ultimately leads to initiation of transcription. Appending of methylation marks to H3K4 in a temporal-specific manner is dependent on CHD1 binding to cognate cis elements and signifies gene activation. Overall, CHD1 is a critical factor for proper development of the follicular epithelium in terms of whole-cell chromatin arrangement.