Structural elucidation of Leuprolide and its analogues in solution: insight into their bioactive conformation

Leuprolide [dLeu⁶, NHEt¹⁰]GnRH, a potent gonadotropin-releasing hormone (GnRH) agonist, is used in a wide variety of hormone-related diseases like cancer and endometriosis. In this report, the conformational behaviour of Leuprolide and its linear synthetic analogues, namely [Tyr⁵(OMe), dLeu⁶, Aze⁹, NHEt¹⁰]GnRH (1) and [Tyr⁵(OMe), dLeu⁶, NHEt¹⁰]GnRH (2) have been studied in DMSO and H₂O solutions by means of 2D nuclear magnetic resonance (NMR) experiments and detailed molecular dynamics (MD) simulations. The aim was to identify the conformational requirements of GnRH analogues for agonistic activity. This approach is of value as no crystallographic data are available for the GnRH receptor (G protein-coupled receptor, GPCR). The NOE data indicate the existence of a β-turn type I in the 2–5 segments of Leuprolide and its linear analogues in the case of using DMSO-d₆ as solvent, whereas a β-turn type II in the 3–6 segments is indicated using D₂O as solvent. The final structures fulfil the conformational requirements that are known, in the literature, to play a significant role in receptor recognition and activation. Finally, the linear analogues (1) and (2) are biologically active when tested against the human breast cancer cell line, MCF-7.     

A COVID moonshot: assessment of ligand binding to the SARS-CoV-2 main protease by saturation transfer difference NMR spectroscopy

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the etiological cause of the coronavirus disease 2019, for which no effective antiviral therapeutics are available. The SARS-CoV-2 main protease (M^pro) is essential for viral replication and constitutes a promising therapeutic target. Many efforts aimed at deriving effective M^pro inhibitors are currently underway, including an international open-science discovery project, codenamed COVID Moonshot. As part of COVID Moonshot, we used saturation transfer difference nuclear magnetic resonance (STD-NMR) spectroscopy to assess the binding of putative M^pro ligands to the viral protease, including molecules identified by crystallographic fragment screening and novel compounds designed as M^pro inhibitors. In this manner, we aimed to complement enzymatic activity assays of M^pro performed by other groups with information on ligand affinity. We have made the M^pro STD-NMR data publicly available. Here, we provide detailed information on the NMR protocols used and challenges faced, thereby placing these data into context. Our goal is to assist the interpretation of M^pro STD-NMR data, thereby accelerating ongoing drug design efforts.

     

Novel stable analogues of the neurotensin C-terminal hexapeptide containing unnatural amino acids

Amino Acids - Neurotensin (NT) (pGlu–Leu–Tyr–Glu–Asn–Lys–Pro–Arg–Arg–Pro–Tyr–Ile–Leu) exerts a dual function as a...     

Towards non-peptide ANG II AT1 receptor antagonists based on urocanic acid: rational design,synthesis and biological evaluation

A series of o-, m- and p-benzyl tetrazole derivatives 11a–c has been designed, synthesized and evaluated as potential Angiotensin II AT1 receptor antagonists, based on urocanic acid. Compound 11b with tetrazole moiety at the m-position showed moderate, however, higher activity compared to the o- and p-counterpart analogues. Molecular modelling techniques were performed in order to extract their putative bioactive conformations and explore their binding modes.     

Structural-Functional Analysis of the Third Transmembrane Domain of the Corticotropin-releasing Factor Type 1 Receptor: ROLE IN ACTIVATION AND ALLOSTERIC ANTAGONISM*

The corticotropin-releasing factor (CRF) type 1 receptor (CRF₁R) for the 41-amino acid peptide CRF is a class B G protein-coupled receptor, which plays a key role in the response of our body to stressful stimuli and the maintenance of homeostasis by regulating neural and endocrine functions. CRF and related peptides, such as sauvagine, bind to the extracellular regions of CRF₁R and activate the receptor. In contrast, small nonpeptide antagonists, which are effective against stress-related disorders, such as depression and anxiety, have been proposed to interact with the helical transmembrane domains (TMs) of CRF₁R and allosterically antagonize peptide binding and receptor activation. Here, we aimed to elucidate the role of the third TM (TM3) in the molecular mechanisms underlying activation of CRF₁R. TM3 was selected because its tilted orientation, relative to the membrane, allows its residues to establish key interactions with ligands, other TM helices, and the G protein. Using a combination of pharmacological, biochemical, and computational approaches, we found that Phe-203^3.40 and Gly-210^3.47 in TM3 play an important role in receptor activation. Our experimental findings also suggest that Phe-203^3.40 interacts with nonpeptide antagonists.     

Cyclization of PLP139-151 peptide reduces its encephalitogenic potential in experimental autoimmune encephalomyelitis

We report the novel synthesis of cyclic PLP_139-151 (cPLP) and its application in SJL/J mice to study its encephalitogenic effects. Our results indicate that the cPLP analog is minimally encephalitogenic when administered to induce experimental autoimmune encephalomyelitis (low disease burden, minimal inflammatory, demyelinating and axonopathic pathology compared to its linear counterpart). Proliferation assays confirmed the low stimulatory potential of the cPLP compared to linPLP (2.5-fold lower proliferation) as well as inducing lower antibody responses. Molecular modeling showed a completely different TCR recognition profile of cPLP in regard to linPLP, where H¹⁴⁷ replaces W¹⁴⁴ and F¹⁵¹-K¹⁵⁰ replace H¹⁴⁷ as TCR contacts, which may explain the difference on each peptide’s response.     

Identification of compounds that bind the centriolar protein SAS-6 and inhibit its oligomerization

Centrioles are key eukaryotic organelles that are responsible for the formation of cilia and flagella, and for organizing the microtubule network and the mitotic spindle in animals. Centriole assembly requires oligomerization of the essential protein spindle assembly abnormal 6 (SAS-6), which forms a structural scaffold templating the organization of further organelle components. A dimerization interaction between SAS-6 N-terminal “head” domains was previously shown to be essential for protein oligomerization in vitro and for function in centriole assembly. Here, we developed a pharmacophore model allowing us to assemble a library of low-molecular-weight ligands predicted to bind the SAS-6 head domain and inhibit protein oligomerization. We demonstrate using NMR spectroscopy that a ligand from this family binds at the head domain dimerization site of algae, nematode, and human SAS-6 variants, but also that another ligand specifically recognizes human SAS-6. Atomistic molecular dynamics simulations starting from SAS-6 head domain crystallographic structures, including that of the human head domain which we now resolve, suggest that ligand specificity derives from favorable Van der Waals interactions with a hydrophobic cavity at the dimerization site.