Cryo-electron microscopy and X-ray crystallography: complementary approaches to structural biology and drug discovery.

The invention of the electron microscope has greatly enhanced the view scientists have of small structural details. Since its implementation, this technology has undergone considerable evolution and the resolution that can be obtained for biological objects has been extended. In addition, the latest generation of cryo-electron microscopes equipped with direct electron detectors and software for the automated collection of images, in combination with the use of advanced image-analysis methods, has dramatically improved the performance of this technique in terms of resolution. While calculating a sub-10 Šresolution structure was an accomplishment less than a decade ago, it is now common to generate structures at sub-5 Šresolution and even better. It is becoming possible to relatively quickly obtain high-resolution structures of biological molecules, in particular large ones (>500 kDa) which, in some cases, have resisted more conventional methods such as X-ray crystallography or nuclear magnetic resonance (NMR). Such newly resolved structures may, for the first time, shed light on the precise mechanisms that are essential for cellular physiological processes. The ability to attain atomic resolution may support the development of new drugs that target these proteins, allowing medicinal chemists to understand the intimacy of the relationship between their molecules and targets. In addition, recent developments in cryo-electron microscopy combined with image analysis can provide unique information on the conformational variability of macromolecular complexes. Conformational flexibility of macromolecular complexes can be investigated using cryo-electron microscopy and multiconformation reconstruction methods. However, the biochemical quality of the sample remains the major bottleneck to routine cryo-electron microscopy-based determination of structures at very high resolution.

Effect of oxime ether incorporation in acyl indole derivatives on PPAR subtype selectivity.

Compounds that simultaneously activate peroxisome proliferator-activated receptor (PPAR) subtypes α and γ have the potential to effectively treat dyslipidemia and type 2 diabetes (T2D) in a single pharmaceutically active molecule. The frequently observed side effects of selective PPARγ agonists, such as edema and weight gain, were expected to be overcome by using additive PPARα activity, leading to dual PPARα/γ agonists with balanced activity for both subtypes. Herein we report the discovery, synthesis, and optimization of a new series of α-ethoxyphenylpropionic acid bearing 5- or 6-substituted indoles. The incorporation of oxime ethers on the carbonyl portion of the benzoyl group can bring the PPARα/γ potency ratio equal to or slightly greater than one, as is the case for compounds 20 c and 21 a. Compound 20 c shows high efficacy in an ob/ob mouse model of T2D and dyslipidemia, similar to that of rosiglitazone and tesaglitazar, but with a significant increase in body weight gain. In contrast, compound 21 a, less potent as a dual PPARα/γ activator than 20 c, showed an interesting pharmacological profile, as it elicits a decrease in body weight relative to reference compounds.

Design and synthesis of naphthalenic derivatives as new ligands at the melatonin binding site MT3.

Naphthalenic analogs of MCA-NAT (5-methoxycarbonylamino-N-acetyltryptamine) have been synthesized and evaluated as melatonin receptor ligands. Introduction of a methoxycarbonylamino substituent at the C-7 position of the naphthalenic nucleus yields MT3 selective ligands. This selectivity can be modulated with suitable variations of the C-7 position and the acyl group on the C-1 side chain. We identified new series of compounds with affinity for the MT3 binding site in the nanomolar range, and singled out a selective ligand, (N-[2-(7-methylsulfamoyl-naphth-1-yl)ethyl]acetamide (17), with a Ki of 4.9 nM and selectivity of 1024 and 2040 versus MT1 and MT2 receptors respectively.

Preparation and pharmacological evaluation of a novel series of 2-(phenylthio)benzo[b]thiophenes as selective MT2 receptor ligands.

A series of N-(2-(5-fluoro-2-(4-fluorophenylthio)benzo[b]thiophen-3-yl)ethyl)acylamides was synthesized and evaluated for binding affinity and intrinsic activity at melatonin receptors. The affinity of each compound for the melatonin receptors was determined by binding studies on cloned human MT1 and MT2 receptors expressed in CHO cells. Agonist and antagonist potency was measured on the [35S]GTPγS binding assay for the most interesting compounds. The new derivatives 8-14 showed modest to high selectivity (between 4 and 220) for MT2 receptors. The most selective compound, N-(2-(5-fluoro-2-(4-fluorophenylthio)benzo[b]thiophen-3-yl)ethyl)but-3-enamide (14), an MT2 ligand with affinity for the MT2 receptor similar to that of melatonin and a 220-fold preference over MT1 receptors, acts as a partial agonist. In addition, N-(2-(5-fluoro-2-(4-fluorophenylthio)benzo[b]thiophen-3-yl)ethyl)propionamide (9), a nanomolar MT2 ligand with a good selectivity ratio (MT1/MT2=51) shows antagonist activity on both melatonin receptors.

4,4-Dimethyl-1,2,3,4-tetrahydroquinoline-based PPARalpha/gamma agonists. Part. II: Synthesis and pharmacological evaluation of oxime and acidic head group structural variations.

Type-2 diabetes (T2D) is a complex metabolic disease characterized by insulin resistance in the liver and peripheral tissues accompanied by a deficiency in pancreatic beta-cells. Since their discovery, three subtypes of peroxisome proliferator activated receptors have been identified, namely PPARalpha, PPARgamma and PPARbeta/(delta). In this study, we were interested in designing novel PPARgamma selective agonists and/or dual PPARalpha/gamma agonists. Based on the typical topology of synthetic PPAR agonists, we focused our design approach on using 4,4-dimethyl-1,2,3,4-tetrahydroquinoline as a novel cyclic scaffold with oxime and acidic head group structural variations.

4,4-Dimethyl-1,2,3,4-tetrahydroquinoline-based PPARalpha/gamma agonists. Part I: synthesis and pharmacological evaluation.

Type-2 diabetes (T2D) is a complex metabolic disease characterized by insulin resistance in the liver and peripheral tissues accompanied by a defect in pancreatic beta-cell. Since their discovery three subtypes of Peroxisomes Proliferators Activated Receptors were identified namely PPARalpha, PPARgamma and PPARbeta/(delta). We were interested in designing novel PPARgamma selective agonists and/or dual PPARalpha/gamma agonists. Based on the typical topology of synthetic PPAR agonists, we focused our design approach on 4,4-dimethyl-1,2,3,4-tetrahydroquinoline as novel cyclic tail.

S18986: a positive modulator of AMPA-receptors enhances (S)-AMPA-mediated BDNF mRNA and protein expression in rat primary cortical neuronal cultures.

The present study describes the effect of (S)-2,3-dihydro-[3,4]cyclopentano-1,2,4-benzothiadiazine-1,1-dioxide (S18986), a positive allosteric modulator of the alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptors, on (S)-AMPA-mediated increases in brain-derived neurotrophic factor (BDNF) mRNA and protein expression in rat primary cortical neuronal cultures. (S)-AMPA (0.01-300 microM) induced a concentration-dependent increase in BDNF mRNA and protein expression (EC(50)=7 microM) with maximal increases (50-fold) compared to untreated cultures observed between 5 and 12 h, whereas for cellular protein levels, maximal expression was detected at 24 h. S18986 alone (< or =300 microM) failed to increase basal BDNF expression. However, S18986 (300 microM) in the presence of increasing concentrations of (S)-AMPA maximally enhanced AMPA-induced expression of BDNF mRNA and protein levels (3-5-fold). S18986 (100-300 microM) potentiated BDNF mRNA induced by 3 microM (S)-AMPA (2-3-fold). Under similar conditions, the AMPA allosteric modulator cyclothiazide induced a potent stimulation of (S)-AMPA-mediated BDNF expression (40-fold; EC(50)=18 microM), whereas IDRA-21 was inactive. Kinetic studies indicated that S18986 (300 microM) in the presence of 3 microM (S)-AMPA was capable of enhancing BDNF mRNA levels for up to 25 h, compared to 3 microM (S)-AMPA alone. On the other hand, S18986 only partially enhanced kainate-mediated expression of BDNF mRNA, but failed to significantly enhance N-methyl-D-aspartate-stimulated BDNF expression levels. In support of these observations, the competitive AMPA receptor antagonist NBQX (1,2,3,4-tetrahydro-6-nitro-2,3-dioxo-benzo[f]quinoxaline-7-sulfonamide) but not the selective NMDA-receptor antagonist, (+)-MK-801 [(5R,10S)-(+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine], abrogated S18986-induced effects on BDNF expression. S18986-mediated enhancement of (S)-AMPA-evoked BDNF protein expression was markedly attenuated in Ca(2+)-free culture conditions. Furthermore, from a series of kinase inhibitors only the Calmodulin-Kinase II/IV inhibitor (KN-62, 25 microM) significantly inhibited (-85%, P<0.001) AMPA+S18986 stimulated expression of BDNF mRNA. The present study supports the observations that AMPA receptor allosteric modulators can enhance the expression of BDNF mRNA and protein expression via the AMPA receptor in cultured primary neurones. Consequently, the long-term elevation of endogenous BDNF expression by pharmacological intervention with this class of compounds represents a potentially promising therapeutic approach for behavioural disorders implicating cognitive deficits.

A convenient 3-step synthesis of 3-acetamido-6-arylpyridazines directed to novel Y5 receptor antagonist.

A 3-step synthesis of 3-acetamido-6-arylpyridazines as potential NPY5 antagonists.