Novel Zinc-Attenuating Compounds as Potent Broad-Spectrum Antifungal Agents with In Vitro and In Vivo Efficacy.

An increase in the incidence of rare but hard-to-treat invasive fungal pathogens as well as resistance to the currently available antifungal drugs calls for new broad-spectrum antifungals with a novel mechanism of action. Here we report the identification and characterization of two novel zinc-attenuating compounds, ZAC307 and ZAC989, which exhibit broad-spectrum in vitro antifungal activity and in vivo efficacy in a fungal kidney burden candidiasis model. The compounds were identified serendipitously as part of a drug discovery process aimed at finding novel inhibitors of the fungal plasma membrane proton ATPase Pma1. Based on their structure, we hypothesized that they might act as zinc chelators. Indeed, both fluorescence-based affinity determination and potentiometric assays revealed these compounds, subsequently termed zinc-attenuating compounds (ZACs), to have strong affinity for zinc, and their growth inhibitory effects on Candida albicans and Aspergillus fumigatus could be inactivated by the addition of exogenous zinc to fungal growth media. We determined the ZACs to be fungistatic, with a low propensity for resistance development. Gene expression analysis suggested that the ZACs interfere negatively with the expression of genes encoding the major components of the A. fumigatus zinc uptake system, thus supporting perturbance of zinc homeostasis as the likely mode of action. With demonstrated in vitro and in vivo antifungal activity, low propensity for resistance development, and a novel mode of action, the ZACs represent a promising new class of antifungal compounds, and their advancement in a drug development program is therefore warranted.

Chloride binding site of neurotransmitter sodium symporters.

Neurotransmitter:sodium symporters (NSSs) play a critical role in signaling by reuptake of neurotransmitters. Eukaryotic NSSs are chloride-dependent, whereas prokaryotic NSS homologs like LeuT are chloride-independent but contain an acidic residue (Glu290 in LeuT) at a site where eukaryotic NSSs have a serine. The LeuT-E290S mutant displays chloride-dependent activity. We show that, in LeuT-E290S cocrystallized with bromide or chloride, the anion is coordinated by side chain hydroxyls from Tyr47, Ser290, and Thr254 and the side chain amide of Gln250. The bound anion and the nearby sodium ion in the Na1 site organize a connection between their coordinating residues and the extracellular gate of LeuT through a continuous H-bond network. The specific insights from the structures, combined with results from substrate binding studies and molecular dynamics simulations, reveal an anion-dependent occlusion mechanism for NSS and shed light on the functional role of chloride binding.

The structural basis of calcium transport by the calcium pump.

The sarcoplasmic reticulum Ca2+-ATPase, a P-type ATPase, has a critical role in muscle function and metabolism. Here we present functional studies and three new crystal structures of the rabbit skeletal muscle Ca2+-ATPase, representing the phosphoenzyme intermediates associated with Ca2+ binding, Ca2+ translocation and dephosphorylation, that are based on complexes with a functional ATP analogue, beryllium fluoride and aluminium fluoride, respectively. The structures complete the cycle of nucleotide binding and cation transport of Ca2+-ATPase. Phosphorylation of the enzyme triggers the onset of a conformational change that leads to the opening of a luminal exit pathway defined by the transmembrane segments M1 through M6, which represent the canonical membrane domain of P-type pumps. Ca2+ release is promoted by translocation of the M4 helix, exposing Glu 309, Glu 771 and Asn 796 to the lumen. The mechanism explains how P-type ATPases are able to form the steep electrochemical gradients required for key functions in eukaryotic cells.

Binding of an octylglucoside detergent molecule in the second substrate (S2) site of LeuT establishes an inhibitor-bound conformation.

The first crystal structure of the neurotransmitter/sodium symporter homolog LeuT revealed an occluded binding pocket containing leucine and 2 Na(+); later structures showed tricyclic antidepressants (TCAs) in an extracellular vestibule approximately 11 A above the bound leucine and 2 Na(+). We recently found this region to be a second binding (S2) site and that binding of substrate to this site triggers Na(+)-coupled substrate symport. Here, we show a profound inhibitory effect of n-octyl-beta-d-glucopyranoside (OG), the detergent used for LeuT crystallization, on substrate binding to the S2 site. In parallel, we determined at 2.8 A the structure of LeuT-E290S, a mutant that, like LeuT-WT, binds 2 substrate molecules. This structure was similar to that of WT and clearly revealed an OG molecule in the S2 site. We also observed electron density at the S2 site in LeuT-WT crystals, and this also was accounted for by an OG molecule in that site. Computational analyses, based on the available crystal structures of LeuT, indicated the nature of structural arrangements in the extracellular region of LeuT that differentiate the actions of substrates from inhibitors bound in the S2 site. We conclude that the current LeuT crystal structures, all of which have been solved in OG, represent functionally blocked forms of the transporter, whereas a substrate bound in the S2 site will promote a different state that is essential for Na(+)-coupled symport.

The Importance of Lipoprotein Lipase Regulation in Atherosclerosis.

Lipoprotein lipase (LPL) plays a major role in the lipid homeostasis mainly by mediating the intravascular lipolysis of triglyceride rich lipoproteins. Impaired LPL activity leads to the accumulation of chylomicrons and very low-density lipoproteins (VLDL) in plasma, resulting in hypertriglyceridemia. While low-density lipoprotein cholesterol (LDL-C) is recognized as a primary risk factor for atherosclerosis, hypertriglyceridemia has been shown to be an independent risk factor for cardiovascular disease (CVD) and a residual risk factor in atherosclerosis development. In this review, we focus on the lipolysis machinery and discuss the potential role of triglycerides, remnant particles, and lipolysis mediators in the onset and progression of atherosclerotic cardiovascular disease (ASCVD). This review details a number of important factors involved in the maturation and transportation of LPL to the capillaries, where the triglycerides are hydrolyzed, generating remnant lipoproteins. Moreover, LPL and other factors involved in intravascular lipolysis are also reported to impact the clearance of remnant lipoproteins from plasma and promote lipoprotein retention in capillaries. Apolipoproteins (Apo) and angiopoietin-like proteins (ANGPTLs) play a crucial role in regulating LPL activity and recent insights into LPL regulation may elucidate new pharmacological means to address the challenge of hypertriglyceridemia in atherosclerosis development.

A first low-resolution difference Fourier map of phosphorus in a membrane protein from near-edge anomalous diffraction.

Crystal diffraction of three membrane proteins (cytochrome bc(1) complex, sarcoplasmic reticulum Ca(2+) ATPase, ADP-ATP carrier) and of one nucleoprotein complex (leucyl tRNA synthetase bound to tRNAleu, leuRS:tRNAleu) was tested at wavelengths near the X-ray K-absorption edge of phosphorus using a new set-up for soft X-ray diffraction at the beamline ID01 of the ESRF. The best result was obtained from crystals of Ca(2+) ATPase [adenosin-5'-(beta,gamma-methylene) triphosphate complex] which diffracted out to 7 A resolution. Data were recorded at a wavelength at which the real resonant scattering factor of phosphorus reaches the extreme value of -20 electron units. The positions of the four triphosphates of the monoclinic unit cell of the ATPase have been obtained from a difference Fourier synthesis based on a limited set of anomalous diffraction data.

Fungal plasma membrane H⁺-ATPase inhibitory activity of o-hydroxybenzylated flavanones and chalcones from Uvaria chamae P. Beauv.

In our ongoing efforts of finding natural fungicides to fight food and feed spoilage during production and storage, the antifungal potential of Ghanaian Uvaria chamae P. Beauv. was investigated, with emphasis on plant metabolites targeting the fungal plasma membrane (PM) H(+)-ATPase. Ethyl acetate extract of U. chamae was subjected to high-resolution fungal PM H(+)-ATPase inhibition screening followed by structural elucidation by high-performance liquid chromatography-high-resolution mass spectrometry-solid-phase extraction-nuclear magnetic resonance spectroscopy (HPLC-HRMS-SPE-NMR). This led to identification of a series of uncommon o-hydroxybenzylated flavanones and chalcones, i.e., chamanetin (8), isochamanetin (9), isouvaretin (10), uvaretin (11), dichamanetin (12), and diuvaretin (15). Preparative-scale isolation of the active metabolites allowed determination of IC50 values for inhibition of the PM H(+)-ATPase, and growth inhibition of Saccharomyces cerevisiae and Candida albicans. These revealed a strong correlation between o-hydroxybenzyl substituents and PM H(+)-ATPase activity, with dichamanetin being the most potent compound, but showing moderate activity in the fungal growth inhibition assays.

High-resolution screening combined with HPLC-HRMS-SPE-NMR for identification of fungal plasma membrane H(+)-ATPase inhibitors from plants.

Crude extracts of 33 plant species were assessed for fungal plasma membrane (PM) H(+)-ATPase inhibition. This led to identification of 18 extracts showing more than 95% inhibition at a concentration of 7.5 mg/mL and/or a concentration-dependent activity profile. These extracts were selected for semi-high-resolution fungal PM H(+)-ATPase inhibition screening, and, on the basis of these results, Haplocoelum foliolosum (Hiern) Bullock and Sauvagesia erecta L. were selected for investigation by high-resolution fungal PM H(+)-ATPase inhibition screening. Structural analysis performed by high-performance liquid chromatography-high-resolution mass spectrometry-solid-phase extraction-nuclear magnetic resonance spectroscopy (HPLC-HRMS-SPE-NMR) led to identification of chebulagic acid (1) and tellimagrandin II (2) from H. foliolosum. Preparative-scale isolation of the two metabolites allowed determination of IC50 values for PM H(+)-ATPase, and growth inhibition of Saccharomyces cerevisiae and Candida albicans. Chebulagic acid and tellimagrandin II are both potent inhibitors of the PM H(+)-ATPase with inhibitory effect on the growth of S. cerevisiae.

What can be learned about the function of a single protein from its various X-ray structures: the example of the sarcoplasmic calcium pump.

Improvements in the handling of membrane proteins for crystallization, combined with better synchrotron sources for X-ray diffraction analysis, are leading to clarification of the structural details of an ever increasing number of membrane transporters and receptors. Here we describe how this development has resulted in the elucidation at atomic resolution of a large number of structures of the sarcoplasmic Ca(2+)-ATPase (SERCA1a) present in skeletal muscle. The structures corresponding to the various intermediary states have been obtained after stabilization with structural analogues of ATP and of metal fluorides as mimicks of inorganic phosphate. From these results it is possible, in accordance with previous biochemical and molecular biology data, to give a detailed structural description of both ATP hydrolysis and Ca(2+) transport through the membrane, to serve as the starting point for a fuller understanding of the pump mechanism and, in future studies, on the regulatory role of this ubiquitous intracellular Ca(2+)-ATPase in cellular Ca(2+) metabolism in normal and pathological conditions.

Crystal structure of D351A and P312A mutant forms of the mammalian sarcoplasmic reticulum Ca(2+) -ATPase reveals key events in phosphorylation and Ca(2+) release.

In recent years crystal structures of the sarcoplasmic reticulum Ca(2+)-ATPase (SERCA1a), stabilized in various conformations with nucleotide and phosphate analogs, have been obtained. However, structural analysis of mutant forms would also be valuable to address key mechanistic aspects. We have worked out a procedure for affinity purification of SERCA1a heterologously expressed in yeast cells, producing sufficient amounts for crystallization and biophysical studies. We present here the crystal structures of two mutant forms, D351A and P312A, to address the issue whether the profound functional changes seen for these mutants are caused by major structural changes. We find that the structure of P312A with ADP and AlF(4)(-) bound (3.5-A resolution) and D351A with AMPPCP or ATP bound (3.4- and 3.7-A resolution, respectively) deviate only slightly from the complexes formed with that of wild-type ATPase. ATP affinity of the D351A mutant was very high, whereas the affinity for cytosolic Ca(2+) was similar to that of the wild type. We conclude from an analysis of data that the extraordinary affinity of the D351A mutant for ATP is caused by the electrostatic effects of charge removal and not by a conformational change. P312A exhibits a profound slowing of the Ca(2+)-translocating Ca(2)E1P-->E2P transition, which seems to be due to a stabilization of Ca(2)E1P rather than a destabilization of E2P. This can be accounted for by the strain that the Pro residue induces in the straight M4 helix of the wild type, which is removed upon the replacement of Pro(312) with alanine in P312A.