Molecular Dynamics Flexible Fitting Simulations Identify New Models of the Closed State of the Cystic Fibrosis Transmembrane Conductance Regulator Protein

Cystic fibrosis (CF) is a lethal, genetic disease found in particular in humans of European origin which is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel. The search for CF therapies acting by modulating the impaired function of mutant CFTR will be greatly advanced by high resolution structures of CFTR in different states. To date, two medium resolution electron microscopy (EM) structures of CFTR are available (one of a distant zebrafish (Danio rerio) CFTR ortholog and one of human CFTR). The two models are nearly identical to one another, and both correspond to the inward-facing, nucleotide binding domains (NBDs) separated, closed state of the channel. In addition, lower resolution structural data are available for human CFTR in an alternative conformation which likely features associated NBDs and thus geometrically resembles the conducting state of the channel. Multiple homology models of human CFTR in multiple states have been developed over the years, yet their correspondence to the existing structural information is unexplored. In this work we use molecular dynamics flexible fitting (MDFF) simulations to refine two previously described CFTR models based on the available cryo-EM map of the <i>human</i> protein. This map was recorded in the absence of ATP and consequently represents closed-state CFTR yet its features likely correspond to an NBD associated conformation of the protein. Accordingly, the resulting models feature dimerized NBDs yet with no membrane traversing pore. Moreover, the open probability of the new models as deduced from the MDFF trajectories is significantly lower than that deduced from control MD trajectories initiated from the starting models. We propose that the new models correspond to a CFTR conformation which to date was largely unexplored yet is one that is relevant to the gating cycle of the protein. In particular this conformation may participate in rapid channel opening and closing through small allosteric movements controlled by nucleotide binding and dissociation events. Analyzing the resulting trajectories (and not only the final models as is usually the case), we demonstrate that the refined models have good stereochemical properties and are also in favorable agreement with multiple experimental data. Moreover, despite different starting points, the final models share many common features. Finally, we propose that the combination of high resolution cryo-EM maps, which are currently emerging from multiple laboratories, and MDFF simulations will be of value for the development of yet more reliable CFTR models as well as for the identification of binding sites for CFTR modulators

Structural basis of heterotetrameric assembly and disease mutations in the human cis-prenyltransferase complex

The human cis-prenyltransferase (hcis-PT) complex synthesizes the precursor of the glycosyl carrier dolichol-phosphate and as such it is essential for protein N-glycosylation. The crystal structure of the complex reveals unusual tetrameric architecture and provides insights into dolichol synthesis mechanism and functional consequences of disease-associated hcis-PT mutations

Chemical Chaperones Modulate the Formation of Metabolite Assemblies

The formation of amyloid-like structures by metabolites is associated with several inborn errors of metabolism (IEMs). These structures display most of the biological, chemical and physical properties of protein amyloids. However, the molecular interactions underlying the assembly remain elusive, and so far, no modulating therapeutic agents are available for clinical use. Chemical chaperones are known to inhibit protein and peptide amyloid formation and stabilize misfolded enzymes. Here, we provide an in-depth characterization of the inhibitory effect of osmolytes and hydrophobic chemical chaperones on metabolite assemblies, thus extending their functional repertoire. We applied a combined in vivo-in vitro-in silico approach and show their ability to inhibit metabolite amyloid-induced toxicity and reduce cellular amyloid content in yeast. We further used various biophysical techniques demonstrating direct inhibition of adenine self-assembly and alteration of fibril morphology by chemical chaperones. Using a scaffold-based approach, we analyzed the physiochemical properties of various dimethyl sulfoxide derivatives and their role in inhibiting metabolite self-assembly. Lastly, we employed whole-atom molecular dynamics simulations to elucidate the role of hydrogen bonds in osmolyte inhibition. Our results imply a dual mode of action of chemical chaperones as IEMs therapeutics, that could be implemented in the rational design of novel lead-like molecules

Highly Selective and Potent Ectonucleotide Pyrophosphatase‑1 (NPP1) Inhibitors Based on Uridine 5′‑P_α,α-Dithiophosphate Analogues

Ectonucleotide pyrophosphatase/phospho­diesterase-1 (NPP1) hydrolyzes phosphodiester bonds of nucleotides such as ATP, resulting mainly in the formation of AMP and pyrophosphate. NPP1 activity plays a deleterious function in calcified aortic valve disease and calcium pyrophosphate deposition disease. Thus, inhibitors of NPP1 represent a medical need. We developed novel NPP1 inhibitors based on uridine 5′-P_α,α-dithiophosphate analogues, <b>9</b>–<b>12</b>. All these analogues potently inhibited hNPP1 (80–100% inhibition) at 100 μM, with no, or minimal, inhibition of NPP3 and other ectonucleotidases (NTPDase1,2,3,8). These compounds showed nearly no activity at uracil-nucleotide sensitive P2Y_2,4,6-receptors and thus represent highly selective NPP1 inhibitors. The most promising inhibitor was diuridine 5′-P_α,α,5″-P_α,α-tetrathio­tetraphosphate, <b>12</b>, exhibiting <i>K</i>_i of 27 nM. Analogues <b>9</b>–<b>12</b> proved to be highly stable to air oxidation and to acidic and basic pH. Docking simulations suggested that the enhanced NPP1 inhibitory activity and selectivity of analogue <b>12</b> could be attributed to the simultaneous occupancy of two sites (the AMP site and an alternative site) of NPP1 by this compound

Highly Selective and Potent Ectonucleotide Pyrophosphatase‑1 (NPP1) Inhibitors Based on Uridine 5′‑P_α,α-Dithiophosphate Analogues

Ectonucleotide pyrophosphatase/phospho­diesterase-1 (NPP1) hydrolyzes phosphodiester bonds of nucleotides such as ATP, resulting mainly in the formation of AMP and pyrophosphate. NPP1 activity plays a deleterious function in calcified aortic valve disease and calcium pyrophosphate deposition disease. Thus, inhibitors of NPP1 represent a medical need. We developed novel NPP1 inhibitors based on uridine 5′-P_α,α-dithiophosphate analogues, <b>9</b>–<b>12</b>. All these analogues potently inhibited hNPP1 (80–100% inhibition) at 100 μM, with no, or minimal, inhibition of NPP3 and other ectonucleotidases (NTPDase1,2,3,8). These compounds showed nearly no activity at uracil-nucleotide sensitive P2Y_2,4,6-receptors and thus represent highly selective NPP1 inhibitors. The most promising inhibitor was diuridine 5′-P_α,α,5″-P_α,α-tetrathio­tetraphosphate, <b>12</b>, exhibiting <i>K</i>_i of 27 nM. Analogues <b>9</b>–<b>12</b> proved to be highly stable to air oxidation and to acidic and basic pH. Docking simulations suggested that the enhanced NPP1 inhibitory activity and selectivity of analogue <b>12</b> could be attributed to the simultaneous occupancy of two sites (the AMP site and an alternative site) of NPP1 by this compound

The CFTR P67L variant reveals a key role for N-terminal lasso helices in channel folding, maturation, and pharmacologic rescue

Patients with cystic fibrosis (CF) harboring the P67L variant in the cystic fibrosis transmembrane conductance regulator (CFTR) often exhibit a typical CF phenotype, including severe respiratory compromise. This rare mutation (reported in <300 patients worldwide) responds robustly to CFTR correctors, such as lumacaftor and tezacaftor, with rescue in model systems that far exceed what can be achieved for the archetypical CFTR mutant F508del. However, the specific molecular consequences of the P67L mutation are poorly characterized. In this study, we conducted biochemical measurements following low-temperature growth and/or intragenic suppression, which suggest a mechanism underlying P67L that (1) shares key pathogenic features with F508del, including off-pathway (nonnative) folding intermediates, (2) is linked to folding stability of nucleotide-binding domains 1 and 2, and (3) demonstrates pharmacologic rescue that requires domains in the carboxyl half of the protein. We also investigated the “lasso” helices 1 and 2, which occur immediately upstream of P67. Based on limited proteolysis, pulse chase, and molecular dynamics analysis of full-length CFTR and a series of deletion constructs, we argue that P67L and other maturational processing (class 2) defects impair the integrity of the lasso motif and confer misfolding of downstream domains. Thus, amino-terminal missense variants elicit a conformational change throughout CFTR that abrogates maturation while providing a robust substrate for pharmacologic repair

The CFTR P67L variant reveals a key role for N-terminal lasso helices in channel folding, maturation, and pharmacologic rescue

Patients with cystic fibrosis (CF) harboring the P67L variant in the cystic fibrosis transmembrane conductance regulator (CFTR) often exhibit a typical CF phenotype, including severe respiratory compromise. This rare mutation (reported in <300 patients worldwide) responds robustly to CFTR correctors, such as lumacaftor and tezacaftor, with rescue in model systems that far exceed what can be achieved for the archetypical CFTR mutant F508del. However, the specific molecular consequences of the P67L mutation are poorly characterized. In this study, we conducted biochemical measurements following low-temperature growth and/or intragenic suppression, which suggest a mechanism underlying P67L that (1) shares key pathogenic features with F508del, including off-pathway (nonnative) folding intermediates, (2) is linked to folding stability of nucleotide-binding domains 1 and 2, and (3) demonstrates pharmacologic rescue that requires domains in the carboxyl half of the protein. We also investigated the “lasso” helices 1 and 2, which occur immediately upstream of P67. Based on limited proteolysis, pulse chase, and molecular dynamics analysis of full-length CFTR and a series of deletion constructs, we argue that P67L and other maturational processing (class 2) defects impair the integrity of the lasso motif and confer misfolding of downstream domains. Thus, amino-terminal missense variants elicit a conformational change throughout CFTR that abrogates maturation while providing a robust substrate for pharmacologic repair

2‑Hexylthio-β,γ-CH₂‑ATP is an Effective and Selective NTPDase2 Inhibitor

NTPDase2 catabolizes nucleoside triphosphates and consequently, through the interaction of nucleotides with P2 receptors, controls multiple biological responses. NTPDase2 inhibitors could modulate responses induced by nucleotides in thrombosis, inflammation, cancer, etc. Here we developed a set of ATP analogues as potential NTPDase inhibitors and identified a subtype-selective and potent NTPDase2 inhibitor, 2-hexylthio-β,γ-methylene-ATP, <b>2</b>. Analogue <b>2</b> was stable to hydrolysis by NTPDase1, -2, -3, and -8. It inhibited hNTPDase2 with <i>K</i>_i 20 μM, while only marginally (5–15%) inhibiting NTPDase1, -3, and -8. Homology models of hNTPDase1 and -2 were constructed. Docking and subsequent linear interaction energy (LIE) simulations provided a correlation with <i>r</i>² = 0.94 between calculated and experimental inhibition data for the triphosphate analogues considered in this work. The origin of selectivity of <b>2</b> for NTPDase2 over NTPDase1 is the thiohexyl moiety of <b>2</b> which is favorably located within a hydrophobic pocket, whereas in NTPDase1 it is exposed to the solvent

Nonhydrolyzable ATP Analogues as Selective Inhibitors of Human NPP1: A Combined Computational/Experimental Study

Elevated nucleotide pyrophosphatase/phosphodiesterase-1 (NPP1) activity is implicated in health disorders including pathological calcification. Specific NPP1 inhibitors would therefore be valuable for studying this enzyme and as potential therapeutic agents. Here we present a combined computational/experimental study characterizing 13 nonhydrolyzable ATP analogues as selective human NPP1 inhibitors. All analogues at 100 μM inhibited (66–99%) the hydrolysis of pnp-TMP by both recombinant NPP1 and cell surface NPP1 activity of osteocarcinoma (HTB-85) cells. These analogues only slightly altered the activity of other ectonucleotidases, NPP3 and NTPDases. The <i>K</i>_i,app values of the seven most potent and selective inhibitors were in the range of 0.5–56 μM, all with mixed type inhibition, predominantly competitive. Those molecules were docked into a newly developed homology model of human NPP1. All adopted ATP-like binding modes, suggesting competitive inhibition with the endogenous ligand. NPP1 selectivity versus NPP3 could be explained in terms of the electrostatic potential of the two proteins that of NPP1 favoring negatively charged ligands. Inhibitor <b>2</b> that had the lowest <i>K</i>_i,app (0.5 μM) was also inactive toward P2Y receptors. Overall, analogue <b>2</b> is the most potent and selective NPP1 inhibitor described so far