Targeting phenylalanine assemblies as a prospective disease-modifying therapy for phenylketonuria.

Phenylketonuria is characterized by the accumulation of phenylalanine, resulting in severe cognitive and neurological disorders if not treated by a remarkably strict diet. There are two approved drugs today, yet both provide only a partial solution. We have previously demonstrated the formation of amyloid-like toxic assemblies by aggregation of phenylalanine, suggesting a new therapeutic target to be further pursued. Moreover, we showed that compounds that halt the formation of these assemblies also prevent their resulting toxicity. Here, we performed high-throughput screening, searching for compounds with inhibitory effects on phenylalanine aggregation. Morin hydrate, one of the most promising hits revealed during the screen, was chosen to be tested in vivo using a phenylketonuria mouse model. Morin hydrate significantly improved cognitive and motor function with a reduction in the number of phenylalanine brain deposits. Moreover, while phenylalanine levels remained high, we observed a recovery in dopaminergic, adrenergic, and neuronal markers. To conclude, the ability of Morin hydrate to halt phenylalanine aggregation without reducing phenylalanine levels implies the toxic role of the phenylalanine assemblies in phenylketonuria and opens new avenues for disease-modifying treatment.

Exploring the Li+ transporting mutant of NCX_Mj for assigning ion binding sites of mitochondrial NCLX.

The plasma membrane (NCX) and mitochondrial (NCLX) Na+/Ca2+ exchangers are structurally related proteins, although they operate under strictly different ionic conditions and membrane potentials. In contrast with NCX, NCLX can transport either Li+ or Na+ in exchange for Ca2+. Whereas the crystal structure of the archaeal NCX (NCX_Mj) describes the binding sites for alternative binding of 3Na+ or 1Ca2+, these features remain elusive for NCLX due to the lack of structural information. To elucidate the ion-binding features of mitochondrial NCLX, we analyzed here the Li+-transporting NCLX_Mj mutant, produced by replacing the ion-coordinating residues in the archaeal NCX (NCX_Mj) to match the ion-coordinating residues of human NCLX. The NCLX_Mj-mediated Na+/Ca2+ or Li+/Ca2+ exchange rates are insensitive to varying voltage, consistent with an electroneutral ion exchange. Molecular dynamics (MD) simulations revealed that NCLX_Mj contains two novel Li+ binding sites with four ion-coordinating residues, derived from the three Na+ binding sites of NCX_Mj. The ion-coordination modes, observed in the MD simulations, were further supported by two-dimensional infrared (2D IR) spectroscopy and by testing the mutational effects on the ion fluxes. Collectively, our results revealed a structural basis for Li+ binding and electroneutral transport (2Na+/Li+:1Ca2+) by NCLX_Mj, meaning that the NCLX-mediated electroneutral transport may predefine mitochondrial Ca2+ and Na+ signaling to modulate cellular functions.

Allosteric inhibitors targeting the calmodulin-PIP2 interface of SK4 K+ channels for atrial fibrillation treatment.

The Ca2+-activated SK4 K+ channel is gated by Ca2+-calmodulin (CaM) and is expressed in immune cells, brain, and heart. A cryoelectron microscopy (cryo-EM) structure of the human SK4 K+ channel recently revealed four CaM molecules per channel tetramer, where the apo CaM C-lobe and the holo CaM N-lobe interact with the proximal carboxyl terminus and the linker S4-S5, respectively, to gate the channel. Here, we show that phosphatidylinositol 4-5 bisphosphate (PIP2) potently activates SK4 channels by docking to the boundary of the CaM-binding domain. An allosteric blocker, BA6b9, was designed to act to the CaM-PIP2-binding domain, a previously untargeted region of SK4 channels, at the interface of the proximal carboxyl terminus and the linker S4-S5. Site-directed mutagenesis, molecular docking, and patch-clamp electrophysiology indicate that BA6b9 inhibits SK4 channels by interacting with two specific residues, Arg191 and His192 in the linker S4-S5, not conserved in SK1-SK3 subunits, thereby conferring selectivity and preventing the Ca2+-CaM N-lobe from properly interacting with the channel linker region. Immunohistochemistry of the SK4 channel protein in rat hearts showed a widespread expression in the sarcolemma of atrial myocytes, with a sarcomeric striated Z-band pattern, and a weaker occurrence in the ventricle but a marked incidence at the intercalated discs. BA6b9 significantly prolonged atrial and atrioventricular effective refractory periods in rat isolated hearts and reduced atrial fibrillation induction ex vivo. Our work suggests that inhibition of SK4 K+ channels by targeting drugs to the CaM-PIP2-binding domain provides a promising anti-arrhythmic therapy.

The contribution of individual residues of an aggregative hexapeptide derived from the human γD-crystallin to its amyloidogenicity.

Human γD-crystallin protein is abundant in the lens and is essential for preserving lens transparency. With age the protein may lose its native structure resulting in the formation of cataract. We recently reported an aggregative peptide, 41Gly-Cys-Trp-Met-Leu-Tyr46 from the human γD-crystallin, termed GDC6, exhibiting amyloidogenic properties in vitro. Here, we aimed to determine the contribution of each residue of the GDC6 to its amyloidogenicity. Molecular dynamic (MD) simulations revealed that the residues Trp, Leu, and Tyr played an important role in the amyloidogenicity of GDC6 by facilitating inter-peptide main-chain hydrogen bonds, and π-π interactions. MD predictions were further validated using single-, double- and triple-alanine-substituted GDC6 peptides in which their amyloidogenic propensity was individually evaluated using complementary biophysical techniques including Thioflavin T assay, turbidity assay, CD spectroscopy, and TEM imaging. Results revealed that the substitution of Trp, Leu, and Tyr together by Ala completely abolished aggregation of GDC6 in vitro, highlighting their importance in the amyloidogenicity of GDC6.

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.

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 (non-native) 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.

A unique mechanism of inactivation gating of the Kv channel family member Kv7.1 and its modulation by PIP2 and calmodulin.

Inactivation of voltage-gated K+ (Kv) channels mostly occurs by fast N-type or/and slow C-type mechanisms. Here, we characterized a unique mechanism of inactivation gating comprising two inactivation states in a member of the Kv channel superfamily, Kv7.1. Removal of external Ca2+ in wild-type Kv7.1 channels produced a large, voltage-dependent inactivation, which differed from N- or C-type mechanisms. Glu295 and Asp317 located, respectively, in the turret and pore entrance are involved in Ca2+ coordination, allowing Asp317 to form H-bonding with the pore helix Trp304, which stabilizes the selectivity filter and prevents inactivation. Phosphatidylinositol 4,5-bisphosphate (PIP2) and Ca2+-calmodulin prevented Kv7.1 inactivation triggered by Ca2+-free external solutions, where Ser182 at the S2-S3 linker relays the calmodulin signal from its inner boundary to the external pore to allow proper channel conduction. Thus, we revealed a unique mechanism of inactivation gating in Kv7.1, exquisitely controlled by external Ca2+ and allosterically coupled by internal PIP2 and Ca2+-calmodulin.

TOR Complex 2- independent mutations in the regulatory PIF pocket of Gad8AKT1/SGK1 define separate branches of the stress response mechanisms in fission yeast.

The Target of rapamycin (TOR) protein kinase forms part of TOR complex 1 (TORC1) and TOR complex 2 (TORC2), two multi-subunit protein complexes that regulate growth, proliferation, survival and developmental processes by phosphorylation and activation of AGC-family kinases. In the fission yeast, Schizosaccharomyces pombe, TORC2 and its target, the AGC kinase Gad8 (an orthologue of human AKT or SGK1) are required for viability under stress conditions and for developmental processes in response to starvation cues. In this study, we describe the isolation of gad8 mutant alleles that bypass the requirement for TORC2 and reveal a separation of function of TORC2 and Gad8 under stress conditions. In particular, osmotic and nutritional stress responses appear to form a separate branch from genotoxic stress responses downstream of TORC2-Gad8. Interestingly, TORC2-independent mutations map into the regulatory PIF pocket of Gad8, a highly conserved motif in AGC kinases that regulates substrate binding in PDK1 (phosphoinositide dependent kinase-1) and kinase activity in several AGC kinases. Gad8 activation is thought to require a two-step mechanism, in which phosphorylation by TORC2 allows further phosphorylation and activation by Ksg1 (an orthologue of PDK1). We focus on the Gad8-K263C mutation and demonstrate that it renders the Gad8 kinase activity independent of TORC2 in vitro and independent of the phosphorylation sites of TORC2 in vivo. Molecular dynamics simulations of Gad8-K263C revealed abnormal high flexibility at T387, the phosphorylation site for Ksg1, suggesting a mechanism for the TORC2-independent Gad8 activity. Significantly, the K263 residue is highly conserved in the family of AGC-kinases, which may suggest a general way of keeping their activity in check when acting downstream of TOR complexes.

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

The human cis-prenyltransferase (hcis-PT) is an enzymatic complex essential for protein N-glycosylation. Synthesizing the precursor of the glycosyl carrier dolichol-phosphate, mutations in hcis-PT cause severe human diseases. Here, we reveal that hcis-PT exhibits a heterotetrameric assembly in solution, consisting of two catalytic dehydrodolichyl diphosphate synthase (DHDDS) and inactive Nogo-B receptor (NgBR) heterodimers. Importantly, the 2.3 Å crystal structure reveals that the tetramer assembles via the DHDDS C-termini as a dimer-of-heterodimers. Moreover, the distal C-terminus of NgBR transverses across the interface with DHDDS, directly participating in active-site formation and the functional coupling between the subunits. Finally, we explored the functional consequences of disease mutations clustered around the active-site, and in combination with molecular dynamics simulations, we propose a mechanism for hcis-PT dysfunction in retinitis pigmentosa. Together, our structure of the hcis-PT complex unveils the dolichol synthesis mechanism and its perturbation in disease.

A novel specific PERK activator reduces toxicity and extends survival in Huntington's disease models.

One of the pathways of the unfolded protein response, initiated by PKR-like endoplasmic reticulum kinase (PERK), is key to neuronal homeostasis in neurodegenerative diseases. PERK pathway activation is usually accomplished by inhibiting eIF2α-P dephosphorylation, after its phosphorylation by PERK. Less tried is an approach involving direct PERK activation without compromising long-term recovery of eIF2α function by dephosphorylation. Here we show major improvement in cellular (STHdhQ111/111) and mouse (R6/2) Huntington's disease (HD) models using a potent small molecule PERK activator that we developed, MK-28. MK-28 showed PERK selectivity in vitro on a 391-kinase panel and rescued cells (but not PERK-/- cells) from ER stress-induced apoptosis. Cells were also rescued by the commercial PERK activator CCT020312 but MK-28 was significantly more potent. Computational docking suggested MK-28 interaction with the PERK activation loop. MK-28 exhibited remarkable pharmacokinetic properties and high BBB penetration in mice. Transient subcutaneous delivery of MK-28 significantly improved motor and executive functions and delayed death onset in R6/2 mice, showing no toxicity. Therefore, PERK activation can treat a most aggressive HD model, suggesting a possible approach for HD therapy and worth exploring for other neurodegenerative disorders.

Chemical Modifications Reduce Auditory Cell Damage Induced by Aminoglycoside Antibiotics.

Although aminoglycoside antibiotics are effective against Gram-negative infections, these drugs often cause irreversible hearing damage. Binding to the decoding site of the eukaryotic ribosomes appears to result in ototoxicity, but there is evidence that other effects are involved. Here, we show how chemical modifications of apramycin and geneticin, considered among the least and most toxic aminoglycosides, respectively, reduce auditory cell damage. Using molecular dynamics simulations, we studied how modified aminoglycosides influence the essential freedom of movement of the decoding site of the ribosome, the region targeted by aminoglycosides. By determining the ratio of a protein translated in mitochondria to that of a protein translated in the cytoplasm, we showed that aminoglycosides can paradoxically elevate rather than reduce protein levels. We showed that certain aminoglycosides induce rapid plasma membrane permeabilization and that this nonribosomal effect can also be reduced through chemical modifications. The results presented suggest a new paradigm for the development of safer aminoglycoside antibiotics.

An Ancient CFTR Ortholog Informs Molecular Evolution in ABC Transporters.

The cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride channel central to the development of secretory diarrhea and cystic fibrosis. The oldest CFTR ortholog identified is from dogfish shark, which retains similar structural and functional characteristics to the mammalian protein, thereby highlighting CFTR's critical role in regulating epithelial ion transport in vertebrates. However, the identification of an early CFTR ortholog with altered structure or function would provide critical insight into the evolution of epithelial anion transport. Here, we describe the earliest known CFTR, expressed in sea lamprey (Petromyzon marinus), with unique structural features, altered kinetics of activation and sensitivity to inhibition, and altered single-channel conductance compared to human CFTR. Our data provide the earliest evolutionary evidence of CFTR, offering insight regarding changes in gene and protein structure that underpin evolution from transporter to anion channel. Importantly, these data provide a unique platform to enhance our understanding of vertebrate phylogeny over a critical period of evolutionary expansion.

Highly Selective and Potent Ectonucleotide Pyrophosphatase-1 (NPP1) Inhibitors Based on Uridine 5'-Pα,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'-Pα,α-dithiophosphate analogues, 9-12. 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 P2Y2,4,6-receptors and thus represent highly selective NPP1 inhibitors. The most promising inhibitor was diuridine 5'-Pα,α,5″-Pα,α-tetrathiotetraphosphate, 12, exhibiting Ki of 27 nM. Analogues 9-12 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 12 could be attributed to the simultaneous occupancy of two sites (the AMP site and an alternative site) of NPP1 by this compound.

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 human 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.

2-Hexylthio-ß,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-ß,γ-methylene-ATP, 2. Analogue 2 was stable to hydrolysis by NTPDase1, -2, -3, and -8. It inhibited hNTPDase2 with Ki 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 r2=0.94 between calculated and experimental inhibition data for the triphosphate analogues considered in this work. The origin of selectivity of 2 for NTPDase2 over NTPDase1 is the thiohexyl moiety of 2 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 Ki,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 2 that had the lowest Ki,app (0.5 µM) was also inactive toward P2Y receptors. Overall, analogue 2 is the most potent and selective NPP1 inhibitor described so far.