A B S T R A C TTwo-pore channels (TPCs) are Ca 2+ -permeable ion channels localised to the endo-lysosomal system where they regulate trafficking of various cargoes including viruses. As a result, TPCs are emerging as important drug targets. However, their pharmacology is ill-defined. There are no approved drugs to target them. And their mechanism of ligand activation is largely unknown. Here, we identify a number of FDA-approved drugs as TPC pore blockers. Using a model of the pore of human TPC2 based on recent structures of mammalian TPCs, we virtually screened a database of~1500 approved drugs. Because TPCs have recently emerged as novel host factors for Ebola virus entry, we reasoned that Ebola virus entry inhibitors may exert their effects through inhibition of TPCs. Cross-referencing hits from the TPC virtual screen with two recent high throughput anti-Ebola screens yielded approved drugs targeting dopamine and estrogen receptors as common hits. These compounds inhibited endogenous NAADP-evoked Ca 2+ release from sea urchin egg homogenates, NAADP-mediated channel activity of TPC2 re-routed to the plasma membrane, and PI(3,5)P 2 -mediated channel activity of TPC2 expressed in enlarged lysosomes. Mechanistically, single channel analyses showed that the drugs reduced mean open time consistent with a direct action on the pore. Functionally, drug potency in blocking TPC2 activity correlated with inhibition of Ebola virus-like particle entry. Our results expand TPC pharmacology through the identification of approved drugs as novel blockers, support a role for TPCs in Ebola virus entry, and provide insight into the mechanisms underlying channel regulation.

No abstract

Lipoteichoic acid synthase (LtaS) is a key enzyme for the cell wall biosynthesis of Gram-positive bacteria. Gram-positive bacteria that lack lipoteichoic acid (LTA) exhibit impaired cell division and growth defects. Thus, LtaS appears to be an attractive antimicrobial target. The pharmacology around LtaS remains largely unexplored with only two small-molecule LtaS inhibitors reported, namely “compound 1771 ” and the Congo red dye. Structure-based drug discovery efforts against LtaS remain unattempted due to the lack of an inhibitor-bound structure of LtaS. To address this, we combined the use of a molecular docking technique with molecular dynamics (MD) simulations to model a plausible binding mode of compound 1771 to the extracellular catalytic domain of LtaS (eLtaS). The model was validated using alanine mutagenesis studies combined with isothermal titration calorimetry. Additionally, lead optimization driven by our computational model resulted in an improved version of compound 1771 , namely, compound 4 which showed greater affinity for binding to eLtaS than compound 1771 in biophysical assays. Compound 4 reduced LTA production in S. aureus dose-dependently, induced aberrant morphology as seen for LTA-deficient bacteria, and significantly reduced bacteria titers in the lung of mice infected with S. aureus . Analysis of our MD simulation trajectories revealed the possible formation of a transient cryptic pocket in eLtaS. Virtual screening (VS) against the cryptic pocket led to the identification of a new class of inhibitors that could potentiate β-lactams against methicillin-resistant S. aureus . Our overall workflow and data should encourage further drug design campaign against LtaS. Finally, our work reinforces the importance of considering protein conformational flexibility to a successful VS endeavor.

Pseudomonas aeruginosa is an opportunistic human pathogen recognized as a critical threat by the World Health Organization because of the dwindling number of effective therapies available to treat infections. Over the past decade, it has become apparent that the glyoxylate shunt plays a vital role in sustaining P. aeruginosa during infection scenarios. The glyoxylate shunt comprises two enzymes: isocitrate lyase and malate synthase isoform G. Inactivation of these enzymes has been reported to abolish the ability of P. aeruginosa to establish infection in a mammalian model system, yet we still lack the structural information to support drug design efforts. In this work, we describe the first X-ray crystal structure of P. aeruginosa malate synthase G in the apo form at 1.62 Å resolution. The enzyme is a monomer composed of four domains and is highly conserved with homologues found in other clinically relevant microorganisms. It is also dependent on Mg 2+ for catalysis. Metal ion binding led to a change in the intrinsic fluorescence of the protein, allowing us to quantitate its affinity for Mg 2+ . We also identified putative drug binding sites in malate synthase G using computational analysis and, because of the high resolution of the experimental data, were further able to characterize its hydration properties. Our data reveal two promising binding pockets in malate synthase G that may be exploited for drug design.

44-12233-34068. Inositol 1, 4, 5-trisphosphate (IP3) binding at the N-terminus (NT) of IP3 receptor (IP3R) allosterically triggers the opening of a Ca 2+ -conducting pore located ~ 100 Å away from the IP3binding core (IBC). However, the precise mechanism of IP3 binding and correlated domain dynamics in the NT that are central to the IP3R activation, remains unknown. Our all-atom molecular dynamics (MD) simulations recapitulate the characteristic twist motion of the suppresser domain (SD) and reveal correlated 'clam closure' dynamics of IBC with IP3-binding, complementing existing suggestions on IP3R activation mechanism. Our study further reveals the existence of inter-domain dynamic correlation in the NT and establishes the SD to be critical for the conformational dynamics of IBC. Also, a tripartite interaction involving Glu283-Arg54-Asp444 at the SD -IBC interface seemed critical for IP3R activation. Intriguingly, during the sub-microsecond long simulation, we observed Arg269 undergoing an SD-dependent flipping of hydrogen bonding between the first and fifth phosphate groups of IP3. This seems to play a major role in determining the IP3 binding affinity of IBC in the presence/absence of the SD. Our study thus provides atomistic details of early molecular events occurring within the NT during and following IP3 binding that lead to channel gating. IBC-β position the IBC relative to the SD, where the SD is located behind the IP3-binding site in the IBC. A recent 4.7 Å full length structure of rat IP3R1 solved through cryoelectron microscopy (cryo-EM) 6 shows that the IBC is located ~100 Å away from the pore region of the receptor. Quite intriguingly, the cryo-EM structure of rat IP3R1 also shows that, unlike the closely related ryanodine receptors (RyRs), the C-terminal tail of the IP3R directly interacts with the SD of NT and forms a connecting link between the pore-forming helices and the IP3-binding NT region. Very recently, crystallographic analysis also shed light on the long-range communication between IBC and the channel domain. The IP3-dependent conformational changes are transmitted to the channel through the three large α-helical domains located between the NT and the transmembrane channel domain 7 . These findings together reaffirm the existence of allosteric modulation of IP3-mediated channel gating in IP3R.Comparison of the apo and IP3-bound crystal structures 4,5,7 provides static snapshots of IP3-evoked major domain movements in NT, where the IP3 binding brings IBC-β and IBC-α domains close together in a 'clam closure' type manner. In agreement with experimental findings 8 , large domain motions in the apo IBC as compared to the IP3-bound form, was observed from the molecular dynamics (MD) simulations of the IBC 9 , supporting this 'clam closure' during IP3 binding. Crystallographic studies further showed IP3-induced translational displacement of SD that causes the latter to rotate towards the IBC, measuring an angular displacement of ~ 9 o between the arm helices (α2/α3 helix, Fig. 1) of the apo...