Niklas G. Johansson

Membrane-bound pyrophosphatases (mPPases) are homodimeric proteins that hydrolyse pyrophosphate and pump H ⁺ /Na ⁺ across membranes. They are crucial for the virulence of protist pathogens, making them attractive drug targets. In this study, we investigate the inhibitory effects of seven distinct bisphosphonates against Thermotoga maritima mPPase to explore their mode of action and assist in future small molecule inhibitor development. We solved two structures of mPPase bound to the inhibitors in the enzyme active sites and probed the conformational dynamics of mPPase under multiple inhibitors and functionally relevant conditions by double electron-electron resonance (DEER) spectroscopy. We found that mPPase adopts distinct conformational equilibria in solution in the presence of different inhibitors, including states consistent with asymmetric binding in the active site (closed-open), but a symmetric apo-like conformation on the periplasmic side (open-open). Combined with solid-supported membrane-based electrophysiology recordings, this revealed that during catalysis, one monomer of the dimer remains open, and Na ⁺ can only be pumped in a closed state. These results further support symmetry-breaking across the membrane, consistent with half-of-the-sites-reactivity.

Membrane-bound pyrophosphatases (mPPases) are homodimeric proteins that hydrolyse pyrophosphate and pump H+/Na+ across membranes. They are crucial for the virulence of protist pathogens, making them attractive drug targets. In this study, we investigate the inhibitory effects of seven distinct bisphosphonates against Thermotoga maritima mPPase to explore their mode of action and assist in future small molecule inhibitor development. We solved two structures of mPPase bound to the inhibitors in the enzyme active sites and probed the conformational dynamics of mPPase under multiple inhibitors and functionally relevant conditions by double electron-electron resonance (DEER) spectroscopy. We found that mPPase adopts distinct conformational equilibria in solution in the presence of different inhibitors, including states consistent with asymmetric binding in the active site (closed-open), but a symmetric apo-like conformation on the periplasmic side (open-open). Combined with solid-supported membrane-based electrophysiology recordings, this revealed that during catalysis, one monomer of the dimer remains open, and Na+ can only be pumped in a closed state. These results further support symmetry-breaking across the membrane, consistent with half-of-the-sites-reactivity.

Membrane-bound pyrophosphatases are integral membrane proteins that catalyze the hydrolysis of pyrophosphate into orthophosphate, while simultaneously facilitating the pumping of protons and/or sodium ions. Since mPPases are absent in humans but play a critical role in the life cycle of protist parasite, they represent promising therapeutic targets. We successfully expressed the Plasmodium falciparum type 1 mPPase in the baculovirus/insect cell expression system and purified the protein, yielding 0.3 mg per liter cell culture. Various detergents were tested for solubilization, with the protein remaining active under all selected detergents. n-dodecyl-β-D-maltoside combined with cholesteryl hemisuccinate provided the highest solubility (88%). Finally, the PfPPase-VP1 was assayed against a set of fourteen antimalarial drugs, along with seven Thermotoga maritima mPPase inhibitors and fourteen compounds of unknown activity against mPPases. Only three compounds, all pyrazolo[1,5-a]pyrimidine-based TmPPase inhibitors, retained micromolar IC₅₀ activity against PfPPase-VP1. The expression and purification of the PfPPase-VP1 will allow to conduct structural studies as well as to develop target-based screens, two steps necessary for the development of inhibitors to combat parasite disease.

In this study, we employed stimulated Raman scattering (SRS) microscopy, augmented with sum frequency generation, to characterize complex solid-state mixtures, containing many solid-state forms of the same compound, for the first time. Five crystalline forms and one amorphous form of lactose were characterized and resolved, including two more recently defined anhydrous solid-state forms. Additionally, the complex solid-state character of several commercially available pharmaceutical tableting and inhalation grades of lactose was profiled. The advanced multimodal label-free microscopy method enabled visualization of the distribution of the solid-state forms with submicron spatial resolution, including the detection of trace levels. In addition, quantitative solid-state compositions of the lactose products were estimated. Overall SRS microscopy allows sensitive and specific spatially resolved solid-state characterization of complex mixtures, beyond what is possible with established (nonspatially resolved) characterization methods.

Membrane-bound pyrophosphatases are integral membrane proteins that catalyze the hydrolysis of pyrophosphate into orthophosphate, while simultaneously facilitating the pumping of protons and/or sodium ions. Since mPPases are absent in humans but play a critical role in the life cycle of protist parasite, they represent promising therapeutic targets. We successfully expressed the Plasmodium falciparum type 1 mPPase in the baculovirus/insect cell expression system and purified the protein, yielding 0.3 mg per liter cell culture. Various detergents were tested for solubilization, with the protein remaining active under all selected detergents. n -dodecyl-β-D-maltoside combined with cholesteryl hemisuccinate provided the highest solubility (88%). Finally, the PfPPase-VP1 was assayed against a set of fourteen antimalarial drugs, along with seven Thermotoga maritima mPPase inhibitors and fourteen compounds of unknown activity against mPPases. Only three compounds, all pyrazolo[1,5- a ]pyrimidine-based TmPPase inhibitors, retained micromolar IC ₅₀ activity against PfPPase-VP1. The expression and purification of the PfPPase-VP1 will allow to conduct structural studies as well as to develop target-based screens, two steps necessary for the development of inhibitors to combat parasite disease.

Membrane-bound pyrophosphatases (mPPases) are homodimeric proteins that hydrolyse pyrophosphate and pump H ⁺ /Na ⁺ across membranes. They are crucial for the virulence of protist pathogens, making them attractive drug targets. In this study, we investigate the inhibitory effects of seven distinct bisphosphonates against Thermotoga maritima mPPase to explore their mode of action and assist in future small molecule inhibitor development. We solved two structures of mPPase bound to the inhibitors in the enzyme active sites and probed the conformational dynamics of mPPase under multiple inhibitors and functionally relevant conditions by double electron-electron resonance (DEER) spectroscopy. We found that mPPase adopts distinct conformational equilibria in solution in the presence of different inhibitors, including states consistent with asymmetric binding in the active site (closed-open), but a symmetric apo-like conformation on the periplasmic side (open-open). Combined with solid-supported membrane-based electrophysiology recordings, this revealed that during catalysis, one monomer of the dimer remains open, and Na ⁺ can only be pumped in a closed state. These results further support symmetry-breaking across the membrane, consistent with half-of-the-sites-reactivity.

Transient receptor potential ankyrin 1 (TRPA1) protein plays an important role in the inflammatory response, and it has been associated with different pain conditions and pain-related diseases, making TRPA1 a valid target for painkillers. In this study, we identified potential TRPA1 inhibitors and located their binding sites utilizing computer-aided drug design (CADD) techniques. The designed 3-phenylcoumarin-based TRPA1 inhibitors were successfully synthesized using a microwave assisted synthetic strategy. 3-(3-Bromophenyl)-7-acetoxycoumarin (5), 7-hydroxy-3-(3-hydroxyphenyl)coumarin (12) and 3-(3-hydroxyphenyl)coumarin (23) all showed inhibitory activity toward TRPA1 in vitro. Compound 5 also decreased the size and formation of breast cancer cells. Hence, targeting TRPA1 may represent a promising alternative for the treatment of pain and inflammation.

Inhibition of membrane‐bound pyrophosphatase (mPPase) with small molecules offer a new approach in the fight against pathogenic protozoan parasites. mPPases are absent in humans, but essential for many protists as they couple pyrophosphate hydrolysis to the active transport of protons or sodium ions across acidocalcisomal membranes. So far, only few nonphosphorus inhibitors have been reported. Here, we explore the chemical space around previous hits using a combination of screening and synthetic medicinal chemistry, identifying compounds with low micromolar inhibitory activities in the Thermotoga maritima mPPase test system. We furthermore provide early structure‐activity relationships around a new scaffold having a pyrazolo[1,5‐a]pyrimidine core. The most promising pyrazolo[1,5‐a]pyrimidine congener was further investigated and found to inhibit Plasmodium falciparum mPPase in membranes as well as the growth of P. falciparum in an ex vivo survival assay.

Light-activated liposomes permit site and time-specific drug delivery to ocular and systemic targets. We combined a light activation technology based on indocyanine green with a hyaluronic acid (HA) coating by synthesizing HA–lipid conjugates. HA is an endogenous vitreal polysaccharide and a potential targeting moiety to cluster of differentiation 44 (CD44)-expressing cells. Light-activated drug release from 100 nm HA-coated liposomes was functional in buffer, plasma, and vitreous samples. The HA-coating improved stability in plasma compared to polyethylene glycol (PEG)-coated liposomes. Liposomal protein coronas on HA- and PEG-coated liposomes after dynamic exposure to undiluted human plasma and porcine vitreous samples were hydrophilic and negatively charged, thicker in plasma (~5 nm hard, ~10 nm soft coronas) than in vitreous (~2 nm hard, ~3 nm soft coronas) samples. Their compositions were dependent on liposome formulation and surface charge in plasma but not in vitreous samples. Compared to the PEG coating, the HA-coated liposomes bound more proteins in vitreous samples and enriched proteins related to collagen interactions, possibly explaining their slightly reduced vitreal mobility. The properties of the most abundant proteins did not correlate with liposome size or charge, but included proteins with surfactant and immune system functions in plasma and vitreous samples. The HA-coated light-activated liposomes are a functional and promising alternative for intravenous and ocular drug delivery.

Membrane-bound pyrophosphatases (mPPases) regulate energy homeostasis in pathogenic protozoan parasites and lack human homologues, which makes them promising targets in e.g. malaria. Yet only few nonphosphorus inhibitors have been reported so far. Here, we explore an isoxazole fragment hit, leading to the discovery of small mPPase inhibitors with 6–10 μM IC₅₀ values in the Thermotoga maritima test system. Promisingly, the compounds retained activity against Plasmodium falciparum mPPase in membranes and inhibited parasite growth.