Polymodal K2P (KCNK) thermo- and mechanosensitive TREK1 potassium channels, generate ‘leak’ currents that regulate neuronal excitability, respond to lipids, temperature, and mechanical stretch, and influence pain, temperature perception, and anesthetic responses1–3. These dimeric voltage-gated ion channel (VGIC) superfamily members have a unique topology comprising two pore forming regions per subunit4–6. Contrasting other potassium channels, K2Ps use a selectivity filter ‘C-type’ gate7–10 as the principal gating site. Despite recent advances3,11,12, K2Ps suffer from a poor pharmacologic profile limiting mechanistic and biological studies. Here, we describe a new small molecule TREK activator class that directly stimulates the C-type gate by acting as molecular wedges that restrict interdomain interface movement behind the selectivity filter. Structures of K2P2.1(TREK-1) alone with two selective K2P2.1(TREK-1) and K2P10.1(TREK-2) activators, an N-aryl-sulfonamide, ML335, and a thiophene-carboxamide, ML402, define a cryptic binding pocket unlike other ion channel small molecule binding sites and, together with functional studies, identify a cation-π interaction that controls selectivity. Together, our data unveil a previously unknown, druggable K2P site that stabilizes the C-type gate ‘leak mode’ and provide direct evidence for K2P selectivity filter gating.

HCN (hyperpolarization-activated cyclic-nucleotide sensitive cation nonselective) channels are activated by voltage and modulated by the direct binding of cAMP to their cytoplasmic C-terminal region named CNBD (cyclic nucleotide binding domain) (Zagotta et al., 2003). HCN channels are further regulated by TRIP8b, a brain-specific auxiliary subunit which controls channel trafficking and gating. In particular, TRIP8b interacts with the HCN channel CNBD and antagonizes the facilitatory effect of cAMP on channel opening (Hu et al., 2013 ). Recently, the cryo-EM structure of human HCN1 was solved in both the cAMP-free and cAMP-bound states (Lee and Mackinnon, 2017). A comparison of the two structures shows that, in the full-length protein, cAMP binding induces all the same changes previously highlighted in the isolated CNBD fragment using NMR (Saponaro et al., 2014), plus the unexpected folding of two additional helices at the C-terminus of the CNBD. These helices were not included in any of the previous experiments conducted using the isolated C-linker/CNBD fragment. To understand whether these two dynamic helices play a role in regulating both cAMP and TRIP8b binding, we have compared, using an in vitro binding assay, two CNBD constructs with or without the newly discovered helices. Surprisingly, their presence increases the affinity for cAMP without affecting TRIP8b binding. This result assigns to the new C-terminal helices of the HCN channel CNBD a specific role in controlling cyclic nucleotide affinity.

P-glycoprotein (P-gp) is an ATP-dependent transport protein that is selectively expressed at entry points of xenobiotics where, acting as an efflux pump, it prevents their entering sensitive organs. The protein also plays a key role in the absorption and blood-brain barrier penetration of many drugs, while its overexpression in cancer cells has been linked to multidrug resistance in tumors. The recent publication of the mouse P-gp crystal structure revealed a large and hydrophobic binding cavity with no clearly defined sub-sites that supports an “induced-fit” ligand binding model. We employed flexible receptor docking to develop a new prediction algorithm for P-gp binding specificity. We tested the ability of this method to differentiate between binders and nonbinders of P-gp using consistently measured experimental data from P-gp efflux and calcein-inhibition assays. We also subjected the model to a blind test on a series of peptidic cysteine protease inhibitors, confirming the ability to predict compounds more likely to be P-gp substrates. Finally, we used the method to predict cellular metabolites that may be P-gp substrates. Overall, our results suggest that many P-gp substrates bind deeper in the cavity than the cyclic peptide in the crystal structure and that specificity in P-gp is better understood in terms of physicochemical properties of the ligands (and the binding site), rather than being defined by specific sub-sites.

We have prepared a series of achiral aminoacetonitriles, bearing tri-ring benzamide moieties and an aminocyclohexanecarboxylate residue at P2. This combination of binding elements resulted in sub-250 pM, reversible, selective, and orally bioavailable cathepsin K inhibitors. Lead compounds displayed single digit nanomolar inhibition in vitro (of rabbit osteoclast-mediated degradation of bovine bone). The best compound in this series, 39n (CRA-013783/L-006235), was orally bioavailable in rats, with a terminal half-life of over 3 h. 39n was dosed orally in ovariectomized rhesus monkeys once per day for 7 days. Collagen breakdown products were reduced by up to 76% dose-dependently. Plasma concentrations of 39n above the bone resorption IC50 after 24 h indicated a correlation between functional cellular and in vivo assays. Inhibition of collagen breakdown by cathepsin K inhibitors suggests this mechanism of action may be useful in osteoporosis and other indications involving bone resorption.

A docking screen identified reversible, non-covalent inhibitors (e.g. 1) of the parasite cysteine protease cruzain. Chemical optimization of 1 led to a series of oxadiazoles possessing interpretable SAR and potencies as much as 500-fold greater than 1. Detailed investigation of the SAR series subsequently revealed that many members of the oxadiazole class (and surprisingly also 1) act via divergent modes of inhibition – competitive or via colloidal aggregation – depending on the assay conditions employed.

Because no drug exists that halts or even slows any neurodegenerative disease, developing effective therapeutics for any prion disorder is urgent. We recently reported two compounds (IND24 and IND81) with the 2-aminothiazole (2-AMT) chemical scaffold that almost doubled the incubation times in scrapie prion-infected, wild-type (wt) FVB mice when given in a liquid diet. Remarkably, oral prophylactic treatment with IND24 beginning 14 days prior to intracerebral prion inoculation extended survival from ∼120 days to over 450 days. In addition to IND24, we evaluated the pharmacokinetics and efficacy of five additional 2-AMTs; one was not followed further because its brain penetration was poor. Of the remaining four new 2-AMTs, IND114338 doubled and IND125 tripled the incubation times of RML-inoculated wt and Tg4053 mice overexpressing wt mouse prion protein (PrP), respectively. Neuropathological examination of the brains from untreated controls showed a widespread deposition of self-propagating, b-sheet-rich "scrapie" isoform (PrP Sc ) prions accompanied by a profound astrocytic gliosis. In contrast, mice treated with 2-AMTs had lower levels of PrP Sc and associated astrocytic gliosis, with each compound resulting in a distinct pattern of deposition. Notably, IND125 prevented both PrP Sc accumulation and astrocytic gliosis in the cerebrum. Progressive central nervous system dysfunction in the IND125-treated mice was presumably due to the PrP Sc that accumulated in their brainstems. Disappointingly, none of the four new 2-AMTs prolonged the lives of mice expressing a chimeric human/ mouse PrP transgene inoculated with Creutzfeldt-Jakob disease prions.

The only small-molecule compound demonstrated to substantially extend survival in prion-infected mice is a biaryl hydrazone termed "Compd B" (4-pyridinecarboxaldehyde,2-[4-(5-oxazolyl)phenyl]hydrazone). However, the hydrazone moiety of Compd B results in toxic metabolites, making it a poor candidate for further drug development. We developed a pharmacophore model based on diverse antiprion compounds identified by high-throughput screening; based on this model, we generated biaryl amide analogs of Compd B. Medicinal chemistry optimization led to multiple compounds with increased potency, increased brain concentrations, and greater metabolic stability, indicating that they could be promising candidates for antiprion therapy. Replacing the pyridyl ring of Compd B with a phenyl group containing an electron-donating substituent increased potency, while adding an aryl group to the oxazole moiety increased metabolic stability. To test the efficacy of Compd B, we applied bioluminescence imaging (BLI), which was previously shown to detect prion disease onset in live mice earlier than clinical signs. In our studies, Compd B showed good efficacy in two lines of transgenic mice infected with the mouse-adapted Rocky Mountain Laboratory (RML) strain of prions, but not in transgenic mice infected with human prions. The BLI system successfully predicted the efficacies in all cases long before extension in survival could be observed. Our studies suggest that this BLI system has good potential to be applied in future antiprion drug efficacy studies.

We describe here the identification of non-peptidic vinylsulfones that inhibit parasite cysteine proteases in vitro and inhibit the growth of T. brucei brucei parasites in culture. A high resolution (1.75Å) co-crystal structure of 8a bound to cruzain reveals how the non-peptidic P2/P3 moiety in such analogs bind the S2 and S3 subsites of the protease, effectively recapitulating important binding interactions present in more traditional peptide-based protease inhibitors and natural substrates.