Activation of human STING by a molecular glue-like compound.

Stimulator of interferon genes (STING) is a dimeric transmembrane adapter protein that plays a key role in the human innate immune response to infection and has been therapeutically exploited for its antitumor activity. The activation of STING requires its high-order oligomerization, which could be induced by binding of the endogenous ligand, cGAMP, to the cytosolic ligand-binding domain. Here we report the discovery through functional screens of a class of compounds, named NVS-STGs, that activate human STING. Our cryo-EM structures show that NVS-STG2 induces the high-order oligomerization of human STING by binding to a pocket between the transmembrane domains of the neighboring STING dimers, effectively acting as a molecular glue. Our functional assays showed that NVS-STG2 could elicit potent STING-mediated immune responses in cells and antitumor activities in animal models.

Successful Strategies for Mitigation of a Preclinical Signal for Phototoxicity in a DGAT1 Inhibitor.

Diacylglycerol O-acyltransferase 1 (DGAT1) inhibitor Pradigastat (1) was shown to be effective at decreasing postprandial triglyceride levels in a patient population with familial chylomicronemia syndrome (FCS). Although pradigastat does not cause photosensitization in humans at the high clinical dose of 40 mg, a positive signal was observed in preclinical models of phototoxicity. Herein, we describe a preclinical phototoxicity mitigation strategy for diarylamine containing molecules utilizing the introduction of an amide or suitable heterocyclic function. This strategy led to the development of two second-generation compounds with low risk of phototoxicity, disparate exposure profiles, and comparable efficacy to 1 in a rodent lipid bolus model for post-prandial plasma triglycerides.

Physician perspectives on education, training, and implementation of complementary and alternative medicine.

Over recent decades, the demand for complementary and alternative medicine (CAM) has continued to rise in the US. Like the practice of traditional Western medicine, CAM is associated with not only significant health benefits but also significant risks. Unlike traditional Western medicine, however, much of CAM use is less regulated and often occurs unbeknownst to a patient's medical doctor. The use of herbals, dietary supplements, and over-the-counter (OTC) medications can result in adverse effects, and many significant interactions can occur when their use is combined with allopathic medications. Even the more peripheral CAM practices (eg, acupuncture, massage, yoga, and Reiki) have associated risk (eg, adverse effects or worsening of physical injury and conditions). There is, however, impetus for change: both patients and physicians favor increasing physician knowledge of CAM and the synergistic implementation of CAM into routine clinical practice. Although improvement has been achieved from contemporary physician educational efforts, recently published results from patient and physician surveys strongly indicate that additional effort to increase physician knowledge of CAM is needed. Utilizing a 37-item survey and convenience-sampling methodology, we collected detailed information from 114 physicians, fellows, and residents from the Ohio State University Medical Center regarding impediments to increasing physician knowledge of CAM and its implementation in routine clinical practice. The aggregate results of our survey data showed that most physicians 1) desired to increase their knowledge of CAM, 2) believed that less than half of their patients were spontaneously reporting their use of CAM therapies, 3) were not aware of available evidence-based resources on CAM, 4) preferred case-based lectures for learning about CAM, and 5) reported insufficient time during patient encounters as the primary barrier for increasing the implementation of CAM in routine clinical practice.

Small-molecule WNK inhibition regulates cardiovascular and renal function.

The With-No-Lysine (K) (WNK) kinases play a critical role in blood pressure regulation and body fluid and electrolyte homeostasis. Herein, we introduce the first orally bioavailable pan-WNK-kinase inhibitor, WNK463, that exploits unique structural features of the WNK kinases for both affinity and kinase selectivity. In rodent models of hypertension, WNK463 affects blood pressure and body fluid and electro-lyte homeostasis, consistent with WNK-kinase-associated physiology and pathophysiology.

A small-molecule dengue virus entry inhibitor.

The incidence of dengue fever epidemics has increased dramatically over the last few decades. However, no vaccine or antiviral therapies are available. Therefore, the need for safe and effective antiviral drugs has become imperative. The entry of dengue virus into a host cell is mediated by its major envelope (E) protein. The crystal structure of the E protein reveals a hydrophobic pocket that is presumably important for low-pH-mediated membrane fusion. High-throughput docking with this hydrophobic pocket was performed, and hits were evaluated in cell-based assays. Compound 6 was identified as one of the inhibitors and had an average 50% effective concentration of 119 nM against dengue virus serotype 2 in a human cell line. Mechanism-of-action studies demonstrated that compound 6 acts at an early stage during dengue virus infection. It arrests dengue virus in vesicles that colocalize with endocytosed dextran and inhibits NS3 expression. The inhibitors described in this report can serve as molecular probes for the study of the entry of flavivirus into host cells.

Yellow fever virus NS3 protease: peptide-inhibition studies.

A recombinant form of yellow fever virus (YFV) NS3 protease, linked via a nonapeptide to the minimal NS2B co-factor sequence (CF40-gly-NS3pro190), was expressed in Escherichia coli and shown to be catalytically active. It efficiently cleaved the fluorogenic tetrapeptide substrate Bz-norleucine-lysine-arginine-arginine-AMC, which was previously optimized for dengue virus NS2B/3 protease. A series of small peptidic inhibitors based on this substrate sequence readily inhibited its enzymic activity. To understand the structure-activity relationship of the inhibitors, they were docked into a homology model of the YFV NS2B/NS3 protease structure. The results revealed that the P1 and P2 positions are most important for inhibitor binding, whilst the P3 and P4 positions have much less effect. These findings indicate that the characteristics of YFV protease are very similar to those reported for dengue and West Nile virus proteases, and suggest that pan-flavivirus NS3 protease drugs may be developed for flaviviral diseases.

Peptide inhibitors of West Nile NS3 protease: SAR study of tetrapeptide aldehyde inhibitors.

A series of inhibitors related to the benzoyl-norleucine-lysine-arginine-arginine (Bz-nKRR) tetrapeptide aldehyde was synthesized. When evaluated against the West Nile virus (WNV) NS3 protease, the measured IC(50) ranges from approximately 1 to 200 microM. Concurrently, a modeling study using the recently published crystal structure of the West Nile NS3/NS2B protease complex (pdb code 2FP7) was conducted. We found that the crystal structure is relevant in explaining the observed SAR for this series of tetrapeptides, with the S1 and S2 pockets being the key peptide recognition sites. In general, a residue capable of both pi-stacking and hydrogen bonding is favored in the S1 pocket, while a positively charged residue is preferred in the S2 pocket. This study not only confirms the importance of the NS2B domain in substrate-based inhibitor binding of WNV, it also suggests that the crystal structure would provide useful guidance in the drug discovery process of related Flavivirus proteases, given the high degree of homology.

Peptide inhibitors of Dengue virus NS3 protease. Part 1: Warhead.

Substrate-based tetrapeptide inhibitors with various warheads were designed, synthesized, and evaluated against the Dengue virus NS3 protease. Effective inhibition was achieved by peptide inhibitors with electrophilic warheads such as aldehyde, trifluoromethyl ketone, and boronic acid. A boronic acid has the highest affinity, exhibiting a K(i) of 43 nM.

Peptide inhibitors of dengue virus NS3 protease. Part 2: SAR study of tetrapeptide aldehyde inhibitors.

With the aim of discovering potent and selective dengue NS3 protease inhibitors, we systematically synthesized and evaluated a series of tetrapeptide aldehydes based on lead aldehyde 1 (Bz-Nle-Lys-Arg-Arg-H, K(i)=5.8 microM). In general, we observe that interactions of P(2) side chain are more important than P(1) followed by P(3) and P(4). Tripeptide and dipeptide aldehyde inhibitors also show low micromolar activity. Additionally, an effective non-basic, uncharged replacement of P(1) Arg is identified.

Finding new medicines for flaviviral targets.

With the incidence of dengue fever increasing all over the world, there is an urgent need for therapies. While drug discovery for any disease is a long and difficult process with uncertain success, dengue fever poses an additional complication in that most of the target patient population is young and lives in developing countries with very limited health care budgets. Recent progress in drug discovery for dengue and an analysis of approaches toward hepatitis C virus (HCV) therapeutics suggest that NS5 polymerase is the most promising target for dengue. Moreover such inhibitors may be useful for several other flaviviral diseases. NS3 proteases will be more challenging targets, especially if oral delivery is desired. Recent work has shown that potent inhibitors can be designed readily, but optimization of pharmacokinetic parameters will probably be a long an arduous task, especially since the primary binding pockets prefer to bind basic amino acids. NS3 helicase can also be considered a viable drug target for flaviviral diseases. It has however proved to be a challenging for HCV and selectivity issues versus human helicases must be overcome.