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Nav1.7 is an extensively investigated target for pain with a strong genetic link in humans, yet in spite of this effort, it remains challenging to identify efficacious, selective, and safe inhibitors. Here, we disclose the discovery and preclinical profile of GDC-0276 (1) and GDC-0310 (2), selective Nav1.7 inhibitors that have completed Phase 1 trials. Our initial search focused on close-in analogues to early compound 3. This resulted in the discovery of GDC-0276 (1), which possessed improved metabolic stability and an acceptable overall pharmacokinetics profile. To further derisk the predicted human pharmacokinetics and enable QD dosing, additional optimization of the scaffold was conducted, resulting in the discovery of a novel series of N-benzyl piperidine Nav1.7 inhibitors. Improvement of the metabolic stability by blocking the labile benzylic position led to the discovery of GDC-0310 (2), which possesses improved Nav selectivity and pharmacokinetic profile over 1.

Clathrin-mediated endocytosis (CME) underlies intra- and extracellular material trafficking in eukaryotes, and is essential to protein metabolism, intercellular signaling, membrane remodeling and other cell regulatory processes. Although CME is usually driven by F-actin polymerization, membrane invagination can also occur through actin independent mechanisms. Here, we show that viscoelastic protein condensates that form via liquid-liquid phase separation at the sites of endocytosis initiation facilitate actin independent CME. Binding energies of the condensate with the membrane and surrounding cytosol provides work required to drive membrane invagination. Our findings expand the repertoire of functions associated with protein condensates that form via liquid-liquid phase separation to include their ability to do work at soft interfaces, thus shaping and organizing cellular matter.

The Bruton's tyrosine kinase (Btk) inhibitor ibrutinib has shown impressive clinical efficacy in a range of B-cell malignancies. However, acquired resistance has emerged, and second generation therapies are now being sought. Ibrutinib is a covalent, irreversible inhibitor that modifies Cys481 in the ATP binding site of Btk and renders the enzyme inactive, thereby blocking B-cell receptor signal transduction. Not surprisingly, Cys481 is the most commonly mutated Btk residue in cases of acquired resistance to ibrutinib. Mutations at other sites, including Thr474, a gatekeeper residue, have also been detected. Herein, we describe noncovalent Btk inhibitors that differ from covalent inhibitors like ibrutinib in that they do not interact with Cys481, they potently inhibit the ibrutinib-resistant Btk C481S mutant in vitro and in cells, and they are exquisitely selective for Btk. Noncovalent inhibitors such as GNE-431 also show excellent potency against the C481R, T474I, and T474M mutants. X-ray crystallographic analysis of Btk provides insight into the unique mode of binding of these inhibitors that explains their high selectivity for Btk and their retained activity against mutant forms of Btk. This class of noncovalent Btk inhibitors may provide a treatment option to patients, especially those who have acquired resistance to ibrutinib by mutation of Cys481 or Thr474.

A series of inhibitors of mTOR kinase based on a quaternary-substituted dihydrofuropyrimidine was designed and synthesized. The most potent compounds in this series inhibited mTOR kinase with K(i)1.0 nM and were highly (100×) selective for mTOR over the closely related PI3 kinases. Compounds in this series showed inhibition of the pathway and antiproliferative activity in cell-based assays. Furthermore, these compounds had excellent mouse PK, and showed a robust PK-PD relationship in a mouse model of cancer.

A Nicotiana benthamiana transient expression system was used to express single antigen and dimeric combinations of the human rotavirus (HRV) VP7 and a truncated VP4 (VP4Δ) proteins fused with Salmonella typhimurium's flagellin fljB subunit. Immunoblot analyses using rabbit antibodies generated against these proteins demonstrated that the constructs were successfully expressed with yields ranging from 0.85 to 31.97 μg of recombinant protein per gram of fresh leaf tissue. Expressing the single and dimeric antigens has no effect on plant growth and development except for VP7 and VP4Δ::VP7, which show mild necrotic lesions. Immunization of mice with proteins from leaves transformed with constructs bearing the fljB moiety elicited an fljB-specific humoral response. The Nicotiana benthamiana transient system is efficient to express multiple combinations of pathogen proteins and demonstrates the potential of generating a Salmonella typhimurium subunit vaccine in plants.

Using Sorafenib as a starting point, a series of potent and selective inhibitors of CDK8 was developed. When cocrystallized with CDK8 and cyclin C, these compounds exhibit a Type-II (DMG-out) binding mode.

Because of the promise of BCL-2 antagonists in combating chronic lymphocytic leukemia (CLL) and non-Hodgkin’s lymphoma (NHL), interest in additional selective antagonists of antiapoptotic proteins has grown. Beginning with a series of selective, potent BCL-XL antagonists containing an undesirable hydrazone functionality, in silico design and X-ray crystallography were utilized to develop alternative scaffolds that retained the selectivity and potency of the starting compounds.

Aberrant activation of the PI3K-Akt-mTOR signaling pathway has been observed in human tumors and tumor cell lines, indicating that these protein kinases may be attractive therapeutic targets for treating cancer. Optimization of advanced lead 1 culminated in the discovery of clinical development candidate 8h, GDC-0349, a potent and selective ATP-competitive inhibitor of mTOR. GDC-0349 demonstrates pathway modulation and dose-dependent efficacy in mouse xenograft cancer models.

The aim of this study was to produce adjuvant with high biosafety, efficacy and low cost. Towards this goal, the plant Nicotiana benthamiana transient expression system was successfully used to express Salmonella typhimurium's flagellin (FljB). The yield of the expressed FljB was 280 mg per kg of fresh weight (FW) leaves. The lyophilized plant powder containing plant expressing FljB was mixed with ovalbumin (OVA) and used for oral immunization of BALB/c mice. The ELISA analysis showed higher and accelerated OVA-specific serum antibody responses in mice given the mixture when compared to animals receiving OVA alone. Furthermore, FljB elicited a mixed Th1/Th2 response as shown by the presence of specific anti-OVA IgG1, IgG2a and IgG2b isotypes. OVA-specific IgAs were also detected in mice given the mixture. Cell-mediated immune response to OVA was induced by FljB as determined by a spleen lymphocyte specific proliferation test. No immune response was generated against FljB. In conclusion, our results showed for the first time the production of FljB in plants and the efficient use of the crude lyophilized extract as an adjuvant for oral immunization.