Potent GCN2 Inhibitor Capable of Reversing MDSC-Driven T Cell Suppression Demonstrates In Vivo Efficacy as a Single Agent and in Combination with Anti-Angiogenesis Therapy.

General control nonderepressible 2 (GCN2) protein kinase is a cellular stress sensor within the tumor microenvironment (TME), whose signaling cascade has been proposed to contribute to immune escape in tumors. Herein, we report the discovery of cell-potent GCN2 inhibitors with excellent selectivity against its closely related Integrated Stress Response (ISR) family members heme-regulated inhibitor kinase (HRI), protein kinase R (PKR), and (PKR)-like endoplasmic reticulum kinase (PERK), as well as good kinome-wide selectivity and favorable PK. In mice, compound 39 engages GCN2 at levels ≥80% with an oral dose of 15 mg/kg BID. We also demonstrate the ability of compound 39 to alleviate MDSC-related T cell suppression and restore T cell proliferation, similar to the effect seen in MDSCs from GCN2 knockout mice. In the LL2 syngeneic mouse model, compound 39 demonstrates significant tumor growth inhibition (TGI) as a single agent. Furthermore, TGI mediated by anti-VEGFR was enhanced by treatment with compound 39 demonstrating the complementarity of these two mechanisms.

Fragment-Based Drug Design of Novel Pyranopyridones as Cell Active and Orally Bioavailable Tankyrase Inhibitors.

Tankyrase activity has been linked to the regulation of intracellular axin levels, which have been shown to be crucial for the Wnt pathway. Deregulated Wnt signaling is important for the genesis of many diseases including cancer. We describe herein the discovery and development of a new series of tankyrase inhibitors. These pyranopyridones are highly active in various cell-based assays. A fragment/structure based optimization strategy led to a compound with good pharmacokinetic properties that is suitable for in vivo studies and further development.

Total synthesis of 6-deoxypladienolide D and Assessment of Splicing Inhibitory Activity in a Mutant SF3B1 cancer cell line.

A total synthesis of the natural product 6-deoxypladienolide D (1) has been achieved. Two noteworthy attributes of the synthesis are (1) a late-stage allylic oxidation which proceeds with full chemo-, regio-, and diastereoselectivity and (2) the development of a scalable and cost-effective synthetic route to support drug discovery efforts. 6-Deoxypladienolide D (1) demonstrates potent growth inhibition in a mutant SF3B1 cancer cell line, high binding affinity to the SF3b complex, and inhibition of pre-mRNA splicing.

Scaffold hopping towards potent and selective JAK3 inhibitors: discovery of novel C-5 substituted pyrrolopyrazines.

The discovery of a novel series of pyrrolopyrazines as JAK inhibitors with comparable enzyme and cellular activity to tofacitinib is described. The series was identified using a scaffold hopping approach aided by structure based drug design using principles of intramolecular hydrogen bonding for conformational restriction and targeting specific pockets for modulating kinase activity.

Strategic use of conformational bias and structure based design to identify potent JAK3 inhibitors with improved selectivity against the JAK family and the kinome.

Using a structure based design approach we have identified a series of indazole substituted pyrrolopyrazines, which are potent inhibitors of JAK3. Intramolecular electronic repulsion was used as a strategy to induce a strong conformational bias within the ligand. Compounds bearing this conformation participated in a favorable hydrophobic interaction with a cysteine residue in the JAK3 binding pocket, which imparted high selectivity versus the kinome and improved selectivity within the JAK family.

Development of amino-pyrimidine inhibitors of c-Jun N-terminal kinase (JNK): kinase profiling guided optimization of a 1,2,3-benzotriazole lead.

A series of amino-pyrimidines was developed based upon an initial kinase cross-screening hit from a CDK2 program. Kinase profiling and structure-based drug design guided the optimization from the initial 1,2,3-benzotriazole hit to a potent and selective JNK inhibitor, compound 24f (JNK1 and 2 IC(50)=16 and 66 nM, respectively), with bioavailability in rats and suitable for further in vivo pharmacological evaluation.

Discovery of novel PI3-kinase δ specific inhibitors for the treatment of rheumatoid arthritis: taming CYP3A4 time-dependent inhibition.

PI3Kδ is a lipid kinase and a member of a larger family of enzymes, PI3K class IA(α, ß, δ) and IB (γ), which catalyze the phosphorylation of PIP2 to PIP3. PI3Kδ is mainly expressed in leukocytes, where it plays a critical, nonredundant role in B cell receptor mediated signaling and provides an attractive opportunity to treat diseases where B cell activity is essential, e.g., rheumatoid arthritis. We report the discovery of novel, potent, and selective PI3Kδ inhibitors and describe a structural hypothesis for isoform (α, ß, γ) selectivity gained from interactions in the affinity pocket. The critical component of our initial pharmacophore for isoform selectivity was strongly associated with CYP3A4 time-dependent inhibition (TDI). We describe a variety of strategies and methods for monitoring and attenuating TDI. Ultimately, a structure-based design approach was employed to identify a suitable structural replacement for further optimization.

8-(4-Methoxyphenyl)pyrazolo[1,5-a]-1,3,5-triazines: selective and centrally active corticotropin-releasing factor receptor-1 (CRF1) antagonists.

This report describes the syntheses and structure-activity relationships of 8-(4-methoxyphenyl)pyrazolo[1,5-a]-1,3,5-triazine corticotropin releasing factor receptor-1 (CRF(1)) receptor antagonists. CRF(1) receptor antagonists may be potential anxiolytic or antidepressant drugs. This research culminated in the discovery of analogue 12-3, which is a potent, selective CRF(1) antagonist (hCRF(1) IC(50) = 4.7 +/- 2.0 nM) with weak affinity for the CRF-binding protein and biogenic amine receptors. This compound also has a good pharmacokinetic profile in dogs. Analogue 12-3 is orally effective in two rat models of anxiety: the defensive withdrawal (situational anxiety) model and the elevated plus maze test. Analogue 12-3 has been advanced to clinical trials.

4-Bromo-2H-1,3-oxazine-2,6(3H)-dione.

The title compound, C(4)H(2)BrNO(3), is one of a series of three substituted oxauracils prepared as precursors in the preparation of 1-aza-1,3-butadienes. Although each structure has identical potential for N-H⋯O inter-molecular hydrogen bonds, each forms a distinctive inter-molecular network. In the title compound, there are two independent mol-ecules in the asymmetric unit, with a non-crystallographic twofold screw-like relationship between them. The two indpendent mol-ecules are linked by an inter-molecular N-H⋯O hydrogen bond. In the crystal structure, this hydrogen-bonded pair is linked to translationally related mol-ecules through further inter-molecular N-H⋯O hydrogen bonds, forming one-dimensional chains along [100]. The crystal structure also has short Br⋯O=C inter-molecular contacts with distances of 2.843 (4) and 2.852 (4) Å.

Fluorogenic peptide sequences--transformation of short peptides into fluorophores under ambient photooxidative conditions.

Long-lived proteins are susceptible to nonenzymatic chemical reactions and the evolution of fluorescence; however, little is known about the sequence-dependence of fluorogenesis. We synthesized a library of over half a million octapeptides and exposed it to light and air in pH 7.4 buffer to identify fluorogenic peptides that evolve under mild oxidative conditions. The bead-based peptide library was composed of the general sequence H(2)N-Ala-(Xxx)(6)-Ala-resin, where Xxx was one of nine representative amino acids: Asp, Gly, His, Leu, Lys, Pro, Ser, Trp, and Tyr. Next, we selected five highly fluorescent beads from the library and subjected them to microsequencing, revealing the sequence of the unreacted peptide. All five of the fluorogenic sequences were ionic; lacked Tyr, His, and Leu; and most of the sequences contained only one Trp. We then synthesized the five soluble peptides corresponding to the fluorogenic peptide sequences and exposed them to photooxidative conditions. In general, the soluble peptides reacted slowly, generating nonfluorescent monooxygenated and dioxygenated products. However, one peptide (H(2)N-AlaLysProTrpGlyGlyAspAla-CONH(2)) evolved into a highly fluorescent photoproduct as well as a nonfluorescent monooxygenated photoproduct. The fluorescent photoproduct consisted of a 2-carboxy-quinolin-4-yl moiety fused to the N-terminus of GlyGlyAspAla. The formation of this photoproduct requires cleavage of the peptide backbone and a dramatic reorganization of tryptophan. This work demonstrates that sequencing unreacted peptide on beads can reveal sequences with unique nonenzymatic reactivity. The study also confirms that peptide fluorogenesis is dependent on sequence and not merely on the presence of tryptophan. The potential importance of fluorogenic peptide sequences is two-fold. First, fluorogenic sequences that arise through mutation could prove to be hot spots for human aging. Second, fluorogenic sequences, particularly those compatible with intracellular conditions, may serve as fluorescent tags for proteins or as fluorescent biomaterials.