Discovery of mobocertinib, a potent, oral inhibitor of EGFR exon 20 insertion mutations in non-small cell lung cancer.

In the treatment of non-small cell lung cancer (NSCLC), patients harboring exon 20 insertion mutations in the epidermal growth factor receptor (EGFR) gene (EGFR) have few effective therapies because this subset of mutants is generally resistant to most currently approved EGFR inhibitors. This report describes the structure-guided design of a novel series of potent, irreversible inhibitors of EGFR exon 20 insertion mutations, including the V769_D770insASV and D770_N771insSVD mutants. Extensive structure-activity relationship (SAR) studies led to the discovery of mobocertinib (compound 21c), which inhibited growth of Ba/F3 cells expressing the ASV insertion with a half-maximal inhibitory concentration of 11 nM and with selectivity over wild-type EGFR. Daily oral administration of mobocertinib induced tumor regression in a Ba/F3 ASV xenograft mouse model at well-tolerated doses. Mobocertinib was approved in September 2021 for the treatment of adult patients with advanced NSCLC with EGFR exon 20 insertion mutations with progression on or after platinum-based chemotherapy.

Optimization of Allosteric With-No-Lysine (WNK) Kinase Inhibitors and Efficacy in Rodent Hypertension Models.

The observed structure-activity relationship of three distinct ATP noncompetitive With-No-Lysine (WNK) kinase inhibitor series, together with a crystal structure of a previously disclosed allosteric inhibitor bound to WNK1, led to an overlay hypothesis defining core and side-chain relationships across the different series. This in turn enabled an efficient optimization through scaffold morphing, resulting in compounds with a good balance of selectivity, cellular potency, and pharmacokinetic profile, which were suitable for in vivo proof-of-concept studies. When dosed orally, the optimized compound reduced blood pressure in mice overexpressing human WNK1, and induced diuresis, natriuresis and kaliuresis in spontaneously hypertensive rats (SHR), confirming that this mechanism of inhibition of WNK kinase activity is effective at regulating cardiovascular homeostasis.

Design, Synthesis, and Structure-Activity Relationships of Pyridine-Based Rho Kinase (ROCK) Inhibitors.

The Rho kinases (ROCK1 and ROCK2) are highly homologous serine/threonine kinases that act on substrates associated with cellular motility, morphology, and contraction and are of therapeutic interest in diseases associated with cellular migration and contraction, such as hypertension, glaucoma, and erectile dysfunction. Beginning with compound 4, an inhibitor of ROCK1 identified through high-throughput screening, systematic exploration of SAR, and application of structure-based design, led to potent and selective ROCK inhibitors. Compound 37 represents significant improvements in inhibition potency, kinase selectivity, and CYP inhibition and possesses pharmacokinetics suitable for in vivo experimentation.

Protein complex interactor analysis and differential activity of KDM3 subfamily members towards H3K9 methylation.

Histone modifications play an important role in chromatin organization and gene regulation, and their interpretation is referred to as epigenetic control. The methylation levels of several lysine residues in histone tails are tightly controlled, and JmjC domain-containing proteins are one class of broadly expressed enzymes catalyzing methyl group removal. However, several JmjC proteins remain uncharacterized, gaps persist in understanding substrate recognition, and the integration of JmjC proteins into signaling pathways is just emerging. The KDM3 subfamily is an evolutionarily conserved group of histone demethylase proteins, thought to share lysine substrate specificity. Here we use a systematic approach to compare KDM3 subfamily members. We show that full-length KDM3A and KDM3B are H3K9me1/2 histone demethylases whereas we fail to observe histone demethylase activity for JMJD1C using immunocytochemical and biochemical approaches. Structure-function analyses revealed the importance of a single amino acid in KDM3A implicated in the catalytic activity towards H3K9me1/2 that is not conserved in JMJD1C. Moreover, we use quantitative proteomic analyses to identify subsets of the interactomes of the 3 proteins. Specific interactor candidates were identified for each of the three KDM3 subfamily members. Importantly, we find that SCAI, a known transcriptional repressor, interacts specifically with KDM3B. Taken together, we identify substantial differences in the biology of KDM3 histone demethylases, namely enzymatic activity and protein-protein interactions. Such comparative approaches pave the way to a better understanding of histone demethylase specificity and protein function at a systems level and are instrumental in identifying the more subtle differences between closely related proteins.

The Discovery of VX-745: A Novel and Selective p38α Kinase Inhibitor.

The synthesis of novel, selective, orally active 2,5-disubstituted 6H-pyrimido[1,6-b]pyridazin-6-one p38α inhibitors is described. Application of structural information from enzyme-ligand complexes guided the selection of screening compounds, leading to the identification of a novel class of p38α inhibitors containing a previously unreported bicyclic heterocycle core. Advancing the SAR of this series led to the eventual discovery of 5-(2,6-dichlorophenyl)-2-(2,4-difluorophenylthio)-6H-pyrimido[1,6-b]pyridazin-6-one (VX-745). VX-745 displays excellent enzyme activity and selectivity, has a favorable pharmacokinetic profile, and demonstrates good in vivo activity in models of inflammation.

Investigation of functionally liver selective glucokinase activators for the treatment of type 2 diabetes.

Type 2 diabetes is a polygenic disease which afflicts nearly 200 million people worldwide and is expected to increase to near epidemic levels over the next 10-15 years. Glucokinase (GK) activators are currently under investigation by a number of pharmaceutical companies with only a few reaching early clinical evaluation. A GK activator has the promise of potentially affecting both the beta-cells of the pancreas, by improving glucose sensitive insulin secretion, as well as the liver, by reducing uncontrolled glucose output and restoring post-prandial glucose uptake and storage as glycogen. Herein, we report our efforts on a sulfonamide chemotype with the aim to generate liver selective GK activators which culminated in the discovery of 3-cyclopentyl-N-(5-methoxy-thiazolo[5,4-b]pyridin-2-yl)-2-[4-(4-methyl-piperazine-1-sulfonyl)-phenyl]-propionamide (17c). This compound activated the GK enzyme (alphaK(a) = 39 nM) in vitro at low nanomolar concentrations and significantly reduced glucose levels during an oral glucose tolerance test in normal mice.

Structure-based design and parallel synthesis of N-benzyl isatin oximes as JNK3 MAP kinase inhibitors.

A series of N-benzylated isatin oximes were developed as inhibitors of the mitogen-activated kinase, JNK3. X-ray crystallographic structures aided in the design and synthesis of novel, selective compounds, that inhibit JNK3, but not p38 MAP kinase and provided key insights into understanding the behavior of gatekeeper residue methionine-146 in determining target selectivity for this series.

Discovery and SAR of novel 4-thiazolyl-2-phenylaminopyrimidines as potent inhibitors of spleen tyrosine kinase (SYK).

A series of SYK inhibitors based on the phenylamino pyrimidine thiazole lead 4 were prepared and evaluated for biological activity. Lead optimization provided compounds with nanomolar K(i)'s against SYK and potent inhibition in mast cell degranulation assays.

Staurosporine-based binding assay for testing the affinity of compounds to protein kinases.

Staurosporine is a broad-spectrum inhibitor of both tyrosine and serine/threonine protein kinases. Excitation of staurosporine and its analogues at 296 nm results in major emission bands centered at 378 and 396 nm. The intensity of the emission bands is enhanced on binding to the adenosine triphosphate (ATP) site of many protein kinases. This property was used to develop a competitive displacement assay for evaluating the binding affinity of small molecules to protein kinases. The assay was validated in both cuvette and plate formats for several phosphorylated and non-phosphorylated protein kinases. The throughput of the assay is high enough to be used in drug discovery for screening as well as lead optimization.

Resveratrol inhibits firefly luciferase.

The potential therapeutic value of resveratrol in age-related disease settings including cancer, diabetes, and Alzheimer's has emerged from a rapidly growing body of experimental evidence. Protection from oxidative stress appears to be a common feature of resveratrol that may be mediated through SirT1, though more specific molecular mechanisms by which resveratrol mediates its effects remain unclear. This has prompted an upsurge in cell-based mechanistic studies, often incorporating reporter assays for pathway elucidation in response to resveratrol treatment. Here, we report that resveratrol potently inhibits firefly luciferase with a K(i) value of 2microM, and caution that this confounding element may lead to compromised data interpretation.

The structure of dimeric ROCK I reveals the mechanism for ligand selectivity.

ROCK or Rho-associated kinase, a serine/threonine kinase, is an effector of Rho-dependent signaling and is involved in actin-cytoskeleton assembly and cell motility and contraction. The ROCK protein consists of several domains: an N-terminal region, a kinase catalytic domain, a coiled-coil domain containing a RhoA binding site, and a pleckstrin homology domain. The C-terminal region of ROCK binds to and inhibits the kinase catalytic domains, and this inhibition is reversed by binding RhoA, a small GTPase. Here we present the structure of the N-terminal region and the kinase domain. In our structure, two N-terminal regions interact to form a dimerization domain linking two kinase domains together. This spatial arrangement presents the kinase active sites and regulatory sequences on a common face affording the possibility of both kinases simultaneously interacting with a dimeric inhibitory domain or with a dimeric substrate. The kinase domain adopts a catalytically competent conformation; however, no phosphorylation of active site residues is observed in the structure. We also determined the structures of ROCK bound to four different ATP-competitive small molecule inhibitors (Y-27632, fasudil, hydroxyfasudil, and H-1152P). Each of these compounds binds with reduced affinity to cAMP-dependent kinase (PKA), a highly homologous kinase. Subtle differences exist between the ROCK- and PKA-bound conformations of the inhibitors that suggest that interactions with a single amino acid of the active site (Ala215 in ROCK and Thr183 in PKA) determine the relative selectivity of these compounds. Hydroxyfasudil, a metabolite of fasudil, may be selective for ROCK over PKA through a reversed binding orientation.

New insights into the structure-function relationships of Rho-associated kinase: a thermodynamic and hydrodynamic study of the dimer-to-monomer transition and its kinetic implications.

The effect of the length of ROCK (Rho-associated kinase) on its oligomerization state has been investigated by analysing full-length protein and four truncated constructs using light-scattering and analytical ultracentrifugation methods. Changes in size correlate with the kinetic properties of the kinase. Sedimentation velocity, sedimentation equilibrium and light-scattering data analyses revealed that protein constructs of size Ser6-Arg415 and larger exist predominantly as dimers, while smaller constructs are predominantly monomeric. The amino acid segments comprising residues 379-415 and 47-78 are shown to be necessary to maintain the dimeric ROCK structure. kcat values ranged from 0.7 to 2.1 s(-1) and from 1.0 to 5.9 s(-1) using ROCK peptide (KKRNRTLSV) and the 20000 Da subunit of myosin light chain respectively as substrate, indicating that the effect of the ROCK oligomerization state on the kcat is minor. Values of ATP K(m) for monomeric constructs were increased by 50-80-fold relative to the dimeric constructs, and K(i) comparisons using the specific competitive ROCK inhibitor Y-27632 also showed increases of at least 120-fold, demonstrating significant perturbations in the ATP binding site. The corresponding K(m) values for the ROCK peptide and myosin light chain substrates increased in the range 1.4-16-fold, demonstrating that substrate binding is less sensitive to the ROCK oligomerization state. These results show that the oligomerization state of ROCK may influence both its kinase activity and its interactions with inhibitors, and suggest that the dimeric structure is essential for normal in vivo function.