Discovery of 5-Azaquinoxaline Derivatives as Potent and Orally Bioavailable Allosteric SHP2 Inhibitors.

SHP2 has emerged as an important target for oncology small-molecule drug discovery. As a nonreceptor tyrosine phosphatase within the MAPK pathway, it has been shown to control cell growth, differentiation, and oncogenic transformation. We used structure-based design to find a novel class of potent and orally bioavailable SHP2 inhibitors. Our efforts led to the discovery of the 5-azaquinoxaline as a new core for developing this class of compounds. Optimization of the potency and properties of this scaffold generated compound 30, that exhibited potent in vitro SHP2 inhibition and showed excellent in vivo efficacy and pharmacokinetic profile.

Preclinical characterization of pirtobrutinib, a highly selective, noncovalent (reversible) BTK inhibitor.

Bruton tyrosine kinase (BTK), a nonreceptor tyrosine kinase, is a major therapeutic target for B-cell-driven malignancies. However, approved covalent BTK inhibitors (cBTKis) are associated with treatment limitations because of off-target side effects, suboptimal oral pharmacology, and development of resistance mutations (eg, C481) that prevent inhibitor binding. Here, we describe the preclinical profile of pirtobrutinib, a potent, highly selective, noncovalent (reversible) BTK inhibitor. Pirtobrutinib binds BTK with an extensive network of interactions to BTK and water molecules in the adenosine triphosphate binding region and shows no direct interaction with C481. Consequently, pirtobrutinib inhibits both BTK and BTK C481 substitution mutants in enzymatic and cell-based assays with similar potencies. In differential scanning fluorimetry studies, BTK bound to pirtobrutinib exhibited a higher melting temperature than cBTKi-bound BTK. Pirtobrutinib, but not cBTKis, prevented Y551 phosphorylation in the activation loop. These data suggest that pirtobrutinib uniquely stabilizes BTK in a closed, inactive conformation. Pirtobrutinib inhibits BTK signaling and cell proliferation in multiple B-cell lymphoma cell lines, and significantly inhibits tumor growth in human lymphoma xenografts in vivo. Enzymatic profiling showed that pirtobrutinib was highly selective for BTK in >98% of the human kinome, and in follow-up cellular studies pirtobrutinib retained >100-fold selectivity over other tested kinases. Collectively, these findings suggest that pirtobrutinib represents a novel BTK inhibitor with improved selectivity and unique pharmacologic, biophysical, and structural attributes with the potential to treat B-cell-driven cancers with improved precision and tolerability. Pirtobrutinib is being tested in phase 3 clinical studies for a variety of B-cell malignancies.

Identification of the Clinical Development Candidate MRTX849, a Covalent KRASG12C Inhibitor for the Treatment of Cancer.

Capping off an era marred by drug development failures and punctuated by waning interest and presumed intractability toward direct targeting of KRAS, new technologies and strategies are aiding in the target's resurgence. As previously reported, the tetrahydropyridopyrimidines were identified as irreversible covalent inhibitors of KRASG12C that bind in the switch-II pocket of KRAS and make a covalent bond to cysteine 12. Using structure-based drug design in conjunction with a focused in vitro absorption, distribution, metabolism and excretion screening approach, analogues were synthesized to increase the potency and reduce metabolic liabilities of this series. The discovery of the clinical development candidate MRTX849 as a potent, selective covalent inhibitor of KRASG12C is described.

Discovery of Tetrahydropyridopyrimidines as Irreversible Covalent Inhibitors of KRAS-G12C with In Vivo Activity.

KRAS is the most frequently mutated driver oncogene in human cancer, and KRAS mutations are commonly associated with poor prognosis and resistance to standard treatment. The ability to effectively target and block the function of mutated KRAS has remained elusive despite decades of research. Recent findings have demonstrated that directly targeting KRAS-G12C with electrophilic small molecules that covalently modify the mutated codon 12 cysteine is feasible. We have discovered a series of tetrahydropyridopyrimidines as irreversible covalent inhibitors of KRAS-G12C with in vivo activity. The PK/PD and efficacy of compound 13 will be highlighted.

PTH modulation of NCC activity regulates TRPV5 Ca2+ reabsorption.

Since parathyroid hormone (PTH) is known to increase transient receptor potential vanilloid (TRPV)5 activity and decrease Na(+)-Cl(-) cotransporter (NCC) activity, we hypothesized that decreased NCC-mediated Na(+) reabsorption contributes to the enhanced TRPV5 Ca(2+) reabsorption seen with PTH. To test this, we used mDCT15 cells expressing functional TRPV5 and ruthenium red-sensitive (45)Ca(2+) uptake. PTH increased (45)Ca(2+) uptake to 8.8 ± 0.7 nmol·mg(-1)·min(-1) (n = 4, P < 0.01) and decreased NCC activity from 75.4 ± 2.7 to 20.3 ± 1.3 nmol·mg(-1)·min(-1) (n = 4, P < 0.01). Knockdown of Ras guanyl-releasing protein (RasGRP)1 had no baseline effect on (45)Ca(2+) uptake but significantly attenuated the response to PTH from a 45% increase (6.0 ± 0.2 to 8.7 ± 0.4 nmol·mg(-1)·min(-1)) in control cells to only 20% in knockdown cells (6.1 ± 0.1 to 7.3 ± 0.2 nmol·mg(-1)·min(-1), n = 4, P < 0.01). Inhibition of PKC and PKA resulted in further attenuation of the PTH effect. RasGRP1 knockdown decreased the magnitude of the TRPV5 response to PTH (7.9 ± 0.1 nmol·mg(-1)·min(-1) for knockdown compared with 9.1 ± 0.1 nmol·mg(-1)·min(-1) in control), and the addition of thiazide eliminated this effect (a nearly identical 9.0 ± 0.1 nmol·mg(-1)·min(-1)). This indicates that functionally active NCC is required for RasGRP1 knockdown to impact the PTH effect on TRPV5 activity. Knockdown of with no lysine kinase (WNK)4 resulted in an attenuation of the increase in PTH-mediated TRPV5 activity. TRPV5 activity increased by 36% compared with 45% in control (n = 4, P < 0.01 between PTH-treated groups). PKC blockade further attenuated the PTH effect, whereas combined PKC and PKA blockade in WNK4KD cells abolished the effect. We conclude that modulation of NCC activity contributes to the response to PTH, implying a role for hormonal modulation of NCC activity in distal Ca(2+) handling.

Mechanisms of angiotensin II stimulation of NCC are time-dependent in mDCT15 cells.

Angiotensin II (ANG II) increases thiazide-sensitive sodium-chloride cotransporter (NCC) activity both acutely and chronically. ANG II has been implicated as a switch that turns WNK4 from an inhibitor of NCC into an activator of NCC, and ANG II's effect on NCC appears to require WNK4. Chronically, ANG II stimulation of NCC results in an increase in total and phosphorylated NCC, but the role of NCC phosphorylation in acute ANG II actions is unclear. Here, using a mammalian cell model with robust native NCC activity, we corroborate the role that ANG II plays in WNK4 regulation and clarify the role of Ste20-related proline alanine-rich kinase (SPAK)-induced NCC phosphorylation in ANG II action. ANG II was noted to have a biphasic effect on NCC, with a peak increase in NCC activity in the physiologic range of 10(-11) M ANG II. This effect was apparent as early as 15 min and remained sustained through 120 min. These changes correlated with significant increases in NCC surface protein expression. Knockdown of WNK4 expression sharply attenuated the effect of ANG II. SPAK knockdown did not affect ANG II action at early time points (15 and 30 min), but it did attenuate the response at 60 min. Correspondingly, NCC phosphorylation did not increase at 15 or 30 min, but increased significantly at 60 min. We therefore conclude that within minutes of an increase in ANG II, NCC is rapidly trafficked to the cell surface in a phosphorylation-independent but WNK4-dependent manner. Then, after 60 min, ANG II induces SPAK-dependent phosphorylation of NCC.

Aldosterone acutely stimulates NCC activity via a SPAK-mediated pathway.

Hypertension is a leading cause of morbidity and mortality worldwide, and disordered sodium balance has long been implicated in its pathogenesis. Aldosterone is perhaps the key regulator of sodium balance and thus blood pressure. The sodium chloride cotransporter (NCC) in the distal convoluted tubule of the kidney is a major site of sodium reabsorption and plays a key role in blood pressure regulation. Chronic exposure to aldosterone increases NCC protein expression and function. However, more acute effects of aldosterone on NCC are unknown. In our salt-abundant modern society where chronic salt deprivation is rare, understanding the acute effects of aldosterone is critical. Here, we examined the acute effects (12-36 h) of aldosterone on NCC in the rodent kidney and in a mouse distal convoluted tubule cell line. Studies demonstrated that aldosterone acutely stimulated NCC activity and phosphorylation without affecting total NCC abundance or surface expression. This effect was dependent upon the presence of the mineralocorticoid receptor and serum- and glucocorticoid-regulated kinase 1 (SGK1). Furthermore, STE20/SPS-1-related proline/alanine-rich kinase (SPAK) phosphorylation also increased, and gene silencing of SPAK eliminated the effect of aldosterone on NCC activity. Aldosterone administration via a minipump in adrenalectomized rodents confirmed an increase in NCC phosphorylation without a change in NCC total protein. These data indicate that acute aldosterone-induced SPAK-dependent phosphorylation of NCC increases individual transporter activity.

A new model of the distal convoluted tubule.

The Na(+)-Cl(-) cotransporter (NCC) in the distal convoluted tubule (DCT) of the kidney is a key determinant of Na(+) balance. Disturbances in NCC function are characterized by disordered volume and blood pressure regulation. However, many details concerning the mechanisms of NCC regulation remain controversial or undefined. This is partially due to the lack of a mammalian cell model of the DCT that is amenable to functional assessment of NCC activity. Previously reported investigations of NCC regulation in mammalian cells have either not attempted measurements of NCC function or have required perturbation of the critical without a lysine kinase (WNK)/STE20/SPS-1-related proline/alanine-rich kinase regulatory pathway before functional assessment. Here, we present a new mammalian model of the DCT, the mouse DCT15 (mDCT15) cell line. These cells display native NCC function as measured by thiazide-sensitive, Cl(-)-dependent (22)Na(+) uptake and allow for the separate assessment of NCC surface expression and activity. Knockdown by short interfering RNA confirmed that this function was dependent on NCC protein. Similar to the mammalian DCT, these cells express many of the known regulators of NCC and display significant baseline activity and dimerization of NCC. As described in previous models, NCC activity is inhibited by appropriate concentrations of thiazides, and phorbol esters strongly suppress function. Importantly, they display release of WNK4 inhibition of NCC by small hairpin RNA knockdown. We feel that this new model represents a critical tool for the study of NCC physiology. The work that can be accomplished in such a system represents a significant step forward toward unraveling the complex regulation of NCC.