Design and Discovery of N-(3-(2-(2-Hydroxyethoxy)-6-morpholinopyridin-4-yl)-4-methylphenyl)-2-(trifluoromethyl)isonicotinamide, a Selective, Efficacious, and Well-Tolerated RAF Inhibitor Targeting RAS Mutant Cancers: The Path to the Clinic.

Direct pharmacological inhibition of RAS has remained elusive, and efforts to target CRAF have been challenging due to the complex nature of RAF signaling, downstream of activated RAS, and the poor overall kinase selectivity of putative RAF inhibitors. Herein, we describe 15 (LXH254, Aversa, R.; et al. Int. Patent WO2014151616A1, 2014), a selective B/C RAF inhibitor, which was developed by focusing on drug-like properties and selectivity. Our previous tool compound, 3 (RAF709; Nishiguchi, G. A.; et al. J. Med. Chem. 2017, 60, 4969), was potent, selective, efficacious, and well tolerated in preclinical models, but the high human intrinsic clearance precluded further development and prompted further investigation of close analogues. A structure-based approach led to a pyridine series with an alcohol side chain that could interact with the DFG loop and significantly improved cell potency. Further mitigation of human intrinsic clearance and time-dependent inhibition led to the discovery of 15. Due to its excellent properties, it was progressed through toxicology studies and is being tested in phase 1 clinical trials.

Design, Synthesis, and Preclinical Characterization of Selective Factor D Inhibitors Targeting the Alternative Complement Pathway.

Complement factor D (FD), a highly specific S1 serine protease, plays a central role in the amplification of the alternative complement pathway (AP) of the innate immune system. Dysregulation of AP activity predisposes individuals to diverse disorders such as age-related macular degeneration, atypical hemolytic uremic syndrome, membranoproliferative glomerulonephritis type II, and paroxysmal nocturnal hemoglobinuria. Previously, we have reported the screening efforts and identification of reversible benzylamine-based FD inhibitors (1 and 2) binding to the open active conformation of FD. In continuation of our drug discovery program, we designed compounds applying structure-based approaches to improve interactions with FD and gain selectivity against S1 serine proteases. We report herein the design, synthesis, and medicinal chemistry optimization of the benzylamine series culminating in the discovery of 12, an orally bioavailable and selective FD inhibitor. 12 demonstrated systemic suppression of AP activation in a lipopolysaccharide-induced AP activation model as well as local ocular suppression in intravitreal injection-induced AP activation model in mice expressing human FD.

Discovery of Highly Potent and Selective Small-Molecule Reversible Factor D Inhibitors Demonstrating Alternative Complement Pathway Inhibition in Vivo.

The highly specific S1 serine protease factor D (FD) plays a central role in the amplification of the complement alternative pathway (AP) of the innate immune system. Genetic associations in humans have implicated AP activation in age-related macular degeneration (AMD), and AP dysfunction predisposes individuals to disorders such as paroxysmal nocturnal hemoglobinuria (PNH) and atypical hemolytic uremic syndrome (aHUS). The combination of structure-based hit identification and subsequent optimization of the center (S)-proline-based lead 7 has led to the discovery of noncovalent reversible and selective human factor D (FD) inhibitors with drug-like properties. The orally bioavailable compound 2 exerted excellent potency in 50% human whole blood in vitro and blocked AP activity ex vivo after oral administration to monkeys as demonstrated by inhibition of membrane attack complex (MAC) formation. Inhibitor 2 demonstrated sustained oral and ocular efficacy in a model of lipopolysaccharide (LPS)-induced systemic AP activation in mice expressing human FD.

Allosteric inhibition of SHP2 phosphatase inhibits cancers driven by receptor tyrosine kinases.

The non-receptor protein tyrosine phosphatase SHP2, encoded by PTPN11, has an important role in signal transduction downstream of growth factor receptor signalling and was the first reported oncogenic tyrosine phosphatase. Activating mutations of SHP2 have been associated with developmental pathologies such as Noonan syndrome and are found in multiple cancer types, including leukaemia, lung and breast cancer and neuroblastoma. SHP2 is ubiquitously expressed and regulates cell survival and proliferation primarily through activation of the RAS­ERK signalling pathway. It is also a key mediator of the programmed cell death 1 (PD-1) and B- and T-lymphocyte attenuator (BTLA) immune checkpoint pathways. Reduction of SHP2 activity suppresses tumour cell growth and is a potential target of cancer therapy. Here we report the discovery of a highly potent (IC50 = 0.071 µM), selective and orally bioavailable small-molecule SHP2 inhibitor, SHP099, that stabilizes SHP2 in an auto-inhibited conformation. SHP099 concurrently binds to the interface of the N-terminal SH2, C-terminal SH2, and protein tyrosine phosphatase domains, thus inhibiting SHP2 activity through an allosteric mechanism. SHP099 suppresses RAS­ERK signalling to inhibit the proliferation of receptor-tyrosine-kinase-driven human cancer cells in vitro and is efficacious in mouse tumour xenograft models. Together, these data demonstrate that pharmacological inhibition of SHP2 is a valid therapeutic approach for the treatment of cancers.

Human mannose-binding lectin inhibitor prevents myocardial injury and arterial thrombogenesis in a novel animal model.

Myocardial infarction and coagulation disorders are leading causes of disability and death in the world. An important role of the lectin complement pathway in myocardial infarction and coagulation has been demonstrated in mice genetically deficient in lectin complement pathway proteins. However, these studies are limited to comparisons between wild-type and deficient mice and lack the ability to examine reversal/inhibition of injury after disease establishment. We developed a novel mouse that expresses functional human mannose-binding lectin (MBL) 2 under the control of Mbl1 promoter. Serum MBL2 concentrations averaged approximately 3 µg/mL in MBL2(+/+)Mbl1(-/-)Mbl2(-/-) [MBL2 knock in (KI)] mice. Serum MBL2 level in MBL2 KI mice significantly increased after 7 (8 µg/mL) or 14 (9 µg/mL) days of hyperglycemia compared to normoglycemic mice (P < 0.001). Monoclonal antibody 3F8 inhibited C3 deposition on mannan-coated plates in MBL2 KI, but not wild-type, mice. Myocardial ischemia/reperfusion in MBL2 KI mice revealed that 3F8 preserved cardiac function and decreased infarct size and fibrin deposition in a time-dependent manner. Furthermore, 3F8 prevented ferric chloride-induced occlusive arterial thrombogenesis in vivo. MBL2 KI mice represent a novel animal model that can be used to study the lectin complement pathway in acute and chronic models of human disease. Furthermore, these novel mice demonstrate the therapeutic window for MBL2 inhibition for effective treatment of disease and its complications.

Murine hyperglycemic vasculopathy and cardiomyopathy: whole-genome gene expression analysis predicts cellular targets and regulatory networks influenced by mannose binding lectin.

Hyperglycemia, in the absence of type 1 or 2 diabetes, is an independent risk factor for cardiovascular disease. We have previously demonstrated a central role for mannose binding lectin (MBL)-mediated cardiac dysfunction in acute hyperglycemic mice. In this study, we applied whole-genome microarray data analysis to investigate MBL's role in systematic gene expression changes. The data predict possible intracellular events taking place in multiple cellular compartments such as enhanced insulin signaling pathway sensitivity, promoted mitochondrial respiratory function, improved cellular energy expenditure and protein quality control, improved cytoskeleton structure, and facilitated intracellular trafficking, all of which may contribute to the organismal health of MBL null mice against acute hyperglycemia. Our data show a tight association between gene expression profile and tissue function which might be a very useful tool in predicting cellular targets and regulatory networks connected with in vivo observations, providing clues for further mechanistic studies.

Absence of mannose-binding lectin prevents hyperglycemic cardiovascular complications.

Diabetes, stress, pharmaceuticals, surgery, and physical trauma can lead to hyperglycemic conditions. A consistent relationship has been found between chronic inflammation and the cardiovascular complications of hyperglycemia. We hypothesized that cardiomyopathy and vasculopathy resulting from acute hyperglycemia are dependent on mannose-binding lectin (MBL) and lectin complement pathway activation. Hyperglycemia was induced in wild-type (WT) C57BL/6 and MBL-null mice after streptozotocin administration. Echocardiographic data and tissue samples were collected after 4, 7, or 14 days of acute hyperglycemia. Hyperglycemic WT mice demonstrated dilated cardiomyopathy with significantly increased short and long axis area measurements during systole and diastole compared to hyperglycemic MBL-null mice. The EC(50) for acetylcholine-induced relaxation of mesenteric arterioles in WT mice after 4 days of hyperglycemia demonstrated a significant loss of nitric oxide-mediated relaxation compared to normoglycemic WT or hyperglycemic MBL-null mice. Myocardial histochemistry and Western blot analysis revealed a significant influx of macrophages, altered morphology, and increased elastin and collagen deposition in hyperglycemic WT hearts compared to MBL-null hearts. Serum transforming growth factor-ß1 levels were significantly lower in hyperglycemic MBL-null compared to WT mice, suggesting decreased profibrotic signaling. Together, these data suggest that MBL and the lectin complement pathway play a significant role in vascular dysfunction and cardiomyopathy after acute hyperglycemia.

Mannose-binding lectin-associated serine protease-1 is a significant contributor to coagulation in a murine model of occlusive thrombosis.

Bleeding disorders and thrombotic complications constitute a major cause of death and disability worldwide. Although it is known that the complement and coagulation systems interact, no studies have investigated the specific role or mechanisms of lectin-mediated coagulation in vivo. FeCl(3) treatment resulted in intra-arterial occlusive thrombogenesis within 10 min in wild-type (WT) and C2/factor B-null mice. In contrast, mannose-binding lectin (MBL)-null and MBL-associated serine protease (MASP)-1/-3 knockout (KO) mice had significantly decreased FeCl(3)-induced thrombogenesis. Reconstitution with recombinant human (rh) MBL restored FeCl(3)-induced thrombogenesis in MBL-null mice to levels comparable to WT mice, suggesting a significant role of the MBL/MASP complex for in vivo coagulation. Additionally, whole blood aggregation demonstrated increased MBL/MASP complex-dependent platelet aggregation. In vitro, MBL/MASP complexes were captured on mannan-coated plates, and cleavage of a chromogenic thrombin substrate (S2238) was measured. We observed no significant differences in S2238 cleavage between WT, C2/factor B-null, MBL-A(-/-), or MBL-C(-/-) sera; however, MBL-null or MASP-1/-3 KO mouse sera demonstrated significantly decreased S2238 cleavage. rhMBL alone failed to cleave S2238, but cleavage was restored when rMASP-1 was added to either MASP-1/-3 KO sera or rhMBL. Taken together, these findings indicate that MBL/MASP complexes, and specifically MASP-1, play a key role in thrombus formation in vitro and in vivo.

The mannose-binding lectin pathway is a significant contributor to reperfusion injury in the type 2 diabetic heart.

The severity of ischaemic heart disease is markedly enhanced in type 2 diabetes. We recently reported that complement activation exacerbates I/R injury in the type 2 diabetic heart. The purpose of this study was to isolate and examine MBL pathway activation following I/R injury in the diabetic heart. ZLC and ZDF rats underwent 30 minutes of left coronary artery occlusion followed by 120 minutes of reperfusion. Two different groups of ZDF rats were treated with either FUT-175, a broad complement inhibitor, or P2D5, a monoclonal antibody raised against rat MBL-A. ZDF rats treated with FUT175 and P2D5 had significantly decreased myocardial infarct size, C3 deposition and neutrophil accumulation compared with untreated ZDF controls. Taken together, these findings indicate that the MBL pathway plays a key role in the severity of complement-mediated I/R injury in the type 2 diabetic heart.

Complement inhibition reduces injury in the type 2 diabetic heart following ischemia and reperfusion.

Chronic inflammation exacerbates the cardiovascular complications of diabetes. Complement activation plays an important role in the inflammatory response and is known to be involved in ischemia-reperfusion (I/R) injury in the nondiabetic heart. The purpose of this study was to determine if increased complement deposition explains, in part, the increased severity of neutrophil-mediated I/R injury in the type 2 diabetic heart. Nondiabetic Zucker lean control (ZLC) and Zucker diabetic fatty (ZDF) rats underwent 30 min of coronary artery occlusion followed by 120 min of reperfusion. Another group of ZDF rats was treated with the complement inhibitor FUT-175 before reperfusion. Left ventricular (LV) tissue samples were stained for complement deposition and neutrophil accumulation following reperfusion. We found significantly more complement deposition in the ZDF LV compared with the ZLC (P < 0.05), and complement deposition was associated with significantly greater neutrophil accumulation. In whole blood samples taken preischemia and at 120 min reperfusion, neutrophils exhibited significantly more CD11b expression in the ZDF group compared with the ZLC group (P < 0.05). Furthermore, intracellular adhesion molecule (ICAM)-1 expression following I/R was increased significantly in ZDF hearts compared with ZLC hearts (P < 0.001). These results indicate that, in the ZDF heart, increased ICAM-1 and polymorphonuclear neutrophil (PMN) CD11b expression play a role in increasing PMN accumulation following I/R. The infarct size of the ZDF was significantly greater than ZLC (P < 0.05), and treatment with FUT-175 significantly decreased infarct size, complement deposition, and PMN accumulation in the diabetic heart. These findings indicate an exacerbated inflammatory response in the type 2 diabetic heart that contributes to the increased tissue injury observed following ischemia and reperfusion.

Alterations to myofibrillar protein function in nonischemic regions of the heart early after myocardial infarction.

Remote-zone left ventricular dysfunction (LVD) contributes to global reductions in contractile function after localized myocardial infarction (MI). However, the molecular mechanisms underlying this form of LVD are not clear. This study tested the hypothesis that myofibrillar protein function is directly affected in remote-zone LVD early after MI. Cardiac myosin and native thin filaments were purified from mouse myocardium taken from both the nonnecrotic zone adjacent to and the nonischemic zone remote from an infarct induced by 1 h of coronary occlusion followed by 24 h of reperfusion. Thin filament velocities were measured using the in vitro motility assay. Results showed that overall function was significantly reduced in samples from both the adjacent (43 +/- 12% of control, n = 7) and remote (53 +/- 8% of control, n = 13) zones when compared with control proteins (P < 0.05). Myosin from the remote zone propelled control thin filaments at reduced velocities similar to those measured above. In contrast, the Ca(2+) sensitivity of remote-zone thin filaments over control myosin was unchanged from control thin filaments (half-maximal at pCa 6.32 +/- 0.08 and 6.27 +/- 0.06, respectively) but showed a 20% increase in velocity at saturating Ca(2+) that parallels an increase in tropomyosin phosphorylation. Myosin dysfunction may be related to oxidation of cysteines in the myosin heavy chains or carbonylation of myosin binding protein-C. We hypothesize that phosphorylation of tropomyosin may serve a compensatory role, augmenting contraction during periods of oxidative stress when myosin function is compromised.