RAF inhibitors that evade paradoxical MAPK pathway activation.

Oncogenic activation of BRAF fuels cancer growth by constitutively promoting RAS-independent mitogen-activated protein kinase (MAPK) pathway signalling. Accordingly, RAF inhibitors have brought substantially improved personalized treatment of metastatic melanoma. However, these targeted agents have also revealed an unexpected consequence: stimulated growth of certain cancers. Structurally diverse ATP-competitive RAF inhibitors can either inhibit or paradoxically activate the MAPK pathway, depending whether activation is by BRAF mutation or by an upstream event, such as RAS mutation or receptor tyrosine kinase activation. Here we have identified next-generation RAF inhibitors (dubbed 'paradox breakers') that suppress mutant BRAF cells without activating the MAPK pathway in cells bearing upstream activation. In cells that express the same HRAS mutation prevalent in squamous tumours from patients treated with RAF inhibitors, the first-generation RAF inhibitor vemurafenib stimulated in vitro and in vivo growth and induced expression of MAPK pathway response genes; by contrast the paradox breakers PLX7904 and PLX8394 had no effect. Paradox breakers also overcame several known mechanisms of resistance to first-generation RAF inhibitors. Dissociating MAPK pathway inhibition from paradoxical activation might yield both improved safety and more durable efficacy than first-generation RAF inhibitors, a concept currently undergoing human clinical evaluation with PLX8394.

Structure-Guided Blockade of CSF1R Kinase in Tenosynovial Giant-Cell Tumor.

BACKGROUND: Expression of the colony-stimulating factor 1 (CSF1) gene is elevated in most tenosynovial giant-cell tumors. This observation has led to the discovery and clinical development of therapy targeting the CSF1 receptor (CSF1R). METHODS: Using x-ray co-crystallography to guide our drug-discovery research, we generated a potent, selective CSF1R inhibitor, PLX3397, that traps the kinase in the autoinhibited conformation. We then conducted a multicenter, phase 1 trial in two parts to analyze this compound. In the first part, we evaluated escalations in the dose of PLX3397 that was administered orally in patients with solid tumors (dose-escalation study). In the second part, we evaluated PLX3397 at the chosen phase 2 dose in an extension cohort of patients with tenosynovial giant-cell tumors (extension study). Pharmacokinetic and tumor responses in the enrolled patients were assessed, and CSF1 in situ hybridization was performed to confirm the mechanism of action of PLX3397 and that the pattern of CSF1 expression was consistent with the pathological features of tenosynovial giant-cell tumor. RESULTS: A total of 41 patients were enrolled in the dose-escalation study, and an additional 23 patients were enrolled in the extension study. The chosen phase 2 dose of PLX3397 was 1000 mg per day. In the extension study, 12 patients with tenosynovial giant-cell tumors had a partial response and 7 patients had stable disease. Responses usually occurred within the first 4 months of treatment, and the median duration of response exceeded 8 months. The most common adverse events included fatigue, change in hair color, nausea, dysgeusia, and periorbital edema; adverse events rarely led to discontinuation of treatment. CONCLUSIONS: Treatment of tenosynovial giant-cell tumors with PLX3397 resulted in a prolonged regression in tumor volume in most patients. (Funded by Plexxikon; ClinicalTrials.gov number, NCT01004861.).

Clinical efficacy of a RAF inhibitor needs broad target blockade in BRAF-mutant melanoma.

B-RAF is the most frequently mutated protein kinase in human cancers. The finding that oncogenic mutations in BRAF are common in melanoma, followed by the demonstration that these tumours are dependent on the RAF/MEK/ERK pathway, offered hope that inhibition of B-RAF kinase activity could benefit melanoma patients. Herein, we describe the structure-guided discovery of PLX4032 (RG7204), a potent inhibitor of oncogenic B-RAF kinase activity. Preclinical experiments demonstrated that PLX4032 selectively blocked the RAF/MEK/ERK pathway in BRAF mutant cells and caused regression of BRAF mutant xenografts. Toxicology studies confirmed a wide safety margin consistent with the high degree of selectivity, enabling Phase 1 clinical trials using a crystalline formulation of PLX4032 (ref. 5). In a subset of melanoma patients, pathway inhibition was monitored in paired biopsy specimens collected before treatment initiation and following two weeks of treatment. This analysis revealed substantial inhibition of ERK phosphorylation, yet clinical evaluation did not show tumour regressions. At higher drug exposures afforded by a new amorphous drug formulation, greater than 80% inhibition of ERK phosphorylation in the tumours of patients correlated with clinical response. Indeed, the Phase 1 clinical data revealed a remarkably high 81% response rate in metastatic melanoma patients treated at an oral dose of 960 mg twice daily. These data demonstrate that BRAF-mutant melanomas are highly dependent on B-RAF kinase activity.

Design and pharmacology of a highly specific dual FMS and KIT kinase inhibitor.

Inflammation and cancer, two therapeutic areas historically addressed by separate drug discovery efforts, are now coupled in treatment approaches by a growing understanding of the dynamic molecular dialogues between immune and cancer cells. Agents that target specific compartments of the immune system, therefore, not only bring new disease modifying modalities to inflammatory diseases, but also offer a new avenue to cancer therapy by disrupting immune components of the microenvironment that foster tumor growth, progression, immune evasion, and treatment resistance. McDonough feline sarcoma viral (v-fms) oncogene homolog (FMS) and v-kit Hardy-Zuckerman 4 feline sarcoma viral oncogene homolog (KIT) are two hematopoietic cell surface receptors that regulate the development and function of macrophages and mast cells, respectively. We disclose a highly specific dual FMS and KIT kinase inhibitor developed from a multifaceted chemical scaffold. As expected, this inhibitor blocks the activation of macrophages, osteoclasts, and mast cells controlled by these two receptors. More importantly, the dual FMS and KIT inhibition profile has translated into a combination of benefits in preclinical disease models of inflammation and cancer.

Scaffold-based discovery of indeglitazar, a PPAR pan-active anti-diabetic agent.

In a search for more effective anti-diabetic treatment, we used a process coupling low-affinity biochemical screening with high-throughput co-crystallography in the design of a series of compounds that selectively modulate the activities of all three peroxisome proliferator-activated receptors (PPARs), PPARalpha, PPARgamma, and PPARdelta. Transcriptional transactivation assays were used to select compounds from this chemical series with a bias toward partial agonism toward PPARgamma, to circumvent the clinically observed side effects of full PPARgamma agonists. Co-crystallographic characterization of the lead molecule, indeglitazar, in complex with each of the 3 PPARs revealed the structural basis for its PPAR pan-activity and its partial agonistic response toward PPARgamma. Compared with full PPARgamma-agonists, indeglitazar is less potent in promoting adipocyte differentiation and only partially effective in stimulating adiponectin gene expression. Evaluation of the compound in vivo confirmed the reduced adiponectin response in animal models of obesity and diabetes while revealing strong beneficial effects on glucose, triglycerides, cholesterol, body weight, and other metabolic parameters. Indeglitazar has now progressed to Phase II clinical evaluations for Type 2 diabetes mellitus (T2DM).

Discovery of a selective inhibitor of oncogenic B-Raf kinase with potent antimelanoma activity.

BRAF(V600E) is the most frequent oncogenic protein kinase mutation known. Furthermore, inhibitors targeting "active" protein kinases have demonstrated significant utility in the therapeutic repertoire against cancer. Therefore, we pursued the development of specific kinase inhibitors targeting B-Raf, and the V600E allele in particular. By using a structure-guided discovery approach, a potent and selective inhibitor of active B-Raf has been discovered. PLX4720, a 7-azaindole derivative that inhibits B-Raf(V600E) with an IC(50) of 13 nM, defines a class of kinase inhibitor with marked selectivity in both biochemical and cellular assays. PLX4720 preferentially inhibits the active B-Raf(V600E) kinase compared with a broad spectrum of other kinases, and potent cytotoxic effects are also exclusive to cells bearing the V600E allele. Consistent with the high degree of selectivity, ERK phosphorylation is potently inhibited by PLX4720 in B-Raf(V600E)-bearing tumor cell lines but not in cells lacking oncogenic B-Raf. In melanoma models, PLX4720 induces cell cycle arrest and apoptosis exclusively in B-Raf(V600E)-positive cells. In B-Raf(V600E)-dependent tumor xenograft models, orally dosed PLX4720 causes significant tumor growth delays, including tumor regressions, without evidence of toxicity. The work described here represents the entire discovery process, from initial identification through structural and biological studies in animal models to a promising therapeutic for testing in cancer patients bearing B-Raf(V600E)-driven tumors.

Association of Combination of Conformation-Specific KIT Inhibitors With Clinical Benefit in Patients With Refractory Gastrointestinal Stromal Tumors: A Phase 1b/2a Nonrandomized Clinical Trial.

IMPORTANCE: Many cancer subtypes, including KIT-mutant gastrointestinal stromal tumors (GISTs), are driven by activating mutations in tyrosine kinases and may initially respond to kinase inhibitors but frequently relapse owing to outgrowth of heterogeneous subclones with resistance mutations. KIT inhibitors commonly used to treat GIST (eg, imatinib and sunitinib) are inactive-state (type II) inhibitors. OBJECTIVE: To assess whether combining a type II KIT inhibitor with a conformation-complementary, active-state (type I) KIT inhibitor is associated with broad mutation coverage and global disease control. DESIGN, SETTING, AND PARTICIPANTS: A highly selective type I inhibitor of KIT, PLX9486, was tested in a 2-part phase 1b/2a trial. Part 1 (dose escalation) evaluated PLX9486 monotherapy in patients with solid tumors. Part 2e (extension) evaluated PLX9486-sunitinib combination in patients with GIST. Patients were enrolled from March 2015 through February 2019; data analysis was performed from May 2020 through July 2020. INTERVENTIONS: Participants received 250, 350, 500, and 1000 mg of PLX9486 alone (part 1) or 500 and 1000 mg of PLX9486 together with 25 or 37.5 mg of sunitinib (part 2e) continuously in 28-day dosing cycles until disease progression, treatment discontinuation, or withdrawal. MAIN OUTCOMES AND MEASURES: Pharmacokinetics, safety, and tumor responses were assessed. Clinical efficacy end points (progression-free survival and clinical benefit rate) were supplemented with longitudinal monitoring of KIT mutations in circulating tumor DNA. RESULTS: A total of 39 PLX9486-naive patients (median age, 57 years [range, 39-79 years]; 22 men [56.4%]; 35 [89.7%] with refractory GIST) were enrolled in the dose escalation and extension parts. The recommended phase 2 dose of PLX9486 was 1000 mg daily. At this dose, PLX9486 could be safely combined with 25 or 37.5 mg daily of sunitinib continuously. Patients with GIST who received PLX9486 at a dose of 500 mg or less, at the recommended phase 2 dose, and with sunitinib had median (95% CI) progression-free survivals of 1.74 (1.54-1.84), 5.75 (0.99-11.0), and 12.1 (1.34-NA) months and clinical benefit rates (95% CI) of 14% (0%-58%), 50% (21%-79%), and 80% (52%-96%), respectively. CONCLUSIONS AND RELEVANCE: In this phase 1b/2a nonrandomized clinical trial, type I and type II KIT inhibitors PLX9486 and sunitinib were safely coadministered at the recommended dose of both single agents in patients with refractory GIST. Results suggest that cotargeting 2 complementary conformational states of the same kinase was associated with clinical benefit with an acceptable safety profile. TRIAL REGISTRATION: ClinicalTrials.gov Identifier: NCT02401815.

Sustained microglial depletion with CSF1R inhibitor impairs parenchymal plaque development in an Alzheimer's disease model.

Many risk genes for the development of Alzheimer's disease (AD) are exclusively or highly expressed in myeloid cells. Microglia are dependent on colony-stimulating factor 1 receptor (CSF1R) signaling for their survival. We designed and synthesized a highly selective brain-penetrant CSF1R inhibitor (PLX5622) allowing for extended and specific microglial elimination, preceding and during pathology development. We find that in the 5xFAD mouse model of AD, plaques fail to form in the parenchymal space following microglial depletion, except in areas containing surviving microglia. Instead, Aß deposits in cortical blood vessels reminiscent of cerebral amyloid angiopathy. Altered gene expression in the 5xFAD hippocampus is also reversed by the absence of microglia. Transcriptional analyses of the residual plaque-forming microglia show they exhibit a disease-associated microglia profile. Collectively, we describe the structure, formulation, and efficacy of PLX5622, which allows for sustained microglial depletion and identify roles of microglia in initiating plaque pathogenesis.

A family of phosphodiesterase inhibitors discovered by cocrystallography and scaffold-based drug design.

Cyclic nucleotide phosphodiesterases (PDEs) comprise a large family of enzymes that regulate a variety of cellular processes. We describe a family of potent PDE4 inhibitors discovered using an efficient method for scaffold-based drug design. This method involves an iterative approach starting with low-affinity screening of compounds followed by high-throughput cocrystallography to reveal the molecular basis underlying the activity of the newly identified compounds. Through detailed structural analysis of the interaction of the initially discovered pyrazole carboxylic ester scaffold with PDE4D using X-ray crystallography, we identified three sites of chemical substitution and designed small selective libraries of scaffold derivatives with modifications at these sites. A 4,000-fold increase in the potency of this PDE4 inhibitor was achieved after only two rounds of chemical synthesis and the structural analysis of seven pyrazole derivatives bound to PDE4B or PDE4D, revealing the robustness of this approach for identifying new inhibitors that can be further developed into drug candidates.

BRD4 Profiling Identifies Critical Chronic Lymphocytic Leukemia Oncogenic Circuits and Reveals Sensitivity to PLX51107, a Novel Structurally Distinct BET Inhibitor.

Bromodomain and extra-terminal (BET) family proteins are key regulators of gene expression in cancer. Herein, we utilize BRD4 profiling to identify critical pathways involved in pathogenesis of chronic lymphocytic leukemia (CLL). BRD4 is overexpressed in CLL and is enriched proximal to genes upregulated or de novo expressed in CLL with known functions in disease pathogenesis and progression. These genes, including key members of the B-cell receptor (BCR) signaling pathway, provide a rationale for this therapeutic approach to identify new targets in alternative types of cancer. Additionally, we describe PLX51107, a structurally distinct BET inhibitor with novel in vitro and in vivo pharmacologic properties that emulates or exceeds the efficacy of BCR signaling agents in preclinical models of CLL. Herein, the discovery of the involvement of BRD4 in the core CLL transcriptional program provides a compelling rationale for clinical investigation of PLX51107 as epigenetic therapy in CLL and application of BRD4 profiling in other cancers.Significance: To date, functional studies of BRD4 in CLL are lacking. Through integrated genomic, functional, and pharmacologic analyses, we uncover the existence of BRD4-regulated core CLL transcriptional programs and present preclinical proof-of-concept studies validating BET inhibition as an epigenetic approach to target BCR signaling in CLL. Cancer Discov; 8(4); 458-77. ©2018 AACR.This article is highlighted in the In This Issue feature, p. 371.

Crystal structures of proto-oncogene kinase Pim1: a target of aberrant somatic hypermutations in diffuse large cell lymphoma.

Pim1, a serine/threonine kinase, is involved in several biological functions including cell survival, proliferation, and differentiation. While pim1 has been shown to be involved in several hematopoietic cancers, it was also recently identified as a target of aberrant somatic hypermutation in diffuse large cell lymphoma (DLCL), the most common form of non-Hodgkin's lymphoma. The crystal structures of Pim1 in apo form and bound with AMPPNP have been solved and several unique features of Pim1 were identified, including the presence of an extra beta-hairpin in the N-terminal lobe and an unusual conformation of the hinge connecting the two lobes of the enzyme. While the apo Pim1 structure is nearly identical with that reported recently, the structure of AMPPNP bound to Pim1 is significantly different. Pim1 is unique among protein kinases due to the presence of a proline residue at position 123 that precludes the formation of the canonical second hydrogen bond between the hinge backbone and the adenine moiety of ATP. One crystal structure reported here shows that changing P123 to methionine, a common residue that offers the backbone hydrogen bond to ATP, does not restore the ATP binding pocket of Pim1 to that of a typical kinase. These unique structural features in Pim1 result in novel binding modes of AMP and a known kinase inhibitor scaffold, as shown by co-crystallography. In addition, the kinase activities of five Pim1 mutants identified in DLCL patients have been determined. In each case, the observed effects on kinase activity are consistent with the predicted consequences of the mutation on the Pim1 structure. Finally, 70 co-crystal structures of low molecular mass, low-affinity compounds with Pim1 have been solved in order to identify novel chemical classes as potential Pim1 inhibitors. Based on the structural information, opportunities for optimization of one specific example are discussed.

Structural basis for the activity of drugs that inhibit phosphodiesterases.

Phosphodiesterases (PDEs) comprise a large family of enzymes that catalyze the hydrolysis of cAMP or cGMP and are implicated in various diseases. We describe the high-resolution crystal structures of the catalytic domains of PDE4B, PDE4D, and PDE5A with ten different inhibitors, including the drug candidates cilomilast and roflumilast, for respiratory diseases. These cocrystal structures reveal a common scheme of inhibitor binding to the PDEs: (i) a hydrophobic clamp formed by highly conserved hydrophobic residues that sandwich the inhibitor in the active site; (ii) hydrogen bonding to an invariant glutamine that controls the orientation of inhibitor binding. A scaffold can be readily identified for any given inhibitor based on the formation of these two types of conserved interactions. These structural insights will enable the design of isoform-selective inhibitors with improved binding affinity and should facilitate the discovery of more potent and selective PDE inhibitors for the treatment of a variety of diseases.

Characterizing and Overriding the Structural Mechanism of the Quizartinib-Resistant FLT3 "Gatekeeper" F691L Mutation with PLX3397.

UNLABELLED: Tyrosine kinase domain mutations are a common cause of acquired clinical resistance to tyrosine kinase inhibitors (TKI) used to treat cancer, including the FLT3 inhibitor quizartinib. Mutation of kinase "gatekeeper" residues, which control access to an allosteric pocket adjacent to the ATP-binding site, has been frequently implicated in TKI resistance. The molecular underpinnings of gatekeeper mutation-mediated resistance are incompletely understood. We report the first cocrystal structure of FLT3 with the TKI quizartinib, which demonstrates that quizartinib binding relies on essential edge-to-face aromatic interactions with the gatekeeper F691 residue, and F830 within the highly conserved Asp-Phe-Gly motif in the activation loop. This reliance makes quizartinib critically vulnerable to gatekeeper and activation loop substitutions while minimizing the impact of mutations elsewhere. Moreover, we identify PLX3397, a novel FLT3 inhibitor that retains activity against the F691L mutant due to a binding mode that depends less vitally on specific interactions with the gatekeeper position. SIGNIFICANCE: We report the first cocrystal structure of FLT3 with a kinase inhibitor, elucidating the structural mechanism of resistance due to the gatekeeper F691L mutation. PLX3397 is a novel FLT3 inhibitor with in vitro activity against this mutation but is vulnerable to kinase domain mutations in the FLT3 activation loop.

Improved human bioavailability of vemurafenib, a practically insoluble drug, using an amorphous polymer-stabilized solid dispersion prepared by a solvent-controlled coprecipitation process.

The present work deals with improving the solubility of vemurafenib, a practically insoluble drug, by converting it into an amorphous-solid dispersion using a solvent-controlled precipitation process. The dispersion containing vemurafenib and hypromellose acetate succinate (HPMCAS), an enteric polymer, is termed microprecipitated bulk powder (MBP), in which the drug is uniformly dispersed within the polymeric substrate. HPMCAS was found to be the most suitable polymer for vemurafenib MBP, among a series of enteric polymers based on superior physical stability and drug-release characteristics of the MBP. The MBP provided a greater rate and extent of dissolution than crystalline drug, reaching an apparent drug concentration of 28-35 µg/mL, almost 30-fold higher than solubility of crystalline drug at 1 µg/mL. The supersaturation was also maintained for more than 4 h. Upon exposure to high temperature and humidity, the MBP was destabilized, resulting in crystallization and lower dissolution rate. The control of moisture and temperature is essential to maintain the stability of the MBP. In a relative human bioavailability study, vemurafenib MBP provided a four- to fivefold increase in exposure compared with crystalline drug. Improving solubility with an amorphous-solid dispersion is a viable strategy for the development of practically insoluble compounds.

Vemurafenib: the first drug approved for BRAF-mutant cancer.

The identification of driver oncogenes has provided important targets for drugs that can change the landscape of cancer therapies. One such example is the BRAF oncogene, which is found in about half of all melanomas as well as several other cancers. As a druggable kinase, oncogenic BRAF has become a crucial target of small-molecule drug discovery efforts. Following a rapid clinical development path, vemurafenib (Zelboraf; Plexxikon/Roche) was approved for the treatment of BRAF-mutated metastatic melanoma in the United States in August 2011 and the European Union in February 2012. This Review describes the underlying biology of BRAF, the technology used to identify vemurafenib and its clinical development milestones, along with future prospects based on lessons learned during its development.

Phosphodiesterase-4 as a potential drug target.

Phosphodiesterase-4 (PDE4) is the predominant enzyme in some specific cell types that is responsible for the degradation of the second messenger, cAMP. Consequently, PDE4 plays a crucial role in cell signalling and, as such, it has been the target of clinical drug development of various indications, ranging from anti-inflammation to memory enhancement. In this review, the fundamental biological role of PDE4 in intracellular signalling, its tissue distribution and regulation are described. The historical development of various chemical classes of PDE4 inhibitors and the challenges that face these inhibitors as therapeutics are also discussed. Finally, recent advances in the structural biology of PDE4 and their complexes with various inhibitors, as well as its potential impact on the rational design of potent and selective PDE4 inhibitors, are presented.

Affinity and intrinsic efficacy (IE) of 5'-carbamoyl adenosine analogues for the A1 adenosine receptor--efforts towards the discovery of a chronic ventricular rate control agent for the treatment of atrial fibrillation (AF).

The SAR for the affinity to the A(1) adenosine receptor and relative intrinsic efficacy (IE, [(35)S]-GTPgammaS binding) of a series of 5'-carbamate and 5'-thionocarbamate derivatives of tecadenoson is described. Based on this SAR, selected compounds were evaluated in guinea pig isolated hearts to determine whether they were partial or full agonists with respect to their negative dromotropism, an A(1) AdoR mediated effect. Progress towards obtaining a partial A(1) AdoR agonist to potentially control ventricular rate during atrial fibrillation has been made with the discovery of several potent partial A(1) AdoR agonists (compounds 13, 14, and 17).

Structure-affinity relationships of 5'-aromatic ethers and 5'-aromatic sulfides as partial A1 adenosine agonists, potential supraventricular anti-arrhythmic agents.

Atrial fibrillation (AF) is the most commonly encountered sustained clinical arrhythmia with an estimated 2.3 million cases in the US (2001). A(1) adenosine receptor agonists can slow the electrical impulse propagation through the atrioventricular (AV) node (i.e., negative dromotropic effect) resulting in prolongation of the stimulus-to-His bundle (S-H) interval to potentially reduce ventricular rate. Compounds that are full agonists of the A(1) adenosine receptor can cause high grade AV block. Therefore, it is envisioned that a compound that is a partial agonist of the A(1) adenosine receptor could avoid this deleterious effect. 5(') Phenyl sulfides (e.g., 17, EC(50)=1.26 microM) and phenyl ethers (e.g., 28, EC(50)=0.2 microM) are partial agonists with respect to their AV nodal effects in guinea pig isolated hearts. Additional affinity, GTPgammaS binding data suggesting partial activity of the A(1) adenosine receptor, and PK results for 5(') modified adenosine derivatives are shown.

Novel inhibitors of fatty acid oxidation as potential metabolic modulators.

We describe the synthesis of novel inhibitors of fatty acid oxidation as potential metabolic modulators for the treatment of stable angina. Replacement of the 2H-benzo[d]1,3-dioxolene ring system in our initial lead 3 with different benzthiazoles, benzoxazoles and introducing small alkyl substituents into the piperazine ring resulted in analogues with enhanced inhibitory activity against 1-(14)[C]-palmitoyl-CoA oxidation in isolated rat heart mitochondria (6, IC(50)=70 nM; 25, IC(50)=23 nM).

CVT-4325: a potent fatty acid oxidation inhibitor with favorable oral bioavailability.

New inhibitors of palmitoyl-CoA oxidation are based on the introduction of nitrogen heterocycles in the 'Western Portion' of the molecule. SAR studies led to the discovery of CVT-4325 (shown), a potent FOXi (IC50=380 nM rat mitochondria) with favorable PK properties (F=93%, t(1/2)=13.6h, dog).