Preclinical Characterization of a Next-Generation Brain Permeable, Paradox Breaker BRAF Inhibitor.

PURPOSE: Disease progression in BRAF V600E/K positive melanomas to approved BRAF/MEK inhibitor therapies is associated with the development of resistance mediated by RAF dimer inducing mechanisms. Moreover, progressing disease after BRAFi/MEKi frequently involves brain metastasis. Here we present the development of a novel BRAF inhibitor (Compound Ia) designed to address the limitations of available BRAFi/MEKi. EXPERIMENTAL DESIGN: The novel, brain penetrant, paradox breaker BRAFi is comprehensively characterized in vitro, ex vivo, and in several preclinical in vivo models of melanoma mimicking peripheral disease, brain metastatic disease, and acquired resistance to first-generation BRAFi. RESULTS: Compound Ia manifested elevated potency and selectivity, which triggered cytotoxic activity restricted to BRAF-mutated models and did not induce RAF paradoxical activation. In comparison to approved BRAFi at clinical relevant doses, this novel agent showed a substantially improved activity in a number of diverse BRAF V600E models. In addition, as a single agent, it outperformed a currently approved BRAFi/MEKi combination in a model of acquired resistance to clinically available BRAFi. Compound Ia presents high central nervous system (CNS) penetration and triggered evident superiority over approved BRAFi in a macro-metastatic and in a disseminated micro-metastatic brain model. Potent inhibition of MAPK by Compound Ia was also demonstrated in patient-derived tumor samples. CONCLUSIONS: The novel BRAFi demonstrates preclinically the potential to outperform available targeted therapies for the treatment of BRAF-mutant tumors, thus supporting its clinical investigation.

Cytochrome P450-Mediated Metabolism and CYP Inhibition for the Synthetic Peroxide Antimalarial OZ439.

OZ439 is a potent synthetic ozonide evaluated for the treatment of uncomplicated malaria. The metabolite profile of OZ439 was characterized in vitro using human liver microsomes combined with LC/MS-MS, chemical derivatization, and metabolite synthesis. The primary biotransformations were monohydroxylation at the three distal carbon atoms of the spiroadamantane substructure, with minor contributions from N-oxidation of the morpholine nitrogen and deethylation cleavage of the morpholine ring. Secondary transformations resulted in the formation of dihydroxylation metabolites and metabolites containing both monohydroxylation and morpholine N-oxidation. With the exception of two minor metabolites, none of the other metabolites had appreciable antimalarial activity. Reaction phenotyping indicated that CYP3A4 is the enzyme responsible for the metabolism of OZ439, and it was found to inhibit CYP3A via both direct and mechanism-based inhibition. Elucidation of the metabolic pathways and kinetics will assist with efforts to predict potential metabolic drug-drug interactions and support physiologically based pharmacokinetic (PBPK) modeling.

Synthesis, Characterization, and in vivo Evaluation of a Novel Potent Autotaxin-Inhibitor.

The autotaxin-lysophosphatidic acid (ATX-LPA) signaling pathway plays a role in a variety of autoimmune diseases, such as rheumatoid arthritis or neurodegeneration. A link to the pathogenesis of glaucoma is suggested by an overactive ATX-LPA axis in aqueous humor samples of glaucoma patients. Analysis of such samples suggests that the ATX-LPA axis contributes to the fibrogenic activity and resistance to aqueous humor outflow through the trabecular meshwork. In order to inhibit or modulate this pathway, we developed a new series of ATX-inhibitors containing novel bicyclic and spirocyclic structural motifs. A potent lead compound (IC50 against ATX: 6 nM) with good in vivo PK, favorable in vitro property, and safety profile was generated. This compound leads to lowered LPA levels in vivo after oral administration. Hence, it was suitable for chronic oral treatment in two rodent models of glaucoma, the experimental autoimmune glaucoma (EAG) and the ischemia/reperfusion models. In the EAG model, rats were immunized with an optic nerve antigen homogenate, while controls received sodium chloride. Retinal ischemia/reperfusion (I/R) was induced by elevating the intraocular pressure (IOP) in one eye to 140 mmHg for 60 min, followed by reperfusion, while the other untreated eye served as control. Retinae and optic nerves were evaluated 28 days after EAG or 7 and 14 days after I/R induction. Oral treatment with the optimized ATX-inhibitor lead to reduced retinal ganglion cell (RGC) loss in both glaucoma models. In the optic nerve, the protective effect of ATX inhibition was less effective compared to the retina and only a trend to a weakened neurofilament distortion was detectable. Taken together, these results provide evidence that the dysregulation of the ATX-LPA axis in the aqueous humor of glaucoma patients, in addition to the postulated outflow impairment, might also contribute to RGC loss. The observation that ATX-inhibitor treatment in both glaucoma models did not result in significant IOP increases or decreases after oral treatment indicates that protection from RGC loss due to inhibition of the ATX-LPA axis is independent of an IOP lowering effect.

Role of the autotaxin-lysophosphatidic acid axis in glaucoma, aqueous humor drainage and fibrogenic activity.

Ocular hypertension due to impaired aqueous humor (AH) drainage through the trabecular meshwork (TM) is a major risk factor for glaucoma, a leading cause of irreversible blindness. However, the etiology of ocular hypertension remains unclear. Although autotaxin, a secreted lysophospholipase D and its catalytic product lysophosphatidic acid (LPA) have been shown to modulate AH drainage through TM, we do not have a complete understanding of their role and regulation in glaucoma patients, TM and AH outflow. This study reports a significant increase in the levels of autotaxin, lysophosphatidylcholine (LPC), LPA and connective tissue growth factor (CTGF) in the AH of Caucasian and African American open angle glaucoma patients relative to age-matched non-glaucoma patients. Treatment of human TM cells with dexamethasone, tumor necrosis factor-α (TNF-α) and interleukin-1ß (IL-1ß) increased the levels of autotaxin protein, a response that was mitigated by inhibitors of glucocorticoid receptor, NF-kB and SMAD3. Dexamethasone, TNF-α, IL-1ß and LPC treatment of TM cells also led to an increase in the levels of CTGF, fibronectin and collagen type 1 in an autotaxin dependent manner. Additionally, in perfused enucleated mouse eyes, autotaxin and LPC were noted to decrease, while inhibition of autotaxin was increased aqueous outflow through the TM. Taken together, these results provide additional evidence for dysregulation of the autotaxin-LPA axis in the AH of glaucoma patients, reveal molecular insights into the regulation of autotaxin expression in TM cells and the consequences of autotaxin inhibitors in suppressing the fibrogenic response and resistance to AH outflow through the TM.

Challenges and Rewards in Medicinal Chemistry Targeting Cardiovascular and Metabolic Diseases.

Medicinal chemistry has been transformed by major technological and conceptual innovations over the last three decades: structural biology and bioinformatics, structure and property based molecular design, the concepts of multidimensional optimization (MDO), in silico and experimental high-throughput molecular property analysis. The novel technologies advanced gradually and in synergy with biology and Roche has been at the forefront. Applications in drug discovery programs towards new medicines in cardiovascular and metabolic diseases are highlighted to show impact and advancement: the early discovery of endothelin antagonists for endothelial dysfunction (Bosentan), 11-beta hydroxysteroid dehydrogenase (11ß-HSD1) inhibitors for dysregulated cellular glucocorticoid tonus (type 2 diabetes and metabolic syndrome) and non-covalent hormone sensitive lipase (HSL) inhibitors to study the scope of direct inhibition of lipolysis in the conceptual frame of lipotoxicity and type 2 diabetes.

Challenges and Rewards in Medicinal Chemistry Targeting Cardiovascular and Metabolic Diseases.

Medicinal chemistry has been transformed by major technological and conceptual innovations over the last three decades: structural biology and bioinformatics, structure and property based molecular design, the concepts of multidimensional optimization (MDO), in silico and experimental high-throughput molecular property analysis. The novel technologies advanced gradually and in synergy with biology and Roche has been at the forefront. Applications in drug discovery programs towards new medicines in cardiovascular and metabolic diseases are highlighted to show impact and advancement: the early discovery of endothelin antagonists for endothelial dysfunction (Bosentan), 11-beta hydroxysteroid dehydrogenase (11ß-HSD1) inhibitors for dysregulated cellular glucocorticoid tonus (type 2 diabetes and metabolic syndrome) and non-covalent hormone sensitive lipase (HSL) inhibitors to study the scope of direct inhibition of lipolysis in the conceptual frame of lipotoxicity and type 2 diabetes.

Aryl- and heteroaryl-substituted aminobenzo[a]quinolizines as dipeptidyl peptidase IV inhibitors.

Synthesis and SAR are described for a structurally distinct class of DPP-IV inhibitors based on aminobenzo[a]quinolizines bearing (hetero-)aromatic substituents in the S1 specificity pocket. The m-(fluoromethyl)-phenyl derivative (S,S,S)-2g possesses the best fit in the S1 pocket. However, (S,S,S)-2i, bearing a more hydrophilic 5-methyl-pyridin-2-yl residue as substituent for the S1 pocket, displays excellent in vivo activity and superior drug-like properties.

Characterization of the two major CYP450 metabolites of ozonide (1,2,4-trioxolane) OZ277.

The antimalarial synthetic ozonide OZ277 (RBx11160) was hydroxylated by human liver microsomes at the distal bridgehead carbon atoms of the spiroadamantane substructure to form two carbinol metabolites devoid of antimalarial activity.

1,3-disubstituted 4-aminopiperidines as useful tools in the optimization of the 2-aminobenzo[a]quinolizine dipeptidyl peptidase IV inhibitors.

In a search for novel DPP-IV inhibitors, 2-aminobenzo[a]quinolizines were identified as submicromolar HTS hits. Due to the difficult synthetic access to this compound class, 1,3-disubstituted 4-aminopiperidines were used as model compounds for optimization. The developed synthetic methodology and the SAR could be transferred to the 2-aminobenzo[a]quinolizine series, leading to highly active DPP-IV inhibitors.

Inhibitors of dipeptidyl peptidase IV--recent advances and structural views.

Prevalence of type 2 diabetes has increased dramatically in the last decades. Current medicines are not yet capable to efficiently prevent or reverse progression of the disease and its associated comorbidities. As a consequence, there is a great need for novel antidiabetic drugs. Treatments of type 2 diabetes that are based on enhanced and sustained action of insulinotropic incretin hormones such as GLP-1 have received much attention in the past years. Treatment strategies include administration of: 1) GLP-1 analogues that are resistant to degradation by the serine protease DPP-IV, and 2) small molecule DPP-IV inhibitors that are able to provide sustained action of endogenous GLP-1, again by preventing its degradation. This review summarizes recent research results for the second approach. It briefly touches upon the advantages that treatment of type 2 diabetes with DPP-IV inhibitors may offer over current medications. In the main section, several important structural classes of DPP-IV inhibitors are described and compared based on literature data. Specific attention is given to the analysis of several X-ray structures of enzyme-inhibitor co-crystals. Finally, as clinical data are steadily emerging for some of the most advanced development candidates, the last section of this review is providing a brief overview of some efficacy data from recent clinical studies with DPP-IV inhibitors.

Stereochemical assignment of intermediates in the rifamycin biosynthetic pathway by precursor-directed biosynthesis.

Natural and semisynthetic rifamycins are clinically important inhibitors of bacterial DNA-dependent RNA polymerase. Although the polyketide-nonribosomal peptide origin of the naphthalene core of rifamycin B is well established, the absolute and relative configuration of both stereocenters introduced by the first polyketide synthase module is obscured by aromatization of the naphthalene ring. To decode the stereochemistry of the rifamycin polyketide precursor, we synthesized all four diastereomers of the biosynthetic substrate for module 2 of the rifamycin synthetase in the form of their N-acetylcysteamine (SNAC) thioester. Only one diastereomer was turned over in vivo into rifamycin B, thus establishing the absolute and relative configuration of the native biosynthetic intermediates.

Identification of an antimalarial synthetic trioxolane drug development candidate.

The discovery of artemisinin more than 30 years ago provided a completely new antimalarial structural prototype; that is, a molecule with a pharmacophoric peroxide bond in a unique 1,2,4-trioxane heterocycle. Available evidence suggests that artemisinin and related peroxidic antimalarial drugs exert their parasiticidal activity subsequent to reductive activation by haem, released as a result of haemoglobin digestion by the malaria-causing parasite. This irreversible redox reaction produces carbon-centred free radicals, leading to alkylation of haem and proteins (enzymes), one of which--the sarcoplasmic-endoplasmic reticulum ATPase PfATP6 (ref. 7)--may be critical to parasite survival. Notably, there is no evidence of drug resistance to any member of the artemisinin family of drugs. The chemotherapy of malaria has benefited greatly from the semi-synthetic artemisinins artemether and artesunate as they rapidly reduce parasite burden, have good therapeutic indices and provide for successful treatment outcomes. However, as a drug class, the artemisinins suffer from chemical (semi-synthetic availability, purity and cost), biopharmaceutical (poor bioavailability and limiting pharmacokinetics) and treatment (non-compliance with long treatment regimens and recrudescence) issues that limit their therapeutic potential. Here we describe how a synthetic peroxide antimalarial drug development candidate was identified in a collaborative drug discovery project.

An aminomethylpyrimidine DPP-IV inhibitor with improved properties.

A recently identified DPP-IV inhibitor (1) was found to induce phospholipidosis and to inhibit CYP3A4. A small series of less lipophilic and less amphiphilic analogues was synthesized in an effort to overcome these issues. One compound from this series was equipotent to 1, did not induce phospholipidosis and showed a reduced CYP3A4 inhibition.

Novel ketolide antibiotics with a fused five-membered lactone ring--synthesis, physicochemical and antimicrobial properties.

In an effort to find novel semisynthetic macrolides with extended antibacterial spectrum and improved activity we prepared a series of compounds based on commercially available clarithromycin, a potent and safe antimicrobial agent of outstanding clinical and commercial interest. According to the literature, improvement of antibacterial activity of erythromycin type antibiotics can be achieved by introduction of fused heterocycles such as cyclic carbonates or carbamates at positions 11 and 12 (such as in telithromycin). In the course of the work presented here, a similar, hitherto unprecedented set of compounds bearing a five-membered lactone ring fused to positions 11 and 12 was prepared based on carbon-carbon bond formation via intramolecular Michael addition of a [(hetero)arylalkylthio]acetic acid ester enolate to an alpha,beta-unsaturated ketone as the key step. Some of the ketolide compounds described in this paper were highly active against a representative set of erythromycin sensitive and erythromycin resistant test strains. The best compound showed a similar antimicrobial spectrum and comparable activity in vitro as well as in vivo as telithromycin. Furthermore, some physicochemical properties of these compounds were determined and are presented here. On the basis of these results, the novel ketolide lactones presented in this paper emerged as valuable lead compounds with comparable properties as the commercial ketolide antibacterial telithromycin (Ketek).

A host-vector system for analysis and manipulation of rifamycin polyketide biosynthesis in Amycolatopsis mediterranei.

Modular polyketide synthases (PKSs) are a large family of multifunctional enzymes responsible for the biosynthesis of numerous bacterial natural products such as erythromycin and rifamycin. Advanced genetic analysis of these remarkable systems is often seriously hampered by the large size (>40 kb) of PKS gene clusters, and, notwithstanding their considerable fundamental and biotechnological significance, by the lack of suitable methods for engineering non-selectable modifications in chromosomally encoded PKS genes. The development of a facile host-vector strategy for genetic engineering of the rifamycin PKS in the producing organism, Amycolatopsis mediterranei S699, is described here. The genes encoding all 10 modules of the rifamycin PKS were replaced with a hygromycin-resistance marker gene. In a similar construction, only the first six modules of the PKS were replaced. The deletion hosts retained the ability to synthesize the primer unit 3-amino-5-hydroxybenzoic acid (AHBA), as judged by co-synthesis experiments with a mutant strain lacking AHBA synthase activity. Suicide plasmids carrying a short fragment from the 5' flanking end of the engineered deletion, an apramycin-resistance marker gene, and suitably engineered PKS genes could be introduced via electroporation into the deletion hosts, resulting in the integration of PKS genes and biosynthesis of a reporter polyketide in quantities comparable to those produced by the wild-type organism. Since this strategy for engineering recombinant PKSs in A. mediterranei requires only a selectable single crossover and eliminates the need for tedious non-selectable double-crossover experiments, it makes rifamycin PKS an attractive target for extensive genetic manipulation.