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Non–small cell lung cancer patients carrying oncogenic EGFR mutations initially respond to EGFR-targeted therapy, but later elicit minimal response due to dose-limiting toxicities and acquired resistance. EGF816 is a novel, irreversible mutant-selective EGFR inhibitor that specifically targets EGFR-activating mutations arising de novo and upon resistance acquisition, while sparing wild-type (WT) EGFR. EGF816 potently inhibited the most common EGFR mutations L858R, Ex19del, and T790M in vitro, which translated into strong tumor regressions in vivo in several patient-derived xenograft models. Notably, EGF816 also demonstrated antitumor activity in an exon 20 insertion mutant model. At levels above efficacious doses, EGF816 treatment led to minimal inhibition of WT EGFR and was well tolerated. In single-dose studies, EGF816 provided sustained inhibition of EGFR phosphorylation, consistent with its ability for irreversible binding. Furthermore, combined treatment with EGF816 and INC280, a cMET inhibitor, resulted in durable antitumor efficacy in a xenograft model that initially developed resistance to first-generation EGFR inhibitors via cMET activation. Thus, we report the first preclinical characterization of EGF816 and provide the groundwork for its current evaluation in phase I/II clinical trials in patients harboring EGFR mutations, including T790M. Cancer Res; 76(6); 1591–602. ©2016 AACR.

This file contains the supplementary Table S1 that describes the characteristics and anti-proliferative activities of EGF816 on a number of patient-derived cell lines

This file contains additional Materials and Methods that are not listed in the main text.

This file shows the Supplementary figures. Figure S1 shows the chemical structure of EGF816 and its binding to EGFR T790M mutant. Figure S2 shows the tolerability of EGF816 in the in vivo xenograft models. Figure S3 shows the in vivo PK and PD after a single dose of EGF816. Figure S4 shows the selectivity profile of EGF816 in the kinome tree. Figure S5 shows the morphology of EGF816 resistance clones.

This file contains the supplementary figure legends. Figure S1 shows the chemical structure of EGF816 and its binding to EGFR T790M mutant. Figure S2 shows the tolerability of EGF816 in the in vivo xenograft models. Figure S3 shows the in vivo PK and PD after a single dose of EGF816. Figure S4 shows the selectivity profile of EGF816 in the kinome tree. Figure S5 shows the morphology of EGF816 resistance clones.

N 6 -methyladenosine (m 6 A) regulates mRNA metabolism. While it has been implicated in the development of the mammalian brain and in cognition, the role of m 6 A in synaptic plasticity, especially during cognitive decline, is not fully understood. In this study, we employed methylated RNA immunoprecipitation sequencing to obtain the m 6 A epitranscriptome of the hippocampal subregions CA1, CA3, and the dentate gyrus and the anterior cingulate cortex (ACC) in young and aged mice. We observed a decrease in m 6 A levels in aged animals. Comparative analysis of cingulate cortex (CC) brain tissue from cognitively intact human subjects and Alzheimer’s disease (AD) patients showed decreased m 6 A RNA methylation in AD patients. m 6 A changes common to brains of aged mice and AD patients were found in transcripts linked to synaptic function including calcium/calmodulin-dependent protein kinase 2 ( CAMKII ) and AMPA-selective glutamate receptor 1 ( Glua1 ). We used proximity ligation assays to show that reduced m 6 A levels result in decreased synaptic protein synthesis as exemplified by CAMKII and GLUA1. Moreover, reduced m 6 A levels impaired synaptic function. Our results suggest that m 6 A RNA methylation controls synaptic protein synthesis and may play a role in cognitive decline associated with aging and AD.

[Image: see text] The selective inhibition of RET kinase as a treatment for relevant cancer types including lung adenocarcinoma has garnered considerable interest in recent years and prompted a variety of efforts toward the discovery of small-molecule therapeutics. Hits uncovered via the analysis of archival kinase data ultimately led to the identification of a promising pyrrolo[2,3-d]pyrimidine scaffold. The optimization of this pyrrolo[2,3-d]pyrimidine core resulted in compound 1, which demonstrated potent in vitro RET kinase inhibition and robust in vivo efficacy in RET-driven tumor xenografts upon multiday dosing in mice. The administration of 1 was well-tolerated at established efficacious doses (10 and 30 mg/kg, po, qd), and plasma exposure levels indicated a minimal risk of KDR or hERG inhibition in vivo, as evaluated by Miles assay and free plasma concentrations, respectively.

Infantile-onset RNaseT2 deficient leukoencephalopathy is characterised by cystic brain lesions, multifocal white matter alterations, cerebral atrophy, and severe psychomotor impairment. The phenotype is similar to congenital cytomegalovirus brain infection and overlaps with type I interferonopathies, suggesting a role for innate immunity in its pathophysiology. To date, pathophysiological studies have been hindered by the lack of mouse models recapitulating the neuroinflammatory encephalopathy found in patients. In this study, we generated Rnaset2−/− mice using CRISPR/Cas9-mediated genome editing. Rnaset2−/− mice demonstrate upregulation of interferon-stimulated genes and concurrent IFNAR1-dependent neuroinflammation, with infiltration of CD8+ effector memory T cells and inflammatory monocytes into the grey and white matter. Single nuclei RNA sequencing reveals homeostatic dysfunctions in glial cells and neurons and provide important insights into the mechanisms of hippocampal-accentuated brain atrophy and cognitive impairment. The Rnaset2−/− mice may allow the study of CNS damage associated with RNaseT2 deficiency and may be used for the investigation of potential therapies., Studies on interferon-driven brain pathology have so far been hampered by the lack of appropriate animal models. Here the authors characterize RNASET2-deficient mice and show that neuroinflammation and brain atrophy are IFNAR1-dependent.

N6-methyladenosine (m6A) plays diverse roles in the regulation of mRNA metabolism. In the mammalian brain it has been linked to developmental processes and memory function. However, the precise role of m6A in the context synaptic plasticity and especially during impaired cognition are not fully understood. Here, we describe the mouse and human brain m6A epi-transcriptome in a tissue-specific manner. We furthermore show that m6A levels undergo a massive decrease across mouse brain regions as a consequence of aging. In addition, Alzheimer's disease in humans correlates with decreased N6-methylation in a similar population of transcripts that are linked to synaptic function and localized to synapses, such as the calcium/calmodulin-dependent kinase II (CaMKII). We furthermore show that reduced m6A levels impair synaptic protein-synthesis of CAMKII. Our results suggest that m6A-RNA-methylation is an important mechanism to control synaptic protein synthesis which is affected early in cognitive diseases.Significance statementThe addition of N6-methyladenosine (m6A) to RNA plays a role in various cellular processes and its de-regulation has been linked to several devastating diseases. The precise role of m6A RNA-methylation in the adult brain is, however, not well understood. In our study, we describe the genome-wide m6A epi-transcriptome in the healthy and diseased brains of mice and humans. Our data demonstrate that a substantial amount of m6A transcripts are conserved. These transcripts are linked to the regulation of synaptic processes and are localized to synapses. In the diseases brain we detect RNA hypomethylation across multiple transcripts in all investigated brain regions and across species. At the mechanistic level we find that reduced m6A levels specifically impairs synaptic protein-synthesis.

RET (REarranged during Transfection) kinase gain-of-function aberrancies have been identified as potential oncogenic drivers in lung adenocarcinoma, along with several other cancer types, prompting the discovery and assessment of selective inhibitors. Internal mining and analysis of relevant kinase data informed the decision to investigate a pyrazolo[1,5-a]pyrimidine scaffold, where subsequent optimization led to the identification of compound WF-47-JS03 (1), a potent RET kinase inhibitor with >500-fold selectivity against KDR (Kinase insert Domain Receptor) in cellular assays. In subsequent mouse in vivo studies, compound 1 demonstrated effective brain penetration and was found to induce strong regression of RET-driven tumor xenografts at a well-tolerated dose (10 mg/kg, po, qd). Higher doses of 1, however, were poorly tolerated in mice, similar to other pyrazolo[1,5-a]pyrimidine compounds at or near the efficacious dose, and indicative of the narrow therapeutic windows seen with this scaffold.

[Image: see text] SPPL2a (Signal Peptide Peptidase Like 2a) is an intramembrane aspartyl protease engaged in the function of B-cells and dendritic cells. Despite being an attractive target for modulation of the immune system, selective SPPL2a inhibitors are barely described in the literature. Recently, we have disclosed a selective, small molecular weight agent SPL-707 which confirmed that pharmacological inhibition of SPPL2a leads to the accumulation of its substrate CD74/p8 and as a consequence to a reduction in the number of B-cells as well as myeloid dendritic cells in mice. In this paper we describe the discovery of novel hydroxyethylamine based SPPL2a inhibitors. Starting from a rather lipophilic screening hit, several iterative optimization cycles allowed for its transformation into a highly potent and selective compound 15 (SPL-410) which inhibited in vivo CD74/p8 fragment processing in mice at 10 mg/kg oral dose.

Neurons are highly compartmentalized cells that depend on local protein synthesis. Thus, messenger RNAs (mRNAs) have been detected in neuronal dendrites and more recently also at the pre- and postsynaptic compartment. Other RNA species, such as microRNAs, have also been described at synapses where they are believed to control mRNA availability for local translation. Nevertheless, a combined dataset analyzing the synaptic coding and non-coding RNAome via next-generation sequencing approaches is missing. Here we isolate synaptosomes from the hippocampus of young wild type mice and provide the coding and non-coding synaptic RNAome. These data are complemented by a novel approach to analyze the synaptic RNAome from primary hippocampal neurons grown in microfluidic chambers. Our data show that synaptic microRNAs control almost the entire synaptic mRNAome and we identified several hub microRNAs. By combining the in vivo synaptosomal data with our novel microfluidic chamber system, we also provide evidence to support the hypothesis that part of the synaptic microRNAome may be supplied to neurons via astrocytes. Moreover, the microfluidic system is suitable to study the dynamics of the synaptic RNAome in response to stimulation. In conclusion, our data provide a valuable resource and hint to several important targets for future experiments.

Neurons are highly compartmentalized cells that depend on local protein synthesis. Messenger RNAs (mRNAs) have thus been detected in neuronal dendrites, and more recently in the pre- and postsynaptic compartments as well. Other RNA species such as microRNAs have also been described at synapses where they are believed to control mRNA availability for local translation. A combined dataset analyzing the synaptic coding and non-coding RNAome via next-generation sequencing approaches is, however, still lacking. Here, we isolate synaptosomes from the hippocampus of young wild-type mice and provide the coding and non-coding synaptic RNAome. These data are complemented by a novel approach for analyzing the synaptic RNAome from primary hippocampal neurons grown in microfluidic chambers. Our data show that synaptic microRNAs control almost the entire synaptic mRNAome, and we identified several hub microRNAs. By combining the in vivo synaptosomal data with our novel microfluidic chamber system, our findings also support the hypothesis that part of the synaptic microRNAome may be supplied to neurons via astrocytes. Moreover, the microfluidic system is suitable for studying the dynamics of the synaptic RNAome in response to stimulation. In conclusion, our data provide a valuable resource and point to several important targets for further research. Supplementary Information The online version contains supplementary material available at 10.1007/s12035-021-02296-y.

Signal peptide peptidase-like 2a (SPPL2a) is an aspartic intramembrane protease which has recently been shown to play an important role in the development and function of antigen presenting cells such as B lymphocytes and dendritic cells. In this paper, we describe the discovery of the first selective and orally active SPPL2a inhibitor (S)-2-cyclopropyl-N1-((S)-5,11-dioxo-10,11-dihydro-1H,3H,5H-spiro[benzo[d]pyrazolo[1,2-a][1,2]diazepine-2,1'-cyclopropan]-10-yl)-N4-(5-fluoro-2-methylpyridin-3-yl)succinamide 40 (SPL-707). This compound shows adequate selectivity against the closely related enzymes γ-secretase and SPP and a good pharmacokinetic profile in mouse and rat. Compound 40 significantly inhibited processing of the SPPL2a substrate CD74/p8 fragment in rodents at doses ≤10 mg/kg b.i.d. po. Oral dosing of 40 for 11 days at ≥10 mg/kg b.i.d. recapitulated the phenotype seen in Sppl2a knockout (ko) and ENU mutant mice (reduced number of specific B cells and myeloid dendritic cells). Thus, we believe that SPPL2a represents an interesting and druggable pharmacological target, potentially providing a novel approach for the treatment of autoimmune diseases by targeting B cells and dendritic cells.

Summary The ability to plan and execute appropriately timed responses to external stimuli is based on a well-orchestrated balance between movement initiation and inhibition. In impulse control disorders involving the prefrontal cortex (PFC) [1], this balance is disturbed, emphasizing the critical role that PFC plays in appropriately timing actions [2–4]. Here, we employed optogenetic and electrophysiological techniques to systematically analyze the functional role of five key subareas of the rat medial PFC (mPFC) and orbitofrontal cortex (OFC) in action control [5–9]. Inactivation of mPFC subareas induced drastic changes in performance, namely an increase (prelimbic cortex, PL) or decrease (infralimbic cortex, IL) of premature responses. Additionally, electrophysiology revealed a significant decrease in neuronal activity of a PL subpopulation prior to premature responses. In contrast, inhibition of OFC subareas (mainly the ventral OFC, i.e., VO) significantly impaired the ability to respond rapidly after external cues. Consistent with these findings, mPFC activity during response preparation predicted trial outcomes and reaction times significantly better than OFC activity. These data support the concept of opposing roles of IL and PL in directing proactive behavior and argue for an involvement of OFC in predominantly reactive movement control. By attributing defined roles to rodent PFC sections, this study contributes to a deeper understanding of the functional heterogeneity of this brain area and thus may guide medically relevant studies of PFC-associated impulse control disorders in this animal model for neural disorders [10–12].

A Correction to this paper has been published: 10.1007/s12035-021-02349-2

Over the past decade, first and second generation EGFR inhibitors have significantly improved outcomes for lung cancer patients with activating mutations in EGFR. However, both resistance through a secondary T790M mutation at the gatekeeper residue and dose-limiting toxicities from wild-type (WT) EGFR inhibition ultimately limit the full potential of these therapies to control mutant EGFR-driven tumors and new therapies are urgently needed. Herein, we describe our approach toward the discovery of 47 (EGF816, nazartinib), a novel, covalent mutant-selective EGFR inhibitor with equipotent activity on both oncogenic and T790M-resistant EGFR mutations. Through molecular docking studies we converted a mutant-selective high-throughput screening hit (7) into a number of targeted covalent EGFR inhibitors with equipotent activity across mutants EGFR and good WT-EGFR selectivity. We used an abbreviated in vivo efficacy study for prioritizing compounds with good tolerability and efficacy that ultimately led to the se...

Over the past decade, first and second generation EGFR inhibitors have significantly improved outcomes for lung cancer patients with activating mutations in EGFR. However, both resistance through a secondary T790M mutation at the gatekeeper residue and dose-limiting toxicities from wild-type (WT) EGFR inhibition ultimately limit the full potential of these therapies to control mutant EGFR-driven tumors and new therapies are urgently needed. Herein, we describe our approach toward the discovery of 47 (EGF816, nazartinib), a novel, covalent mutant-selective EGFR inhibitor with equipotent activity on both oncogenic and T790M-resistant EGFR mutations. Through molecular docking studies we converted a mutant-selective high-throughput screening hit (7) into a number of targeted covalent EGFR inhibitors with equipotent activity across mutants EGFR and good WT-EGFR selectivity. We used an abbreviated in vivo efficacy study for prioritizing compounds with good tolerability and efficacy that ultimately led to the selection of 47 as the clinical candidate.

The discovery, synthesis, and optimization of compound 1 from a high-throughput screening hit to highly potent and selective peroxisome proliferator-activated receptor delta (PPARdelta) agonists are reported. The synthesis and structure-activity relationship in this series are described in detail. On the basis of a general schematic PPAR pharmacophore model, scaffold 1 was divided into headgroup, linker, and tailgroup and successively optimized for PPAR activation using in vitro PPAR transactivation assays. A (2-methylphenoxy)acetic acid headgroup, a flexible linker, and a five-membered heteroaromatic center ring with two hydrophobic aryl substituents were required for efficient and selective PPARdelta activation. The fine-tuning of these aryl substituents led to an array of highly potent and selective compounds such as compound 38c, displaying an excellent pharmacokinetic profile in mouse. In an in vivo acute dosing model, selected members of this array were shown to induce the expression of pyruvate dehydrogenase kinase-4 (PDK4) and uncoupling protein-3 (UCP3), genes that are known to be involved in energy homeostasis and regulated by PPARdelta in skeletal muscle.

The epidermal growth factor receptor (EGFR)-directed tyrosine kinase inhibitors (TKIs) gefitinib, erlotinib and afatinib are approved treatments for non-small cell lung cancers harbouring activating mutations in the EGFR kinase, but resistance arises rapidly, most frequently owing to the secondary T790M mutation within the ATP site of the receptor. Recently developed mutant-selective irreversible inhibitors are highly active against the T790M mutant, but their efficacy can be compromised by acquired mutation of C797, the cysteine residue with which they form a key covalent bond. All current EGFR TKIs target the ATP-site of the kinase, highlighting the need for therapeutic agents with alternative mechanisms of action. Here we describe the rational discovery of EAI045, an allosteric inhibitor that targets selected drug-resistant EGFR mutants but spares the wild-type receptor. The crystal structure shows that the compound binds an allosteric site created by the displacement of the regulatory C-helix in an inactive conformation of the kinase. The compound inhibits L858R/T790M-mutant EGFR with low-nanomolar potency in biochemical assays. However, as a single agent it is not effective in blocking EGFR-driven proliferation in cells owing to differential potency on the two subunits of the dimeric receptor, which interact in an asymmetric manner in the active state. We observe marked synergy of EAI045 with cetuximab, an antibody therapeutic that blocks EGFR dimerization, rendering the kinase uniformly susceptible to the allosteric agent. EAI045 in combination with cetuximab is effective in mouse models of lung cancer driven by EGFR(L858R/T790M) and by EGFR(L858R/T790M/C797S), a mutant that is resistant to all currently available EGFR TKIs. More generally, our findings illustrate the utility of purposefully targeting allosteric sites to obtain mutant-selective inhibitors.

A series of PPARdelta-selective agonists was investigated and optimized for a favorable in vivo pharmacokinetic profile. Isoxazole LCI765 (17d) was found to be a potent and selective PPARdelta agonist with good in vivo PK properties in mouse (C(max)=5.1 microM, t(1/2)=3.1 h). LCI765 regulated expression of genes involved in energy homeostasis in relevant tissues when dosed orally in C57BL6 mice. A co-crystal structure of compound LCI765 and the LBD of PPARdelta is discussed.

The synthesis and structure-activity relationship of a series of arylaminoethyl amide cathepsin S inhibitors are reported. Optimization of P3 and P2 groups to improve overall physicochemical properties resulted in significant improvements in oral bioavailability over early lead compounds. An X-ray structure of compound 37 bound to the active site of cathepsin S is also reported.

A series of N α -2-benzoxazolyl-α-amino acid-(arylaminoethyl)amides were identified as potent, selective, and noncovalent inhibitors of cathepsin S. Structure–activity relationships including strategies for modulating the selectivities among cathepsins S, K, and L, and in vivo pharmacokinetics are discussed. A X-ray structure of compound 3 bound to the active site of cathepsin S is also reported.

A series of Nα-acyl-α-amino acid-(arylaminoethyl)amides were found to be potent and noncovalent cathepsin S inhibitors. Compound 20 possessed high cathepsin S affinity (Ki = 3.3 nM) and showed excellent selectivity over cathepsin K, L, F, and V. Molecular modeling, design, synthesis, and in vitro activity are described.

A screen of over 500 diversely functionalized additives in osmium-catalyzed dihydroxylation has uncovered that electron-deficient olefins are converted into the corresponding diols much more efficiently when the pH of the reaction medium is maintained on the acidic side. Further studies have identified citric acid as the additive of choice, for it allows preparation of very pure diols in yields generally exceeding 90%. As described here, a much wider range of olefin classes can now be successfully dihydroxylated. The process is experimentally simple, in most cases involving little more than dissolving the reactants in water or a water/tert-butyl alcohol mixture, stirring them, and filtering off the pure diol product.

Systematic SAR optimization of the GPR119 agonist lead 1, derived from an internal HTS campaign, led to compound 29. Compound 29 displays significantly improved in vitro activity and oral exposure, leading to GLP1 elevation in acutely dosed mice and reduced glucose excursion in an OGTT study in rats at doses ⩾10 mg/kg.

Screening hit 5 was identified in a biochemical screen for GPR119 agonists. Compound 5 was structurally novel, displayed modest biochemical activity and no oral exposure, but was structurally distinct from typical GPR119 agonist scaffolds. Systematic optimization led to compound 36 with significantly improved in vitro activity and oral exposure, to elevate GLP1 acutely in an in vivo mouse model at a dose of 10mg/kg.

DNA photolyases are repair enzymes which split (repair) UV-induced cyclobutane DNA lesions. Critical steps in the light-driven repair reaction are the absorption of light by a deazaflavin or methen...

DNA photolyases are enzymes that catalyze the light-dependend repair of cis-syn-cyclobutane-thymine dimer UV lesions in a variety of organisms. The basis of the repairreaction is an electron transfer from a reduced and deprotonated flavin cofactor to the dimer unit, which splits spontaneously as its radical anion. A second cofactor, which is either an 8-hydroxy-5-deazaflavin or a methenyl-tetrahydrofolate is required as a photo antenna and ensures efficient light absorption. With the help of model compounds that are able to mimic all crucial steps of the repair reaction, detailed mechanistic insights into the repair reaction could be obtained. It became clear, that the enzyme requires the reduced flavin in its deprotonated form and that the repair reaction proceeds most efficiently in polar media, which is in agreement with the observed highly polar flavin binding pocket. Investigations with flavin- and deazaflavin-containing model compounds confirmed that the deazaflavin functions solely as a photo antenna and allowed to study the dependencies of the antenna function on the protonation state of the 8-hydroxy-5-deazaflavin. The ability to mimic the repair reaction with small model compounds allowed finally the development of flavin cofactor functionalized oligo-peptides. Cofactor peptides with the sequence of the DNA-binding domain of the transcription factor MyoD were shown to be able to repair UV light lesions of DNA within a DNA single strand.

A distinct energy transfer between the deazaflavin and the flavin is observed in compounds that mimic the DNA repair function of DNA-photolase enzymes (see sketch). Studies on these models prove that the deazaflavin acts solely as photoantenna and suggest that the two cofactors have to be separated within the enzyme to suppress a competitive intercofactor electron transfer reaction.

Ein deutlicher Energietransfer wird zwischen der Desazaflavin- und der Flavineinheit von Modellverbindungen (siehe rechts) gemessen, die die DNA-Reparaturreaktion im aktiven Zentrum von DNA-Photolyasen nachahmen. Modellstudien belegen die ausschliesliche Antennenfunktion des Desazaflavins und deuten an, das die beiden Chromophore separiert werden mussen, um einen konkurrierenden Elektronentransfer zwischen ihnen zu verhindern.

Herein, we report a convenient method for the preparation of four benzyl-ester-protected, (carboxymethyl)-functionalized cyclobuta-fused uracil dimers 1–4, including the desired cis-cisoid-cis,syn- configured isomer 1, a compound which is suitable for the preparation of catalytic antibodies, synthetic receptors, and model compounds, required for the investigation of the DNA-lesion, recognition step and DNA-repair mechanisms. A comprehensive structure determination of each isomer by 1H-NMR, and in the case of the cis-cisoid-cis,syn- and the cis-traftsoid-cis,Syn-dimers by X-ray crystal-structure analysis, is also provided.

Herein, we report a convenient method for the preparation of four benzyl-ester-protected, (carboxymethyl)-functionalized cyclobuta-fused uracil dimers 1–4, including the desired cis-cisoid-cis,syn- configured isomer 1, a compound which is suitable for the preparation of catalytic antibodies, synthetic receptors, and model compounds, required for the investigation of the DNA-lesion, recognition step and DNA-repair mechanisms. A comprehensive structure determination of each isomer by 1H-NMR, and in the case of the cis-cisoid-cis,syn- and the cis-traftsoid-cis,Syn-dimers by X-ray crystal-structure analysis, is also provided.

DNA photolyases are enzymes that catalyze the light-dependend repair of cis-syn-cyclobutane-thymine dimer UV lesions in a variety of organisms. The basis of the repairreaction is an electron transfer from a reduced and deprotonated flavin cofactor to the dimer unit, which splits spontaneously as its radical anion. A second cofactor, which is either an 8-hydroxy-5-deazaflavin or a methenyl-tetrahydrofolate is required as a photo antenna and ensures efficient light absorption. With the help of model compounds that are able to mimic all crucial steps of the repair reaction, detailed mechanistic insights into the repair reaction could be obtained. It became clear, that the enzyme requires the reduced flavin in its deprotonated form and that the repair reaction proceeds most efficiently in polar media, which is in agreement with the observed highly polar flavin binding pocket. Investigations with flavin- and deazaflavin-containing model compounds confirmed that the deazaflavin functions solely as a photo antenna and allowed to study the dependencies of the antenna function on the protonation state of the 8-hydroxy-5-deazaflavin. The ability to mimic the repair reaction with small model compounds allowed finally the development of flavin cofactor functionalized oligo-peptides. Cofactor peptides with the sequence of the DNA-binding domain of the transcription factor MyoD were shown to be able to repair UV light lesions of DNA within a DNA single strand.

A screen of over 500 diversely functionalized additives in osmium-catalyzed dihydroxylation has uncovered that electron-deficient olefins are converted into the corresponding diols much more efficiently when the pH of the reaction medium is maintained on the acidic side. Further studies have identified citric acid as the additive of choice, for it allows preparation of very pure diols in yields generally exceeding 90%. As described here, a much wider range of olefin classes can now be successfully dihydroxylated. The process is experimentally simple, in most cases involving little more than dissolving the reactants in water or a water/tert-butyl alcohol mixture, stirring them, and filtering off the pure diol product.

Peptidic, non-covalent inhibitors of lysosomal cysteine protease cathepsin S (1 and 2) were investigated due to low oral bioavailability, leading to an improved series of peptidomimetic inhibitors. Utilizing phenyl succinamides as the P2 residue increased the oral exposure of this lead series of compounds, while retaining selective inhibition of the cathepsin S isoform. Concurrent investigation of the P1 and P2 subsites resulted in the discovery of several potent and selective inhibitors of cathepsin S with good pharmacokinetic properties due to the elimination of saturated aliphatic P2 residues.

Epidermal growth factor receptor (EGFR) is a validated therapeutic target for lung cancer. First and second generation EGFR inhibitors (e.g., gefitinib, erlotinib and afatinib) have revolutionized treatment paradigms of non-small cell lung cancer (NSCLC) patients with oncogenic EGFR mutations. The use of EGFR tyrosine kinase inhibitors (TKI) provides superior efficacy compared to chemotherapy in patients with EGFR L858R or exon 19 deletion tumors. However, resistance inevitably develops after 8-12 months of treatment; most commonly via a secondary T790M point mutation at the gatekeeper residue of EGFR. Furthermore, responses are hindered due to treatment intolerance in the form of rash and diarrhea that are mediated by simultaneous inhibition of wild-type (WT) EGFR at doses required for mutant EGFR suppression. To overcome these limitations, we initiated a project to identify mutant-selective EGFR inhibitors that potently inhibit both activating and T790M resistance EGFR mutations while sparing WT EGFR. In this presentation, we report our medicinal chemistry approach and optimization that led to the discovery of EGF816, a selective and potent covalent mutant-selective EGFR inhibitor with single digit nanomolar cellular target modulation on both activating and T790M resistance mutations. In addition, we will also report validated clinical efficacy data from the first patient treated with EGF816. Citation Format: Gerald Lelais, Robert Epple, Pierre-Yves Michellys, Thomas H. Marsilje, Yun Long, Matthew McNeill, Bei Chen, Wenshuo Lu, Badry Bursulaya, Michael DiDonato, Yong Jia, Shailaja Kasibhatla, Chun Li, Igor Matushansky, Steven Bender. Discovery of a potent covalent mutant-selective EGFR inhibitor - the journey from high throughput screening to EGF816. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2585. doi:10.1158/1538-7445.AM2015-2585

A 6-oxa-1-aza-bicyclo[3.2.1]octan-7-one system inhibits the proteolytic activity of several cysteine proteases belonging to the papain family. In vitro mechanistic studies and in silico calculations suggest that the minimal pi-overlap between the bridgehead nitrogen and the carbonyl leads to a considerable weakening of the urethane system, making it susceptible to nucleophilic attack from the active site thiol group. The resulting covalent adduct is slowly hydrolyzed, releasing the hydroxypiperidine product of the inhibitor. Synthesis and testing of a set of analogs led to variable protease subtype selectivities ranging from micromolar to nanomolar potencies.

A series of highly potent and selective PPARdelta agonists is described using the known non-selective ligand GW2433 as a structural template. Compound 1 is bioavailable, potent (10 nM), and shows no cross-activity with other PPAR subtypes up to 10 microM, making it a useful tool in studying the biological effects of selective PPARdelta activation.

EGFR is a major oncogene in NSCLC. Patients with the oncogenic mutations L858R and Ex19Del are responsive to the first generation pan-EGFR tyrosine kinase inhibitors (TKIs) erlotinib and gefitinib. The associated dose-limiting toxicities (DLTs) are severe rash and GI tolerability due to WT EGFR inhibition. However, therapy is universally limited by the development of acquired drug resistance where EGFR gatekeeper mutation T790M accounts for 50% of incidence. Second generation irreversible pan-EGFR TKI afatinib was developed to overcome T790M resistance. Though effective in animal models, the efficacy of afatinib on T790M patients is largely limited by its DLTs due to potent inhibition of WT EGFR. A strong medical need still exists for better tolerated therapy to treat NSCLC patients harboring EGFR mutations. Here we report the discovery and development of a potent third generation, irreversible mutant-selective EGFR TKI that is expected to improve/maintain efficacy on oncogenic EGFR mutant patients while demonstrating reduced side effects. EGF816 potently inhibits both activating (L858R and Ex19Del) and T790M resistant mutations in various cellular assays; it is selective against a large panel of kinases in both Ambit and BaF3 profiling, and more importantly is selective against WT EGFR. EGF816 is efficacious in mutant EGFR-driven xenograft models, is well tolerated in IND-enabling toxicology studies, and is entering phase 1 trials. Citation Format: Yong Jia, Jose Juarez, Mari Manuia, Gerald Lelais, Shailaja Kasibhatla, Oliver Long, Matthew McNeill, Michael DiDonato, Badry Bursulaya, Debbie Liao, Eric Murphy, Robert Epple, Thomas Marsilje, Nuzhat Pathan, Pierre-Yves Michellys, Steven Bender, Jennifer Harris. In vitro characterization of EGF816, a third-generation mutant-selective EGFR inhibitor. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1734. doi:10.1158/1538-7445.AM2014-1734

Cyclobutane uridine and thymidine dimers with cis-syn-structure are DNA lesions, which are efficiently repaired in many species by DNA photolyases. The essential step of the repair reaction is a light driven electron transfer from a reduced FAD cofactor (FADH ) to the dimer lesion, which splits spontaneously into the monomers. Repair studies with UV-light damaged DNA revealed significant rate differences for the various dimer lesions. In particular the effect of the almost eclipsed positioned methyl groups at the thymidine cyclobutane dimer moiety on the splitting rates is unknown. In order to investigate the cleavage vulnerability of thymine and uracil cyclobutane photodimers outside the protein environment, two model compounds, containing a thymine or a uracil dimer and a covalently connected flavin, were prepared and comparatively investigated. Cleavage investigations under internal competition conditions revealed, in contrast to all previous findings, faster repair of the sterically less encumbered uracil dimer. Stereoelectronic effects are offered as a possible explanation. Ab initio calculations and X-ray crystal structure data reveal a different cyclobutane ring pucker of the uracil dimer, which leads to a better overlap of the pi*-C(4)-O(4)-orbital with the sigma*-C(5)-C(5')-orbital. Enzymatic studies with a DNA photolyase (A. nidulans) and oligonucleotides, which contain either a uridine or a thymidine dimer analogue, showed comparable repair efficiencies for both dimer lesions. Under internal competition conditions significantly faster repair of uridine dimers is observed.