Metabolism-driven in vitro/in vivo disconnect of an oral ERɑ VHL-PROTAC.

Targeting the estrogen receptor alpha (ERα) pathway is validated in the clinic as an effective means to treat ER+ breast cancers. Here we present the development of a VHL-targeting and orally bioavailable proteolysis-targeting chimera (PROTAC) degrader of ERα. In vitro studies with this PROTAC demonstrate excellent ERα degradation and ER antagonism in ER+ breast cancer cell lines. However, upon dosing the compound in vivo we observe an in vitro-in vivo disconnect. ERα degradation is lower in vivo than expected based on the in vitro data. Investigation into potential causes for the reduced maximal degradation reveals that metabolic instability of the PROTAC linker generates metabolites that compete for binding to ERα with the full PROTAC, limiting degradation. This observation highlights the requirement for metabolically stable PROTACs to ensure maximal efficacy and thus optimisation of the linker should be a key consideration when designing PROTACs.

The Next-Generation Oral Selective Estrogen Receptor Degrader Camizestrant (AZD9833) Suppresses ER+ Breast Cancer Growth and Overcomes Endocrine and CDK4/6 Inhibitor Resistance.

Oral selective estrogen receptor degraders (SERD) could become the backbone of endocrine therapy (ET) for estrogen receptor-positive (ER+) breast cancer, as they achieve greater inhibition of ER-driven cancers than current ETs and overcome key resistance mechanisms. In this study, we evaluated the preclinical pharmacology and efficacy of the next-generation oral SERD camizestrant (AZD9833) and assessed ER-co-targeting strategies by combining camizestrant with CDK4/6 inhibitors (CDK4/6i) and PI3K/AKT/mTOR-targeted therapy in models of progression on CDK4/6i and/or ET. Camizestrant demonstrated robust and selective ER degradation, modulated ER-regulated gene expression, and induced complete ER antagonism and significant antiproliferation activity in ESR1 wild-type (ESR1wt) and mutant (ESR1m) breast cancer cell lines and patient-derived xenograft (PDX) models. Camizestrant also delivered strong antitumor activity in fulvestrant-resistant ESR1wt and ESR1m PDX models. Evaluation of camizestrant in combination with CDK4/6i (palbociclib or abemaciclib) in CDK4/6-naive and -resistant models, as well as in combination with PI3Kαi (alpelisib), mTORi (everolimus), or AKTi (capivasertib), indicated that camizestrant was active with CDK4/6i or PI3K/AKT/mTORi and that antitumor activity was further increased by the triple combination. The response was observed independently of PI3K pathway mutation status. Overall, camizestrant shows strong and broad antitumor activity in ER+ breast cancer as a monotherapy and when combined with CDK4/6i and PI3K/AKT/mTORi. SIGNIFICANCE: Camizestrant, a next-generation oral SERD, shows promise in preclinical models of ER+ breast cancer alone and in combination with CDK4/6 and PI3K/AKT/mTOR inhibitors to address endocrine resistance, a current barrier to treatment.

Discovery of a Potent and Orally Bioavailable Zwitterionic Series of Selective Estrogen Receptor Degrader-Antagonists.

Herein, we report the optimization of a meta-substituted series of selective estrogen receptor degrader (SERD) antagonists for the treatment of ER+ breast cancer. Structure-based design together with the use of modeling and NMR to favor the bioactive conformation led to a highly potent series of basic SERDs with promising physicochemical properties. Issues with hERG activity resulted in a strategy of zwitterion formation and ultimately in the identification of 38. This compound was shown to be a highly potent SERD capable of effectively degrading ERα in both MCF-7 and CAMA-1 cell lines. The low lipophilicity and zwitterionic nature led to a SERD with a clean secondary pharmacology profile and no hERG activity. Favorable physicochemical properties resulted in good oral bioavailability in preclinical species and potent in vivo activity in a mouse xenograft model.

Automation and Expansion of EMMA Assembly for Fast-Tracking Mammalian System Engineering.

With applications from functional genomics to the production of therapeutic biologics, libraries of mammalian expression vectors have become a cornerstone of modern biological investigation and engineering. Multiple modular vector platforms facilitate the rapid design and assembly of vectors. However, such systems approach a technical bottleneck when a library of bespoke vectors is required. Utilizing the flexibility and robustness of the Extensible Mammalian Modular Assembly (EMMA) toolkit, we present an automated workflow for the library-scale design, assembly, and verification of mammalian expression vectors. Vector design is simplified using our EMMA computer-aided design tool (EMMA-CAD), while the precision and speed of acoustic droplet ejection technology are applied in vector assembly. Our pipeline facilitates significant reductions in both reagent usage and researcher hands-on time compared with manual assembly, as shown by system Q-metrics. To demonstrate automated EMMA performance, we compiled a library of 48 distinct plasmid vectors encoding either CRISPR interference or activation modalities. Characterization of the workflow parameters shows that high assembly efficiency is maintained across vectors of various sizes and design complexities. Our system also performs strongly compared with manual assembly efficiency benchmarks. Alongside our automated pipeline, we present a straightforward strategy for integrating gRNA and Cas modules into the EMMA platform, enabling the design and manufacture of valuable genome editing resources.

Regulation of Gene Expression and the Elucidative Role of CRISPR-Based Epigenetic Modifiers and CRISPR-Induced Chromosome Conformational Changes.

In complex multicellular systems, gene expression is regulated at multiple stages through interconnected complex molecular pathways and regulatory networks. Transcription is the first step in gene expression and is subject to multiple layers of regulation in which epigenetic mechanisms such as DNA methylation, histone tail modifications, and chromosomal conformation play an essential role. In recent years, CRISPR-Cas9 systems have been employed to unearth this complexity and provide new insights on the contribution of chromatin dysregulation in the development of genetic diseases, as well as new tools to prevent or reverse this dysregulation. In this review, we outline the recent development of a variety of CRISPR-based epigenetic editors for targeted DNA methylation/demethylation, histone modification, and three-dimensional DNA conformational change, highlighting their relative performance and impact on gene regulation. Finally, we provide insights on the future developments aimed to accelerate our understanding of the causal relationship between epigenetic marks, genome organization, and gene regulation.

Discovery of AZD9833, a Potent and Orally Bioavailable Selective Estrogen Receptor Degrader and Antagonist.

Herein we report the optimization of a series of tricyclic indazoles as selective estrogen receptor degraders (SERD) and antagonists for the treatment of ER+ breast cancer. Structure based design together with systematic investigation of each region of the molecular architecture led to the identification of N-[1-(3-fluoropropyl)azetidin-3-yl]-6-[(6S,8R)-8-methyl-7-(2,2,2-trifluoroethyl)-6,7,8,9-tetrahydro-3H-pyrazolo[4,3-f]isoquinolin-6-yl]pyridin-3-amine (28). This compound was demonstrated to be a highly potent SERD that showed a pharmacological profile comparable to fulvestrant in its ability to degrade ERα in both MCF-7 and CAMA-1 cell lines. A stringent control of lipophilicity ensured that 28 had favorable physicochemical and preclinical pharmacokinetic properties for oral administration. This, combined with demonstration of potent in vivo activity in mouse xenograft models, resulted in progression of this compound, also known as AZD9833, into clinical trials.

Negative Regulation of TLR Signaling by BCAP Requires Dimerization of Its DBB Domain.

The B cell adaptor protein (BCAP) is a multimodular regulator of inflammatory signaling in diverse immune system cells. BCAP couples TLR signaling to phosphoinositide metabolism and inhibits MyD88-directed signal transduction. BCAP is recruited to the TLR signalosome forming multitypic interactions with the MAL and MyD88 signaling adaptors. In this study, we show that indirect dimerization of BCAP TIR is required for negative regulation of TLR signaling. This regulation is mediated by a transcription factor Ig (TIG/IPT) domain, a fold found in the NF-κB family of transcription factors. We have solved the crystal structure of the BCAP TIG and find that it is most similar to that of early B cell factor 1 (EBF1). In both cases, the dimer is stabilized by a helix-loop-helix motif at the C terminus and interactions between the ß-sheets of the Ig domains. BCAP is exclusively localized in the cytosol and is unable to bind DNA. Thus, the TIG domain is a promiscuous dimerization module that has been appropriated for a range of regulatory functions in gene expression and signal transduction.

Phosphorylation of the multifunctional signal transducer B-cell adaptor protein (BCAP) promotes recruitment of multiple SH2/SH3 proteins including GRB2.

B-cell adaptor protein (BCAP) is a multimodular, multifunctional signal transducer that regulates signal transduction pathways in leukocytes, including macrophages, B-cells, and T-cells. In particular, BCAP suppresses inflammatory signaling by Toll-like receptors (TLRs). However, how BCAP itself is regulated and what its interaction partners are is unclear. Here, using human immune cell lines, including THP-1 cells, we characterized the complex phosphorylation patterns of BCAP and used a novel protein complex trapping strategy, called virotrap, to identify its interaction partners. This analysis identified known interactions of BCAP with phosphoinositide 3-kinase (PI3K) p85 subunit and NCK adaptor protein (NCK), together with previously unknown interactions of BCAP with Src homology 2 (SH2) and SH3 domain-containing adaptor proteins, notably growth factor receptor-bound protein 2 (GRB2) and CRK-like proto-oncogene, adaptor protein (CRKL). We show that the SH3 domain of GRB2 can bind to BCAP independently of BCAP phosphorylation status, suggesting that the SH2 domains mediate interactions with activated receptor tyrosine kinase complexes including the CD19 subunit of the B-cell receptor. Our results also suggested that the PI3K p85 subunit binds to BCAP via SH3 domains forming an inactive complex that is then activated by sequential binding with the SH2 domains. Taken together, our results indicate that BCAP is a complex hub that processes signals from multiple pathways in diverse cell types of the immune system.

Systematic Evaluation of CRISPRa and CRISPRi Modalities Enables Development of a Multiplexed, Orthogonal Gene Activation and Repression System.

The ability to manipulate the expression of mammalian genes using synthetic transcription factors is highly desirable in both fields of basic research and industry for diverse applications, including stem cell reprogramming and differentiation, tissue engineering, and drug discovery. Orthogonal CRISPR systems can be used for simultaneous transcriptional upregulation of a subset of target genes while downregulating another subset, thus gaining control of gene regulatory networks, signaling pathways, and cellular processes whose activity depends on the expression of multiple genes. We have used a rapid and efficient modular cloning system to build and test in parallel diverse CRISPRa and CRISPRi systems and develop an efficient orthogonal gene regulation system for multiplexed and simultaneous up- and downregulation of endogenous target genes.

Protein Expression via Transient Transfection of Mammalian Cells in a WAVE Bioreactor.

Large culture volumes are often required when expression constructs are particularly low-yielding or when end-uses require significant amounts of material. In these cases, a single homogenous culture is usually more convenient, in terms of both consistency of expression and labour/resource requirements, than multiple parallel cultures. Using a WAVE Bioreactor culture volumes as high as 500 L may be achieved in a single vessel. Here we describe the transfection of 293-6E cells in a disposable 50 L Cellbag on a WAVE Bioreactor platform to produce recombinant protein. The methods described herein may be adapted, with suitable optimizations, for other suspension-adapted mammalian cell lines.

A protein chimera strategy supports production of a model "difficult-to-express" recombinant target.

Due in part to the needs of the biopharmaceutical industry, there has been an increased drive to generate high quality recombinant proteins in large amounts. However, achieving high yields can be a challenge as the novelty and increased complexity of new targets often makes them 'difficult-to-express'. This study aimed to define the molecular features that restrict the production of a model 'difficult-to-express' recombinant protein, Tissue Inhibitor Metalloproteinase-3 (TIMP-3). Building from experimental data, computational approaches were used to rationalize the redesign of this recombinant target to generate a chimera with enhanced secretion. The results highlight the importance of early identification of unfavourable sequence attributes, enabling the generation of engineered protein forms that bypass 'secretory' bottlenecks and result in efficient recombinant protein production.

Use of a protein engineering strategy to overcome limitations in the production of "Difficult to Express" recombinant proteins.

Certain recombinant proteins are deemed "difficult to express" in mammalian expression systems requiring significant cell and/or process engineering to abrogate expression bottlenecks. With increasing demand for the production of recombinant proteins in mammalian cells, low protein yields can have significant consequences for industrial processes. To investigate the molecular mechanisms that restrict expression of recombinant proteins, naturally secreted model proteins were analyzed from the tissue inhibitors of metalloproteinase (TIMP) protein family. In particular, TIMP-2 and TIMP-3 were subjected to detailed study. TIMP proteins share significant sequence homology (∼50% identity and ∼70% similarity in amino acid sequence). However, they show marked differences in secretion in mammalian expression systems despite this extensive sequence homology. Using these two proteins as models, this study characterized the molecular mechanisms responsible for poor recombinant protein production. Our results reveal that both TIMP-2 and TIMP-3 are detectable at mRNA and protein level within the cell but only TIMP-2 is secreted effectively into the extracellular medium. Analysis of protein localization and the nature of intracellular protein suggest TIMP-3 is severely limited in its post-translational processing. To overcome this challenge, modification of the TIMP-3 sequence to include a furin protease-cleavable pro-sequence resulted in secretion of the modified TIMP-3 protein, however, incomplete processing was observed. Based on the TIMP-3 data, the protein engineering approach was optimized and successfully applied in combination with cell engineering, the overexpression of furin, to another member of the TIMP protein family (the poorly expressed TIMP-4). Use of the described protein engineering strategy resulted in successful secretion of poorly (TIMP-4) and non-secreted (TIMP-3) targets, and presents a novel strategy to enhance the production of "difficult" recombinant targets. Biotechnol. Bioeng. 2017;114: 2348-2359. © 2017 Wiley Periodicals, Inc.

Structures of closed and open conformations of dimeric human ATM.

ATM (ataxia-telangiectasia mutated) is a phosphatidylinositol 3-kinase-related protein kinase (PIKK) best known for its role in DNA damage response. ATM also functions in oxidative stress response, insulin signaling, and neurogenesis. Our electron cryomicroscopy (cryo-EM) suggests that human ATM is in a dynamic equilibrium between closed and open dimers. In the closed state, the PIKK regulatory domain blocks the peptide substrate-binding site, suggesting that this conformation may represent an inactive or basally active enzyme. The active site is held in this closed conformation by interaction with a long helical hairpin in the TRD3 (tetratricopeptide repeats domain 3) domain of the symmetry-related molecule. The open dimer has two protomers with only a limited contact interface, and it lacks the intermolecular interactions that block the peptide-binding site in the closed dimer. This suggests that the open conformation may be more active. The ATM structure shows the detailed topology of the regulator-interacting N-terminal helical solenoid. The ATM conformational dynamics shown by the structures represent an important step in understanding the enzyme regulation.

Development of a Medium-Throughput Targeted LCMS Assay to Detect Endogenous Cellular Levels of Malonyl-CoA to Screen Fatty Acid Synthase Inhibitors.

The fatty acid synthase (FAS) enzyme in mammalian cells is a large multidomain protein responsible for de novo synthesis of fatty acids. The steps catalyzed by FAS involve the condensation of acetyl-CoA and malonyl-CoA moieties in the presence of NADPH until palmitate is formed. Inhibition of FAS causes an accumulation of intracellular malonyl-CoA, as this metabolite is essentially committed to fatty acid synthesis once formed. Detection of intracellular metabolites for screening can be problematic due to a lack of appropriate tools, but here we describe a targeted liquid chromatography-mass spectroscopy (LCMS) method to directly measure endogenous levels of malonyl-CoA to drive a drug development structure-activity relationship (SAR) screening cascade. Our process involves preparation of samples at 96-well scale, normalization postpermeabilization via use of a whole-well imaging platform, and the LCMS detection methodology. The assay is amenable to multiplexing cellular endpoints, has a typical Z' of >0.6, and has high reproducibility of EC50 values.

Cellular assay optimization: part I: the use of large-scale transiently transfected cryobanks and introduction of a c-Myc tag to design a standardized ELISA process.

This study investigated the use of large-scale transiently transfected cryopreserved cells for medium-throughput cellular screening. The data generated indicated that preprepared transiently transfected cryobanks can be used for cell-based assays and in fact can greatly enhance the consistency of data generated by cellular screens. In addition to this, a generic enzyme-linked immunosorbent assay method was designed that introduced a c-Myc tag to four different targets and allowed all four cell assays to be run using a standardized process. These process improvements yielded cost savings and greatly reduced the required resource, as well as reducing timelines for developing cellular assays.

Correlation of intracellular diadenosine triphosphate (Ap3A) with apoptosis in Fhit-positive HEK293 cells.

The pro-apoptotic Fhit tumor suppressor protein binds and hydrolyses diadenosine triphosphate (Ap3A) and diadenosine tetraphosphate (Ap4A) in vitro. We have measured the level of both these nucleotides in Fhit-positive HEK293 cells exposed to various apoptosis inducers. Cold shock, anti-Fas, cadmium ions and etoposide all increased the basal level of Ap4A of 0.500pmol/10(6)cells by about 50%. However, the corresponding increases in Ap3A from a basal 0.079pmol/10(6)cells correlated closely with the degree of apoptosis produced, up to a maximum of 0.510pmol/10(6)cells with etoposide. These results support the view that Ap3A is the in vivo Fhit ligand and that an inhibition of Fhit activity is a key element in Fhit-mediated apoptosis.

Characterization of the Mn2+-stimulated (di)adenosine polyphosphate hydrolase encoded by the Deinococcus radiodurans DR2356 nudix gene.

The DR2356 nudix hydrolase gene from Deinococcus radiodurans has been cloned and the product expressed as an 18 kDa histidine-tagged protein. The enzyme hydrolysed adenosine and diadenosine polyphosphates, always generating ATP as one of the initial products. ATP and other (deoxy)nucleoside triphosphates were also substrates, yielding (d)NDP and Pi as products. The DR2356 protein was most active at pH 8.6-9.0 and showed a strong preference for Mn(2+) as activating cation. Mg(2+) ions at 15 mM supported only 5% of the activity achieved with 2 mM Mn(2+). K (m) and k (cat) values for diadenosine tetra-, penta- and hexaphosphates were 2.0, 2.4 and 1.1 microM and 11.4, 28.6 and 12.0 s(-1), respectively, while for GTP they were 20.3 microM and 1.8 s(-1), respectively. The K (m )for adenosine 5'-pentaphosphate was <1 microM. Expression analysis showed the DR2356 gene to be induced eight- to ninefold in stationary phase and in cells subjected to slow dehydration plus rehydration. Superoxide (but not peroxide) treatment and rapid dehydration caused a two-to threefold induction. The Mn-requirement and induction in stationary phase suggest that DR2356 may have a specific role in maintenance mode metabolism in stationary phase as Mn(2+) accumulates.

Characterization of a nudix hydrolase from Deinococcus radiodurans with a marked specificity for (deoxy)ribonucleoside 5'-diphosphates.

BACKGROUND: Nudix hydrolases form a protein family whose function is to hydrolyse intracellular nucleotides and so regulate their levels and eliminate potentially toxic derivatives. The genome of the radioresistant bacterium Deinococcus radiodurans encodes 25 nudix hydrolases, an unexpectedly large number. These may contribute to radioresistance by removing mutagenic oxidised and otherwise damaged nucleotides. Characterisation of these hydrolases is necessary to understand the reason for their presence. Here, we report the cloning and characterisation of the DR0975 gene product, a nudix hydrolase that appears to be unique to this organism. RESULTS: The DR0975 gene was cloned and expressed as a 20 kDa histidine-tagged recombinant product in Escherichia coli. Substrate analysis of the purified enzyme showed it to act primarily as a phosphatase with a marked preference for (deoxy)nucleoside 5'-diphosphates (dGDP > ADP > dADP > GDP > dTDP > UDP > dCDP > CDP). Km for dGDP was 110 microM and kcat was 0.18 s-1 under optimal assay conditions (pH 9.4, 7.5 mM Mg2+). 8-Hydroxy-2'-deoxyguanosine 5'-diphosphate (8-OH-dGDP) was also a substrate with a Km of 170 microM and kcat of 0.13 s-1. Thus, DR0975 showed no preference for 8-OH-dGDP over dGDP. Limited pyrophosphatase activity was also observed with NADH and some (di)adenosine polyphosphates but no other substrates. Expression of the DR0975 gene was undetectable in logarithmic phase cells but was induced at least 30-fold in stationary phase. Superoxide, but not peroxide, stress and slow, but not rapid, dehydration both caused a slight induction of the DR0975 gene. CONCLUSION: Nucleotide substrates for nudix hydrolases conform to the structure NDP-X, where X can be one of several moieties. Thus, a preference for (d)NDPs themselves is most unusual. The lack of preference for 8-OH-dGDP over dGDP as a substrate combined with the induction in stationary phase, but not by peroxide or superoxide, suggests that the function of DR09075 may be to assist in the recycling of nucleotides under the very different metabolic requirements of stationary phase. Thus, if DR0975 does contribute to radiation resistance, this contribution may be indirect.

Nudix hydrolases that degrade dinucleoside and diphosphoinositol polyphosphates also have 5-phosphoribosyl 1-pyrophosphate (PRPP) pyrophosphatase activity that generates the glycolytic activator ribose 1,5-bisphosphate.

A total of 17 Nudix hydrolases were tested for their ability to hydrolyze 5-phosphoribosyl 1-pyrophosphate (PRPP). All 11 enzymes that were active toward dinucleoside polyphosphates with 4 or more phosphate groups as substrates were also able to hydrolyze PRPP, whereas the 6 that could not and that have coenzyme A, NDP-sugars, or pyridine nucleotides as preferred substrates did not degrade PRPP. The products of hydrolysis were ribose 1,5-bisphosphate and P(i). Active PRPP pyrophosphatases included the diphosphoinositol polyphosphate phosphohydrolase (DIPP) subfamily of Nudix hydrolases, which also degrade the non-nucleotide diphosphoinositol polyphosphates. K(m) and k(cat) values for PRPP hydrolysis for the Deinococcus radiodurans DR2356 (di)nucleoside polyphosphate hydrolase, the human diadenosine tetraphosphate hydrolase, and human DIPP-1 (diadenosine hexaphosphate and diphosphoinositol polyphosphate hydrolase) were 1 mm and 1.5 s(-1), 0.13 mm and 0.057 s(-1), and 0.38 mm and 1.0 s(-1), respectively. Active site mutants of the Caenorhabditis elegans diadenosine tetraphosphate hydrolase had no activity, confirming that the same active site is responsible for nucleotide and PRPP hydrolysis. Comparison of the specificity constants for nucleotide, diphosphoinositol polyphosphate, and PRPP hydrolysis suggests that PRPP is a significant substrate for the D. radiodurans DR2356 enzyme and for the DIPP subfamily. In the latter case, generation of the glycolytic activator ribose 1,5-bisphosphate may be a new function for these enzymes.