Time-Resolved Analysis Reveals Rapid Dynamics and Broad Scope of the CBP/p300 Acetylome.

The acetyltransferases CBP and p300 are multifunctional transcriptional co-activators. Here, we combined quantitative proteomics with CBP/p300-specific catalytic inhibitors, bromodomain inhibitor, and gene knockout to reveal a comprehensive map of regulated acetylation sites and their dynamic turnover rates. CBP/p300 acetylates thousands of sites, including signature histone sites and a multitude of sites on signaling effectors and enhancer-associated transcriptional regulators. Time-resolved acetylome analyses identified a subset of CBP/p300-regulated sites with very rapid (<30 min) acetylation turnover, revealing a dynamic balance between acetylation and deacetylation. Quantification of acetylation, mRNA, and protein abundance after CBP/p300 inhibition reveals a kinetically competent network of gene expression that strictly depends on CBP/p300-catalyzed rapid acetylation. Collectively, our in-depth acetylome analyses reveal systems attributes of CBP/p300 targets, and the resource dataset provides a framework for investigating CBP/p300 functions and for understanding the impact of small-molecule inhibitors targeting its catalytic and bromodomain activities.

An Insulin-Responsive Sensor in the SIRT1 Disordered Region Binds DBC1 and PACS-2 to Control Enzyme Activity.

Current models of SIRT1 enzymatic regulation primarily consider the effects of fluctuating levels of its co-substrate NAD+, which binds to the stably folded catalytic domain. By contrast, the roles of the sizeable disordered N- and C-terminal regions of SIRT1 are largely unexplored. Here we identify an insulin-responsive sensor in the SIRT1 N-terminal region (NTR), comprising an acidic cluster (AC) and a 3-helix bundle (3HB), controlling deacetylase activity. The allosteric assistor DBC1 removes a distal N-terminal shield from the 3-helix bundle, permitting PACS-2 to engage the acidic cluster and the transiently exposed helix 3 of the 3-helix bundle, disrupting its structure and inhibiting catalysis. The SIRT1 activator (STAC) SRT1720 binds and stabilizes the 3-helix bundle, protecting SIRT1 from inhibition by PACS-2. Identification of the SIRT1 insulin-responsive sensor and its engagement by the DBC1 and PACS-2 regulatory hub provides important insight into the roles of disordered regions in enzyme regulation and the mode by which STACs promote metabolic fitness.

Inhibition of NLRP3 inflammasome by MCC950 under hypoxia alleviates photoreceptor apoptosis via inducing autophagy in Müller glia.

NLRP3 inflammasome activation has emerged as a critical initiator of inflammatory response in ischemic retinopathy. Here, we identified the effect of a potent, selective NLRP3 inhibitor, MCC950, on autophagy and apoptosis under hypoxia. Neonatal mice were exposed to hyperoxia for 5 days to establish oxygen-induced retinopathy (OIR) model. Intravitreal injection of MCC950 was given, and then autophagy and apoptosis markers were assessed. Retinal autophagy, apoptosis, and related pathways were evaluated by western blot, immunofluorescent labeling, transmission electron microscopy, and TUNEL assay. Autophagic activity in Müller glia after NLRP3 inflammasome inhibition, together with its influence on photoreceptor death, was studied using western blot, immunofluorescence staining, mRFP-GFP-LC3 adenovirus transfection, cell viability, proliferation, and apoptosis assays. Results showed that activation of NLRP3 inflammasome in Müller glia was detected in OIR model. MCC950 could improve impaired retinal autophagic flux and attenuate retinal apoptosis while it regulated the retinal AMPK/mTOR/ULK-1 pathway. Suppressed autophagy and depressed proliferation capacity resulting from hypoxia was promoted after MCC950 treatment in Müller glia. Inhibition of AMPK and ULK-1 pathway significantly interfered with the MCC950-induced autophagy activity, indicating MCC950 positively modulated autophagy through AMPK/mTOR/ULK-1 pathway in Müller cells. Furthermore, blockage of autophagy in Müller glia significantly induced apoptosis in the cocultured 661W photoreceptor cells, whereas MCC950 markedly preserved the density of photoreceptor cells. These findings substantiated the therapeutic potential of MCC950 against impaired autophagy and subsequent apoptosis under hypoxia. Such protective effect might involve the modulation of AMPK/mTOR/ULK-1 pathway. Targeting NLRP3 inflammasome in Müller glia could be beneficial for photoreceptor survival under hypoxic conditions.

Design, synthesis, and biological evaluation of novel benzo[6,7]indolo[3,4-c]isoquinolines as anticancer agents with topoisomerase I inhibition.

A novel series of benzo[6,7]indolo[3,4-c]isoquinolines 3a-3f was designed by scaffold hopping of topoisomerase I inhibitor benzo[g][1]benzopyrano[4,3-b]indol-6(13H)-ones (BBPIs), which were developed by structural modification of the natural marine product lamellarin. The unconventional pentacycle was constructed by Bischler-Napieralski-type condensation of amide 11 and subsequent intramolecular Heck reaction. In vitro anticancer activity of the synthesized benzo[6,7]indolo[3,4-c]isoquinolines was evaluated on a panel of 39 human cancer cell lines (JFCR39). Among the compounds tested, N-(3-morpholinopropyl) derivative 3e showed the most potent antiproliferative activity, with a mean GI50 value of 39 nM. This compound inhibited topoisomerase I activity by stabilizing the enzyme-DNA complex.

SIRT1 as a potential therapeutic target in pelvic organ prolapse due to protective effects against oxidative stress and cellular senescence in human uterosacral ligament fibroblasts.

INTRODUCTION: The pathogenesis of pelvic organ prolapse (POP), an age-related disease, has not been fully elucidated. Therapeutic targets of POP are limited. Silencing information regulator 2 related enzyme 1 (SIRT1), a gene considered capable of regulating oxidative stress and cellular senescence, has been widely demonstrated involved in aging and age-related diseases. The present study aimed to explore the role of SIRT1 in POP in vivo and in vitro. METHODS: Expression levels of SIRT1 in uterosacral ligament (USL) tissues from patients with or without POP were measured using immunohistochemical assays. SRT1720, a SIRT1 agonist, was used to upregulate SIRT1, and hydrogen peroxide (H2O2) was used to establish an oxidative stress model in human uterosacral ligament fibroblasts (hUSLFs). The effects of SIRT1 on cell viability, apoptosis, senescence, and reactive oxygen species (ROS) levels were detected, respectively. Western blot assays were used to examine expression levels of apoptosis- and senescence-associated biomarkers. Unpaired Student's t test, Mann-Whitney U test, χ2 test, and one-way ANOVA were performed for determining statistically significant differences. RESULTS: Compared to the control group, expression levels of SIRT1 were downregulated in USL tissues and hUSLFs from patients with POP, and associated with stage (p < 0.05). hUSLFs of patients with POP had lower growth rates (p < 0.0001) than those of the control group, which were improved by upregulating SIRT1 (p < 0.05). The senescent proportion was higher in the POP group than the control group (43.63 ± 10.62% vs. 4.84 ± 5.32%, p < 0.0001), which could be reduced by upregulating SIRT1 (p < 0.0001). High ROS levels in the POP group were also alleviated by SRT1720. H2O2 exposure increased ROS levels, inhibited proliferation, and triggered apoptosis and senescence in hUSLFs of patients without POP in a concentration-dependent manner. Further, these damages were alleviated by pretreatment with SRT1720. CONCLUSIONS: SIRT1 is downregulated in patients with POP, and the development of SIRT1 activators or agonists may have applications in the treatment and prevention of POP through antioxidative stress and antisenescence effects.

Effect of single-administration of D-sorbitol pretreatment on the bitterness and continued willingness to take asenapine: a randomized, single-blind, placebo-controlled, crossover trial.

BACKGROUND: Asenapine has unique orally-related side effects, such as a bitter taste induced by sublingual administration, which often results in discontinuation of the medication. While the FDA has approved black-cherry-flavored asenapine, several countries have prescribed only unflavored versions. Specifically, Asians commonly report experiencing the bitterness of asenapine because they are more sensitive to bitter tastes than other ethnic groups. In this study, with the aim of improving adherence by reducing the bitterness of asenapine, we investigated the effects of D-sorbitol, which reduced the bitterness parameters of taste sensors in our previous basic study on the bitterness and continuity of asenapine among patients with schizophrenia. METHODS: Twenty adult patients with schizophrenia were included in this single-blind, placebo-controlled, crossover trial. Participants rinsed their mouths with single-administration of D-sorbitol or a placebo prior to each administration of asenapine. We then conducted the questionnaires and assessed changes in the bitterness of asenapine (primary end point) and willingness to continue its use (secondary end point). RESULTS: D-sorbitol significantly improved the bitterness of asenapine (p = 0.038). Although it did not significantly increase the willingness to continue asenapine (p = 0.180), it did show improvement over the placebo in enhancing willingness to continue, especially in patients who were not accustomed to its taste. CONCLUSION: Our findings indicate that single-administration of D-sorbitol significantly reduces the bitterness of asenapine. In countries where flavored asenapine is not available, this finding could benefit patients who were not accustomed to its bitter taste. TRIAL REGISTRATION: This study was registered in the Japan Registry of Clinical Trials (jRCTs041210019) on May 14, 2021.

The small-molecule BMH-21 directly inhibits transcription elongation and DNA occupancy of RNA polymerase I in vivo and in vitro.

Cancer cells are dependent upon an abundance of ribosomes to maintain rapid cell growth and proliferation. The rate-limiting step of ribosome biogenesis is ribosomal RNA (rRNA) synthesis by RNA polymerase I (Pol I). Therefore, a goal of the cancer therapeutic field is to develop and characterize Pol I inhibitors. Here, we elucidate the mechanism of Pol I inhibition by a first-in-class small-molecule BMH-21. To characterize the effects of BMH-21 on Pol I transcription, we leveraged high-resolution in vitro transcription assays and in vivo native elongating transcript sequencing (NET-seq). We find that Pol I transcription initiation, promoter escape, and elongation are all inhibited by BMH-21 in vitro. In particular, the transcription elongation phase is highly sensitive to BMH-21 treatment, as it causes a decrease in transcription elongation rate and an increase in paused Pols on the ribosomal DNA (rDNA) template. In vivo NET-seq experiments complement these findings by revealing a reduction in Pol I occupancy on the template and an increase in sequence-specific pausing upstream of G-rich rDNA sequences after BMH-21 treatment. Collectively, these data reveal the mechanism of action of BMH-21, which is a critical step forward in the development of this compound and its derivatives for clinical use.

Toward "E-Ring-Free" Lamellarin Analogues: Synthesis and Preliminary Biological Evaluation.

Since the discovery of anticancer properties of a naturally occurring hexacyclic marine alkaloid Lamellarin D, the attempts have been made to prepare its synthetic analogues and elucidate the effects of each structural component on their activity profile. While F-ring-free, A-ring-free and B-ring-open lamellarins are known, E-ring-free analogues have never been investigated. In this work, we developed a facile and straightforward synthetic method toward E-ring-free lamellarin analogues based on the [3+2]-cycloaddition. For the first time, we prepared several pentacyclic lamellarin analogues without E-ring in their structure and assessed their cytotoxicity in a panel of cancer cell lines in comparison with several hexacyclic lamellarins. E-ring-free lamellarins were devoid of cytotoxicity due to their poor solubility in cellular environment.

Shear stress-induced cellular senescence blunts liver regeneration through Notch-sirtuin 1-P21/P16 axis.

BACKGROUND AND AIMS: The mechanisms involved in liver regeneration after partial hepatectomy (pHx) are complicated. Cellular senescence, once linked to aging, plays a pivotal role in wound repair. However, the regulatory effects of cellular senescence on liver regeneration have not been fully elucidated. APPROACH AND RESULTS: Mice subjected to pHx were analyzed 14 days after surgery. The incomplete remodeling of liver sinusoids affected shear stress-induced endothelial nitric oxide synthase (eNOS) signaling on day 14, resulting in the accumulation of senescent LSECs. Removing macrophages to augment LSEC senescence led to a malfunction of the regenerating liver. A dynamic fluctuation in Notch activity accompanied senescent LSEC accumulation during liver regeneration. Endothelial Notch activation by using Cdh5-CreERT NICeCA mice triggered LSEC senescence and senescence-associated secretory phenotype, which disrupted liver regeneration. Blocking the Notch by γ-secretase inhibitor (GSI) diminished senescence and promoted LSEC expansion. Mechanically, Notch-hairy and enhancer of split 1 signaling inhibited sirtuin 1 (Sirt1) transcription by binding to its promoter region. Activation of Sirt1 by SRT1720 neutralized the up-regulation of P53, P21, and P16 caused by Notch activation and eliminated Notch-driven LSEC senescence. Finally, Sirt1 activator promoted liver regeneration by abrogating LSEC senescence and improving sinusoid remodeling. CONCLUSIONS: Shear stress-induced LSEC senescence driven by Notch interferes with liver regeneration after pHx. Sirt1 inhibition accelerates liver regeneration by abrogating Notch-driven senescence, providing a potential opportunity to target senescent cells and facilitate liver repair after injury.

The emerging landscape of RORγt biology.

The transcription factor retinoid-related orphan receptor gamma t (RORγt) has emerged as an exciting target for inflammatory diseases. Xiao et al. (2014) show that a new class of RORγt antagonists can inhibit the inflammatory function of T helper 17 cells without altering RORγt occupancy on its target genes.

Small-molecule RORγt antagonists inhibit T helper 17 cell transcriptional network by divergent mechanisms.

We identified three retinoid-related orphan receptor gamma t (RORγt)-specific inhibitors that suppress T helper 17 (Th17) cell responses, including Th17-cell-mediated autoimmune disease. We systemically characterized RORγt binding in the presence and absence of drugs with corresponding whole-genome transcriptome sequencing. RORγt acts as a direct activator of Th17 cell signature genes and a direct repressor of signature genes from other T cell lineages; its strongest transcriptional effects are on cis-regulatory sites containing the RORα binding motif. RORγt is central in a densely interconnected regulatory network that shapes the balance of T cell differentiation. Here, the three inhibitors modulated the RORγt-dependent transcriptional network to varying extents and through distinct mechanisms. Whereas one inhibitor displaced RORγt from its target loci, the other two inhibitors affected transcription predominantly without removing DNA binding. Our work illustrates the power of a system-scale analysis of transcriptional regulation to characterize potential therapeutic compounds that inhibit pathogenic Th17 cells and suppress autoimmunity.

PDE1 Inhibition Modulates Cav1.2 Channel to Stimulate Cardiomyocyte Contraction.

[Figure: see text].

Sparstolonin B inhibits pancreatic adenocarcinoma through the NF-κB signaling pathway.

Pancreatic adenocarcinoma is a highly lethal malignant gastrointestinal tumor. Sparstolonin B is an isocoumarin whose anticancer activity has recently received increasing attention. This study aimed to investigate Sparstolonin B's potential antitumor effect on pancreatic adenocarcinoma. The effect of Sparstolonin B on pancreatic cancer target genes and molecular mechanism was predicted via network pharmacology; Sparstolonin B significantly decreased Panc-1 and SW1990 cell viability and effectively suppressed the proliferation, migration, and invasion of pancreatic cancer cells as shown by CCK-8, colony formation, and Transwell assays. Flow cytometry showed that it induced cell cycle arrest and apoptosis. Sparstolonin B also upregulated Bax levels but decreased those of MMP2 and Bcl-2, downregulated IκBα expression, and upregulated p65 and IκBα phosphorylation; however, it had no effect on total NF-κB p65 levels. The NF-κB pathway inhibitor QNZ reversed these effects. The treatment group (26 µmol/L) had reduced graft volume and weight and fewer Ki-67-positive cells than the control group. Therefore, Sparstolonin B can inhibit the growth and induce the apoptosis of pancreatic cancer cells via the NF-κB signaling pathway and may be a potential novel drug for pancreatic cancer treatment.

Discovery of a selective catalytic p300/CBP inhibitor that targets lineage-specific tumours.

The dynamic and reversible acetylation of proteins, catalysed by histone acetyltransferases (HATs) and histone deacetylases (HDACs), is a major epigenetic regulatory mechanism of gene transcription and is associated with multiple diseases. Histone deacetylase inhibitors are currently approved to treat certain cancers, but progress on the development of drug-like histone actyltransferase inhibitors has lagged behind. The histone acetyltransferase paralogues p300 and CREB-binding protein (CBP) are key transcriptional co-activators that are essential for a multitude of cellular processes, and have also been implicated in human pathological conditions (including cancer). Current inhibitors of the p300 and CBP histone acetyltransferase domains, including natural products, bi-substrate analogues and the widely used small molecule C646, lack potency or selectivity. Here, we describe A-485, a potent, selective and drug-like catalytic inhibitor of p300 and CBP. We present a high resolution (1.95 Å) co-crystal structure of a small molecule bound to the catalytic active site of p300 and demonstrate that A-485 competes with acetyl coenzyme A (acetyl-CoA). A-485 selectively inhibited proliferation in lineage-specific tumour types, including several haematological malignancies and androgen receptor-positive prostate cancer. A-485 inhibited the androgen receptor transcriptional program in both androgen-sensitive and castration-resistant prostate cancer and inhibited tumour growth in a castration-resistant xenograft model. These results demonstrate the feasibility of using small molecule inhibitors to selectively target the catalytic activity of histone acetyltransferases, which may provide effective treatments for transcriptional activator-driven malignancies and diseases.

Inhibition of PIKfyve kinase prevents infection by Zaire ebolavirus and SARS-CoV-2.

Virus entry is a multistep process. It initiates when the virus attaches to the host cell and ends when the viral contents reach the cytosol. Genetically unrelated viruses can subvert analogous subcellular mechanisms and use similar trafficking pathways for successful entry. Antiviral strategies targeting early steps of infection are therefore appealing, particularly when the probability for successful interference through a common step is highest. We describe here potent inhibitory effects on content release and infection by chimeric vesicular stomatitis virus (VSV) containing the envelope proteins of Zaire ebolavirus (VSV-ZEBOV) or severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) (VSV-SARS-CoV-2) elicited by Apilimod and Vacuolin-1, small-molecule inhibitors of the main endosomal phosphatidylinositol-3-phosphate/phosphatidylinositol 5-kinase, PIKfyve. We also describe potent inhibition of SARS-CoV-2 strain 2019-nCoV/USA-WA1/2020 by Apilimod. These results define tools for studying the intracellular trafficking of pathogens elicited by inhibition of PIKfyve kinase and suggest the potential for targeting this kinase in developing small-molecule antivirals against SARS-CoV-2.

Osteogenic Activities of Trifolirhizin as a Bioactive Compound for the Differentiation of Osteogenic Cells.

Plant extracts are widely used as traditional medicines. Sophora flavescens Aiton-derived natural compounds exert various beneficial effects, such as anti-inflammatory, anticancer, antioxidant, and antiregenerative activities, through their bioactive compounds, including flavonoids and alkaloids. In the present study, we investigated the biological effects of an S. flavescens-derived flavonoid, trifolirhizin (trifol), on the stimulation of osteogenic processes during osteoblast differentiation. Trifol (>98% purity) was successfully isolated from the root of S. flavescens and characterized. Trifol did not exhibit cellular toxicity in osteogenic cells, but promoted alkaline phosphatase (ALP) staining and activity, with enhanced expression of the osteoblast differentiation markers, including Alp, ColI, and Bsp. Trifol induced nuclear runt-related transcription factor 2 (RUNX2) expression during the differentiation of osteogenic cells, and concomitantly stimulated the major osteogenic signaling proteins, including GSK3ß, ß-catenin, and Smad1/5/8. Among the mitogen-activated protein kinases (MAPKs), Trifol activated JNK, but not ERK1/2 and p38. Trifol also increased the osteoblast-mediated bone-forming phenotypes, including transmigration, F-actin polymerization, and mineral apposition, during osteoblast differentiation. Overall, trifol exhibits bioactive activities related to osteogenic processes via differentiation, migration, and mineralization. Collectively, these results suggest that trifol may serve as an effective phytomedicine for bone diseases such as osteoporosis.

Second-Generation Human Immunodeficiency Virus Integrase Inhibitors Induce Differentiation Dysregulation and Exert Toxic Effects in Human Embryonic Stem Cell and Mouse Models.

BACKGROUND: Each year, approximately 1.1 million children are exposed in utero to human immunodeficiency virus antiretrovirals, yet their safety is often not well characterized during pregnancy. The Tsepamo study reported a neural tube defect signal in infants exposed to the integrase strand transfer inhibitor (InSTI) dolutegravir from conception, suggesting that exposure during early fetal development may be detrimental. METHODS: The effects of InSTIs on 2 human embryonic stem cell (hESC) lines were characterized with respect to markers of pluripotency, early differentiation, and cellular health. In addition, fetal resorptions after exposure to InSTIs from conception were analyzed in pregnant mice. RESULTS: At subtherapeutic concentrations, second-generation InSTIs bictegravir, cabotegravir, and dolutegravir decreased hESC counts and pluripotency and induced dysregulation of genes involved in early differentiation. At therapeutic concentrations, bictegravir induced substantial hESC death and fetal resorptions. It is notable that first-generation InSTI raltegravir did not induce any hESC toxicity or differentiation, at any concentration tested. CONCLUSIONS: Exposure to some InSTIs, even at subtherapeutic concentrations, can induce adverse effects in hESCs and pregnant mice. Given the increasingly prevalent use of second-generation InSTIs, including in women of reproductive age, it is imperative to further elucidate the effect of InSTIs on embryonic development, as well as their long-term safety after in utero exposure.

A clinical review of HIV integrase strand transfer inhibitors (INSTIs) for the prevention and treatment of HIV-1 infection.

Integrase strand transfer inhibitors (INSTIs) have improved the treatment of human immunodeficiency virus (HIV). There are currently four approved for use in treatment-naïve individuals living with HIV; these include first generation raltegravir, elvitegravir, and second generation dolutegravir and bictegravir. The most recent INSTI, cabotegravir, is approved for (1) treatment of HIV infection in adults to replace current antiretroviral therapy in individuals who maintain virologic suppression on a stable antiretroviral regimen without history of treatment failure and no known resistance to its components and (2) pre-exposure prophylaxis in individuals at risk of acquiring HIV-1 infection. Cabotegravir can be administered intramuscularly as a monthly or bi-monthly injection depending on the indication. This long-acting combination has been associated with treatment satisfaction in clinical studies and may be helpful for individuals who have difficulty taking daily oral medications. Worldwide, second generation INSTIs are preferred for treatment-naïve individuals. Advantages of these INSTIs include their high genetic barrier to resistance, limited drug-drug interactions, excellent rates of virologic suppression, and favorable tolerability. Few INSTI resistance-associated mutations have been reported in clinical trials involving dolutegravir, bictegravir and cabotegravir. Other advantages of specific INSTIs include their use in various populations such as infants and children, acute HIV infection, and individuals of childbearing potential. The most common adverse events observed in clinical studies involving INSTIs included diarrhea, nausea, insomnia, fatigue, and headache, with very low rates of treatment discontinuation versus comparator groups. The long-term clinical implications of weight gain associated with second generation INSTIs dolutegravir and bictegravir warrants further study. This review summarizes key clinical considerations of INSTIs in terms of clinical pharmacology, drug-drug interactions, resistance, and provides perspective on clinical decision-making. Additionally, we summarize major clinical trials evaluating the efficacy and safety of INSTIs in treatment-naïve patients living with HIV as well as individuals at risk of acquiring HIV infection.

NecroX-5 ameliorates bleomycin-induced pulmonary fibrosis via inhibiting NLRP3-mediated epithelial-mesenchymal transition.

BACKGROUND: Pulmonary fibrosis is a progressive and usually lethal pulmonary disease. Despite considerable research efforts, no effective therapeutic strategy for pulmonary fibrosis has been developed. NecroX-5 has been reported to possess anti-inflammatory, anti-oxidative and anti-tumor activities. In the present study, we aimed to determine whether NecroX-5 exhibits antifibrotic property in bleomycin (BLM)-induced pulmonary fibrosis. RESULTS: We found that pre-treatment with NecroX-5 alleviated inflammatory response, reduced oxidative stress, inhibited epithelial-mesenchymal transition (EMT), and ameliorated pulmonary fibrosis in vivo and in vitro. Our data further indicated that NecroX-5 substantially reduced activation of NLRP3 inflammasome and TGF-ß1/Smad2/3 signaling in vivo and in vitro. Additionally, NLRP3 overexpression significantly reversed the protective effects of NecroX-5 in lung epithelial cells exposed to BLM. CONCLUSIONS: Overall, our results demonstrate the potent antifibrotic properties of NecroX-5 and its therapeutic potential for pulmonary fibrosis.

Recent applications of seven-membered rings in drug design.

This short review aims at highlighting recent design strategies hinged on using seven-membered rings. Analyses of the different selected examples coupled with torsion profiles derived from the CCDC suggest some of these strategies could have broad applications.