17 research outputs found
Recommended from our members
De novo design of drug-binding proteins with predictable binding energy and specificity
The de novo design of small molecule-binding proteins has seen exciting recent progress; however, high-affinity binding and tunable specificity typically require laborious screening and optimization after computational design. We developed a computational procedure to design a protein that recognizes a common pharmacophore in a series of poly(ADP-ribose) polymerase-1 inhibitors. One of three designed proteins bound different inhibitors with affinities ranging from <5 nM to low micromolar. X-ray crystal structures confirmed the accuracy of the designed protein-drug interactions. Molecular dynamics simulations informed the role of water in binding. Binding free energy calculations performed directly on the designed models were in excellent agreement with the experimentally measured affinities. We conclude that de novo design of high-affinity small molecule-binding proteins with tuned interaction energies is feasible entirely from computation
Preliminary development of an assay for detection of TERT expression, telomere length, and telomere elongation in single cells.
The telomerase enzyme enables unlimited proliferation of most human cancer cells by elongating telomeres and preventing replicative senescence. Despite the critical importance of telomerase in cancer biology, challenges detecting telomerase activity and expression in individual cells have hindered the ability to study patterns of telomerase expression and function across heterogeneous cell populations. While sensitive assays to ascertain telomerase expression and function exist, these approaches have proven difficult to implement at the single cell level. Here, we validate in situ RNAscope detection of the telomerase TERT mRNA and couple this assay with our recently described TSQ1 method for in situ detection of telomere elongation. This approach enables detection of TERT expression, telomere length, and telomere elongation within individual cells of the population. Using this assay, we show that the heterogeneous telomere elongation observed across a HeLa cell population is in part driven by variable expression of the TERT gene. Furthermore, we show that the absence of detectable telomere elongation in some TERT-positive cells is the result of inhibition by the telomeric shelterin complex. This combined assay provides a new approach for understanding the integrated expression, function, and regulation of telomerase at the single cell level
A Whole-Genome CRISPR Screen Identifies AHR Loss as a Mechanism of Resistance to a PARP7 Inhibitor.
Inhibitors directed toward PARP1 and PARP2 are approved agents for the treatment of BRCA1 and BRCA2-related cancers. Other members of the PARP family have also been implicated in cancer and are being assessed as therapeutic targets in cancer and other diseases. Recently, an inhibitor of PARP7 (RBN-2397) has reached early-stage human clinical trials. Here, we performed a genome-wide CRISPR screen for genes that modify the response of cells to RBN-2397. We identify the polycyclic aromatic hydrocarbon receptor AHR and multiple components of the cohesin complex as determinants of resistance to this agent. Activators and inhibitors of AHR modulate the cellular response to PARP7 inhibition, suggesting potential combination therapy approaches
Recommended from our members
Genome-wide CRISPR Screens in Primary Human T Cells Reveal Key Regulators of Immune Function
Human T cells are central effectors of immunity and cancer immunotherapy. CRISPR-based functional studies in T cells could prioritize novel targets for drug development and improve the design of genetically reprogrammed cell-based therapies. However, large-scale CRISPR screens have been challenging in primary human cells. We developed a new method, single guide RNA (sgRNA) lentiviral infection with Cas9 protein electroporation (SLICE), to identify regulators of stimulation responses in primary human T cells. Genome-wide loss-of-function screens identified essential T cell receptor signaling components and genes that negatively tune proliferation following stimulation. Targeted ablation of individual candidate genes characterized hits and identified perturbations that enhanced cancer cell killing. SLICE coupled with single-cell RNA sequencing (RNA-seq) revealed signature stimulation-response gene programs altered by key genetic perturbations. SLICE genome-wide screening was also adaptable to identify mediators of immunosuppression, revealing genes controlling responses to adenosine signaling. The SLICE platform enables unbiased discovery and characterization of functional gene targets in primary cells
Recommended from our members
Design and Synthesis of Poly(ADP-ribose) Polymerase Inhibitors: Impact of Adenosine Pocket-Binding Motif Appendage to the 3‑Oxo-2,3-dihydrobenzofuran-7-carboxamide on Potency and Selectivity
Poly(adenosine 5'-diphosphate-ribose) polymerase (PARP) inhibitors are a class of anticancer drugs that block the catalytic activity of PARP proteins. Optimization of our lead compound 1 (( Z)-2-benzylidene-3-oxo-2,3-dihydrobenzofuran-7-carboxamide; PARP-1 IC50 = 434 nM) led to a tetrazolyl analogue (51, IC50 = 35 nM) with improved inhibition. Isosteric replacement of the tetrazole ring with a carboxyl group (60, IC50 = 68 nM) gave a promising new lead, which was subsequently optimized to obtain analogues with potent PARP-1 IC50 values (4-197 nM). PARP enzyme profiling revealed that the majority of compounds are selective toward PARP-2 with IC50 values comparable to clinical inhibitors. X-ray crystal structures of the key inhibitors bound to PARP-1 illustrated the mode of interaction with analogue appendages extending toward the PARP-1 adenosine-binding pocket. Compound 81, an isoform-selective PARP-1/-2 (IC50 = 30 nM/2 nM) inhibitor, demonstrated selective cytotoxic effect toward breast cancer gene 1 ( BRCA1)-deficient cells compared to isogenic BRCA1-proficient cells
Recommended from our members
PLX038: A Long-Acting Topoisomerase I Inhibitor With Robust Antitumor Activity in ATM-Deficient Tumors and Potent Synergy With PARP Inhibitors.
Alterations in the ATM gene are among the most common somatic and hereditary cancer mutations, and ATM-deficient tumors are hypersensitive to DNA-damaging agents. A synthetic lethal combination of DNA-damaging agents and DNA repair inhibitors could have widespread utility in ATM-deficient cancers. However, overlapping normal tissue toxicities from these drug classes have precluded their clinical translation. We investigated PLX038, a releasable polyethylene glycol-conjugate of the topoisomerase I inhibitor SN-38, in ATM wild-type and null isogenic xenografts and in a BRCA1-deficient xenograft. PLX038 monotherapy and combination with PARP inhibition potently inhibited the growth of both BRCA1- and ATM-deficient tumors. A patient with an ATM-mutated breast cancer treated with PLX038 and the PARP inhibitor rucaparib achieved rapid, symptomatic, and radiographic complete response lasting 12 months. Single-agent PLX038 or PLX038 in combination with DNA damage response inhibitors are novel therapeutic paradigms for patients with ATM-loss cancers
Replication stress is a potent driver of functional decline in ageing haematopoietic stem cells
Haematopoietic stem cells (HSCs) self-renew for life, thereby making them one of the few blood cells that truly age(1,2). Paradoxically, although HSCs numerically expand with age, their functional activity declines over time, resulting in degraded blood production and impaired engraftment following transplantation(2). While many drivers of HSC ageing have been proposed(2-5), the reason why HSC function degrades with age remains unknown. Here we show that cycling old HSCs in mice have heightened levels of replication stress associated with cell cycle defects and chromosome gaps or breaks, which are due to decreased expression of mini-chromosome maintenance (MCM) helicase components and altered dynamics of DNA replication forks. Nonetheless, old HSCs survive replication unless confronted with a strong replication challenge, such as transplantation. Moreover, once old HSCs re-establish quiescence, residual replication stress on ribosomal DNA (rDNA) genes leads to the formation of nucleolar-associated gamma H2AX signals, which persist owing to ineffective H2AX dephosphorylation by mislocalized PP4c phosphatase rather than ongoing DNA damage. Persistent nucleolar gamma H2AX also acts as a histone modification marking the transcriptional silencing of rDNA genes and decreased ribosome biogenesis in quiescent old HSCs. Our results identify replication stress as a potent driver of functional decline in old HSCs, and highlight the MCM DNA helicase as a potential molecular target for rejuvenation therapies
Recommended from our members
Resistance to ATR Inhibitors Is Mediated by Loss of the Nonsense-Mediated Decay Factor UPF2.
Over one million cases of gastric cancer are diagnosed each year globally, and the metastatic disease continues to have a poor prognosis. A significant proportion of gastric tumors have defects in the DNA damage response pathway, creating therapeutic opportunities through synthetic lethal approaches. Several small-molecule inhibitors of ATR, a key regulator of the DNA damage response, are now in clinical development as targeted agents for gastric cancer. Here, we performed a large-scale CRISPR interference screen to discover genetic determinants of response and resistance to ATR inhibitors (ATRi) in gastric cancer cells. Among the top hits identified as mediators of ATRi response were UPF2 and other components of the nonsense-mediated decay (NMD) pathway. Loss of UPF2 caused ATRi resistance across multiple gastric cancer cell lines. Global proteomic, phosphoproteomic, and transcriptional profiling experiments revealed that cell-cycle progression and DNA damage responses were altered in UPF2-mutant cells. Further studies demonstrated that UPF2-depleted cells failed to accumulate in G1 following treatment with ATRi. UPF2 loss also reduced transcription-replication collisions, which has previously been associated with ATRi response, thereby suggesting a possible mechanism of resistance. Our results uncover a novel role for NMD factors in modulating response to ATRi in gastric cancer, highlighting a previously unknown mechanism of resistance that may inform the clinical use of these drugs.SignificanceLoss of NMD proteins promotes resistance to ATR inhibitors in gastric cancer cells, which may provide a combination of therapeutic targets and biomarkers to improve the clinical utility of these drugs
In situ visualization of telomere elongation patterns in human cells
The telomerase enzyme plays a critical role in human aging and cancer biology by maintaining telomere length and extending the proliferative lifespan of most stem cells and cancer cells. Despite the importance of this enzyme, our understanding of the mechanisms that regulate its activity and establish telomere length homeostasis in mammalian cells is incomplete, in part because the perfect repetitive nature of telomeric sequence hampers in situ detection of telomere elongation patterns. Here, we describe a novel assay using a mutant telomerase that adds a well-tolerated variant telomeric repeat sequence to telomere ends. By specifically detecting the addition of these variant repeats, we can directly visualize telomere elongation events in human cells. We validate this approach by in situ mapping of telomere elongation patterns within individual nuclei and across a population of cells
A single inactivating amino acid change in the SARS-CoV-2 NSP3 Mac1 domain attenuates viral replication in vivo.
Despite unprecedented efforts, our therapeutic arsenal against SARS-CoV-2 remains limited. The conserved macrodomain 1 (Mac1) in NSP3 is an enzyme exhibiting ADP-ribosylhydrolase activity and a possible drug target. To determine the role of Mac1 catalytic activity in viral replication, we generated recombinant viruses and replicons encoding a catalytically inactive NSP3 Mac1 domain by mutating a critical asparagine in the active site. While substitution to alanine (N40A) reduced catalytic activity by ~10-fold, mutations to aspartic acid (N40D) reduced activity by ~100-fold relative to wild-type. Importantly, the N40A mutation rendered Mac1 unstable in vitro and lowered expression levels in bacterial and mammalian cells. When incorporated into SARS-CoV-2 molecular clones, the N40D mutant only modestly affected viral fitness in immortalized cell lines, but reduced viral replication in human airway organoids by 10-fold. In mice, the N40D mutant replicated at >1000-fold lower levels compared to the wild-type virus while inducing a robust interferon response; all animals infected with the mutant virus survived infection. Our data validate the critical role of SARS-CoV-2 NSP3 Mac1 catalytic activity in viral replication and as a promising therapeutic target to develop antivirals