64 research outputs found
Small Molecule Inhibitor of CBFbeta-RUNX Binding for RUNX Transcription Factor Driven Cancers
Transcription factors have traditionally been viewed with skepticism as viable drug targets, but they offer the potential for completely novel mechanisms of action that could more effectively address the stem cell like properties, such as self-renewal and chemo-resistance, that lead to the failure of traditional chemotherapy approaches. Core binding factor is a heterodimeric transcription factor comprised of one of 3 RUNX proteins (RUNX1-3) and a CBFbeta binding partner. CBFbeta enhances DNA binding of RUNX subunits by relieving auto-inhibition. Both RUNX1 and CBFbeta are frequently mutated in human leukemia. More recently, RUNX proteins have been shown to be key players in epithelial cancers, suggesting the targeting of this pathway could have broad utility. In order to test this, we developed small molecules which bind to CBFbeta and inhibit its binding to RUNX. Treatment with these inhibitors reduces binding of RUNX1 to target genes, alters the expression of RUNX1 target genes, and impacts cell survival and differentiation. These inhibitors show efficacy against leukemia cells as well as basal-like (triple-negative) breast cancer cells. These inhibitors provide effective tools to probe the utility of targeting RUNX transcription factor function in other cancers
The NOESY Jigsaw: Automated Protein Secondary Structure and Main-Chain Assignment from Sparse, Unassigned NMR Data
High-throughput, data-directed computational protocols for Structural Genomics (or Proteomics) are required in order to evaluate the protein products of genes for structure and function at rates comparable to current gene-sequencing technology. This paper presents the Jigsaw algorithm, a novel high-throughput, automated approach to protein structure characterization with nuclear magnetic resonance (NMR). Jigsaw consists of two main components: (1) graph-based secondary structure pattern identification in unassigned heteronuclear NMR data, and (2) assignment of spectral peaks by probabilistic alignment of identified secondary structure elements against the primary sequence. Jigsaw\u27s deferment of assignment until after secondary structure identification differs greatly from traditional approaches, which begin by correlating peaks among dozens of experiments. By deferring assignment, Jigsaw not only eliminates this bottleneck, it also allows the number of experiments to be reduced from dozens to four, none of which requires 13C-labeled protein. This in turn dramatically reduces the amount and expense of wet lab molecular biology for protein expression and purification, as well as the total spectrometer time to collect data.
Our results for three test proteins demonstrate that we are able to identify and align approximately 80 percent of alpha-helical and 60 percent of beta-sheet structure. Jigsaw is extremely fast, running in minutes on a Pentium-class Linux workstation. This approach yields quick and reasonably accurate (as opposed to the traditional slow and extremely accurate) structure calculations, utilizing a suite of graph analysis algorithms to compensate for the data sparseness. Jigsaw could be used for quick structural assays to speed data to the biologist early in the process of investigation, and could in principle be applied in an automation-like fashion to a large fraction of the proteome
On the mechanism of autoinhibition of the RhoA-specific nucleotide exchange factor PDZRhoGEF
<p>Abstract</p> <p>Background</p> <p>The Dbl-family of guanine nucleotide exchange factors (GEFs) activate the cytosolic GTPases of the Rho family by enhancing the rate of exchange of GTP for GDP on the cognate GTPase. This catalytic activity resides in the DH (Dbl-homology) domain, but typically GEFs are multidomain proteins containing other modules. It is believed that GEFs are autoinhibited in the cytosol due to supramodular architecture, and become activated in diverse signaling pathways through conformational change and exposure of the DH domain, as the protein is translocated to the membrane. A small family of RhoA-specific GEFs, containing the RGSL (regulators of G-protein signaling-like) domain, act as effectors of select GPCRs <it>via </it>Gα<sub>12/13</sub>, although the molecular mechanism by which this pathway operates is not known. These GEFs include p115, LARG and PDZRhoGEF (PRG).</p> <p>Results</p> <p>Here we show that the autoinhibition of PRG is caused largely by an interaction of a short negatively charged sequence motif, immediately upstream of the DH-domain and including residues Asp706, Glu708, Glu710 and Asp712, with a patch on the catalytic surface of the DH-domain including Arg867 and Arg868. In the absence of both PDZ and RGSL domains, the DH-PH tandem with additional 21 residues upstream, is 50% autoinhibited. However, within the full-length protein, the PDZ and/or RGSL domains significantly restore autoinhibition.</p> <p>Conclusion</p> <p>Our results suggest a mechanism for autoinhibition of RGSL family of GEFs, in which the RGSL domain and a unique sequence motif upstream of the DH domain, act cooperatively to reduce the ability of the DH domain to bind the nucleotide free RhoA. The activation mechanism is likely to involve two independent steps, i.e. displacement of the RGSL domain and conformational change involving the autoinhibitory sequence motif containing several negatively charged residues.</p
A small-molecule inhibitor of the aberrant transcription factor CBFβ-SMMHC delays leukemia in mice
This is the author’s version of the work. It is posted here by permission of the AAAS for personal use, not for redistribution. The definitive version was published in Science on 2015 February 13; 347(6223): 779–784, DOI: 10.1126/science.aaa0314.Acute myeloid leukemia (AML) is the most common form of adult leukemia. The transcription factor fusion CBFβ-SMMHC (core binding factor β and the smooth-muscle myosin heavy chain), expressed in AML with the chromosome inversion inv(16)(p13q22), outcompetes wild-type CBFβ for binding to the transcription factor RUNX1, deregulates RUNX1 activity in hematopoiesis, and induces AML. Current inv(16) AML treatment with nonselective cytotoxic chemotherapy results in a good initial response but limited long-term survival. Here, we report the development of a protein-protein interaction inhibitor, AI-10-49, that selectively binds to CBFβ-SMMHC and disrupts its binding to RUNX1. AI-10-49 restores RUNX1 transcriptional activity, displays favorable pharmacokinetics, and delays leukemia progression in mice. Treatment of primary inv(16) AML patient blasts with AI-10-49 triggers selective cell death. These data suggest that direct inhibition of the oncogenic CBFβ-SMMHC fusion protein may be an effective therapeutic approach for inv(16) AML, and they provide support for transcription factor targeted therapy in other cancers
Gene regulatory network analysis predicts cooperating transcription factor regulons required for FLT3-ITD+ AML growth
Acute myeloid leukemia (AML) is a heterogeneous disease caused by different mutations. Previously, we showed that each mutational subtype develops its specific gene regulatory network (GRN) with transcription factors interacting within multiple gene modules, many of which are transcription factor genes themselves. Here, we hypothesize that highly connected nodes within such networks comprise crucial regulators of AML maintenance. We test this hypothesis using FLT3-ITD-mutated AML as a model and conduct an shRNA drop-out screen informed by this analysis. We show that AML-specific GRNs predict crucial regulatory modules required for AML growth. Furthermore, our work shows that all modules are highly connected and regulate each other. The careful multi-omic analysis of the role of one (RUNX1) module by shRNA and chemical inhibition shows that this transcription factor and its target genes stabilize the GRN of FLT3-ITD+ AML and that its removal leads to GRN collapse and cell death.</p
Pharmacological inhibition of RAS overcomes FLT3 inhibitor resistance in FLT3-ITD+ AML through AP-1 and RUNX1
AML is characterized by mutations in genes associated with growth regulation such as internal tandem duplications (ITD) in the receptor kinase FLT3. Inhibitors targeting FLT3 (FLT3i) are being used to treat patients with FLT3-ITD+ but most relapse and become resistant. To elucidate the resistance mechanism, we compared the gene regulatory networks (GRNs) of leukemic cells from patients before and after relapse, which revealed that the GRNs of drug-responsive patients were altered by rewiring their AP-1-RUNX1 axis. Moreover, FLT3i induces the upregulation of signaling genes, and we show that multiple cytokines, including interleukin-3 (IL-3), can overcome FLT3 inhibition and send cells back into cycle. FLT3i leads to loss of AP-1 and RUNX1 chromatin binding, which is counteracted by IL-3. However, cytokine-mediated drug resistance can be overcome by a pan-RAS inhibitor. We show that cytokines instruct AML growth via the transcriptional regulators AP-1 and RUNX1 and that pan-RAS drugs bypass this barrier
The disruption of protein-protein interactions as a therapeutic strategy for prostate cancer
This is an accepted manuscript of an article published by Elsevier in Pharmacological Research on 16/08/2020, available online: https://doi.org/10.1016/j.phrs.2020.105145
The accepted version of the publication may differ from the final published version.Prostate cancer (PCa) is one of the most common male-specific cancers worldwide, with high morbidity and mortality rates associated with advanced disease stages. The current treatment options of PCa are prostatectomy, hormonal therapy, chemotherapy or radiotherapy, the selection of which is usually dependent upon the stage of the disease. The development of PCa to a castration-resistant phenotype (CRPC) is associated with a more severe prognosis requiring the development of a new and effective therapy. Protein-protein interactions (PPIs) have been recognised as an emerging drug modality and targeting PPIs is a promising therapeutic approach for several diseases, including cancer. The efficacy of several compounds in which target PPIs and consequently impair disease progression were validated in phase I/II clinical trials for different types of cancer. In PCa, various small molecules and peptides proved successful in inhibiting important PPIs, mainly associated with the androgen receptor (AR), Bcl-2 family proteins, and kinases/phosphatases, thus impairing the growth of PCa cells in vitro. Moreover, a majority of these compounds require further validation in vivo and, preferably, in clinical trials. In addition, several other PPIs associated with PCa progression have been identified and now require experimental validation as potential therapeutic loci.
In conclusion, we consider the disruption of PPIs to be a promising though challenging therapeutic strategy for Pca. Agents which modulate PPIs might be employed as a monotherapy or as an adjunct to classical chemotherapeutics to overcome drug resistance and improve efficacy. The discovery of new PPIs with important roles in disease progression, and of novel optimized strategies to target them, are major challenges for the scientific and pharmacological communities.We thank the Portuguese Foundation for Science and Technology(FCT), European Union, QREN, FEDER and COMPETE for funding iBiMED (UIDB/04501/2020, POCI-01-0145-FEDER-23007628 and UID/BIM/04501/2019) and an individual scholarship from BM (SFRH/BD/146032/2019)
Molecular Basis and Targeted Inhibition of CBFbeta-SMMHC Acute Myeloid Leukemia
Acute myeloid leukemia (AML) is characterized by recurrent chromosomal rearrangements that encode for fusion proteins which drive leukemia initiation and maintenance. The inv(16) (p13q22) rearrangement is a founding mutation and the associated CBFbeta-SMMHC fusion protein is essential for the survival of inv(16) AML cells. This Chapter will discuss our understanding of the function of this fusion protein in disrupting hematopoietic homeostasis and creating pre-leukemic blasts, in its cooperation with other co-occurring mutations during leukemia initiation, and in leukemia maintenance. In addition, this chapter will discuss the current approaches used for the treatment of inv(16) AML and the recent development of AI-10-49, a selective targeted inhibitor of CBFbeta-SMMHC/RUNX1 binding, the first candidate targeted therapy for inv(16) AML
Charged Gels as Orienting Media for Measurement of Residual Dipolar Couplings in Soluble and Integral Membrane Proteins
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