Papers by Cristina C Clement
Cheng SY, Vargas A, Lee JY, Clement CC, Champeil E.
Mitomycin C (MC) is a well-known DNA alkylat... more Cheng SY, Vargas A, Lee JY, Clement CC, Champeil E.
Mitomycin C (MC) is a well-known DNA alkylating agent. MC analog, 10-decarbamoyl mitomycin C (DMC), unlike MC, has stronger effects on cancer with p53 mutation. We previously demonstrated that MC/DMC could activate p21WAF1/CIP1 in MCF-7 (p53-proficient) and K562 (p53-deficient) cells in a p53-independent mode. This study aimed to elucidate the upstream signaling pathway of p21WAF1/CIP1 activation triggered by MC/DMC. Besides p53, Akt plays an important role on deactivating p21WAF1/CIP1 . The results showed that MC/DMC inhibited Akt in MCF-7 cells, but not in K562 cells. By knocking down p53, the Akt inhibition in MCF-7 cells was alleviated. This implied that the deactivated Akt caused by MC/DMC was p53-dependent. With Akt activator (SC79), p21WAF1/CIP1 activation triggered by MC/DMC in MCF-7 cells was not reduced. This indicated that Akt inhibition triggered by MC/DMC was not associated with MC/DMC-induced p21WAF1/CIP1 activation. Label-free quantitative proteomic profiling analysis revealed that DMC has a stronger effect on downregulating the PI3K/Akt signaling pathway in MCF-7 cells as compared with MC. No significant effect of MC/DMC on PI3K/Akt in K562 cells was observed. In summary, MC/DMC regulate Akt activation in a p53-dependent manner. This Akt deactivation is not associated with p21WAF1/CIP1 activation in response to MC/DMC. This article is protected by copyright. All rights reserved.
Transport of tissue-derived lymphatic fluid and clearance by draining lymph nodes are pivotal for... more Transport of tissue-derived lymphatic fluid and clearance by draining lymph nodes are pivotal for maintenance of fluid homeostasis in the body and for immune-surveillance of the self-and non-self-proteomes. Yet a quantitative analysis of nodal filtration of the tissue-derived proteome present in lymphatic fluid has not been reported. Here we quantified the efficiency of nodal clearance of the composite proteomic load using label-free and isotope-labeling proteomic analysis of pre-nodal and post-nodal samples collected by direct cannulation. These results were extended by quantitation of the filtration efficiency of fluorophore-labeled proteins, bacteria, and beads infused at physiological flow rates into pre-nodal lymphatic collectors and collected by post-nodal cannulation. We developed a linear model of nodal filtration efficiency dependent on pre-nodal protein concentrations and molecular weight, and uncovered criteria for disposing the proteome incoming from defined anatomical districts under physiological conditions. These findings are pivotal to understanding the maximal antigenic load sustainable by a draining node, and promote understanding of pathogen spreading and nodal filtration of tumor metastasis, potentially helping to improve design of vaccination protocols, immunization strategies and drug delivery. Blood circulating throughout the capillary bed is not in direct contact with the cellular layers of each parenchymal tissue. Thus lipids, proteins, and small molecules need to extravasate in order to provide cellular nutrients and to hydrate tissue cells 1,2. Extravasation is mediated by hydrostatic pressure inside the blood capillaries and by the Starling forces that drive the ultrafiltration process, moving proteins, macromolecules, and associated water into the interstitial space. A fraction of the extravasated fluid will be absorbed back into the capillary bed, but most will remain in the interstitial tissue 1,2. These products of capillary extravasation, combined with secreted products deriving from cellular metabolism and catabolism, make up the interstitial fluid that baths every parenchymal organ 3. Under physiological conditions, in humans, around 8–10 liters of interstitial fluid are formed daily, which need to be returned to the blood circulation to prevent tissue edema 1. However, the vast majority of the interstitial fluid will not directly be reabsorbed into the blood system, but rather will be collected into the lymphatic capillar-ies as lymph, and will pass through one or more of the 600–800 draining lymph nodes disseminated throughout the human body, before circulating into the thoracic duct and then the vena cava 2. There are several possible explanations why interstitial fluid does not drain directly into the general blood circulation but instead is filtered through the lymph nodes. First, lymphatic passage through the nodes ensures that tissue-invading pathogens do not directly enter into the bloodstream but can be captured by dendritic cells and macrophages residing in the lymph node. Second, the collection of products of tissue remodeling, cellular secretion/processing, and extracellular debris by lymphatic fluid ensures that nodal immune cells are constantly exposed to the self-proteome from each parenchymal organ, helping to maintain peripheral tolerance 2,4–7. Third, immune cells patrolling peripheral tissues can use lymph flow as a fast and direct conduit to lymph nodes. Fourth, lymph composition at different times and locations can vary widely in protein concentration, electrolytes composition , pH and cellular composition, as opposed to blood, where these parameters are tightly controlled. Thus, the lymph, as observed in both physiological and pathological conditions, can withstand changes occurring in Published: xx xx xxxx OPEN
Tuberculosis (TB) is caused by Mycobacterium tuberculosis, and remains a major, worldwide health ... more Tuberculosis (TB) is caused by Mycobacterium tuberculosis, and remains a major, worldwide health concern. The emergence of M. tuberculosis strains that are resistant to front line antimicrobacterial drugs such as isoniazid and rifampicin have greatly complicated efforts to control the spread of TB. Of the various resistance mechanisms, the most effective involves the production of β-lactamases that hydrolyze β-lactam antibiotics. One approach to overcoming resistance to β-lactam antibiotics is to find new drugs or additives that can prevent the β-lactam antibiotic from being hydrolyzed by β-lactamases. The spectrum of anti-TB drugs consisting of non β-lactam scaffolds has been expanded by the development of new boronic acid derivatives and cationic peptidic inhibitors that proved effective in neutralizing bacterial resistance, especially when administered in combination with antibiotics. The intrinsic resistance to β-lactam antibiotics was due mainly to the presence of a chromosomally-encoded gene (blaC) in M. tuberculosis for a Class A, Ambler β-lactamase (BlaC). The BlaC enzyme has already been validated as one of the leading targets of tuberculosis therapy. In the search for new β-lactamase inhibitors we employed the on-line server Pharmit (pharmit.csb.pitt.edu) to discover new 3-D pharmacophores and search different databases of chemical libraries, and commercial compounds. Autodock Vina was employed to perform molecular docking of selected compounds from ChEMBL/MolPort/ZINC databases using the M. tuberculosis β-lactamase 3M6B.pdb as the target protein. This combination of 3-D pharmacophore screening and molecular docking lead to the discovery of natural products, and novel, small molecules inhibitors of Y-49 β-lactamase from Mycobacterium tuberculosis. Notably new 3D-pharmacophores derived from hydroxyphenylglycine and fragments from the family of biphenyls, aromatic ketones, and stilbenes emerged as potential inhibitors of the recombinant Y-49 β-lactamase exhibiting in vitro inhibitory constants (Ki), in the low 5uM-200 uM range. These newly discovered pharmacophore features enabled by in silico screening are extending the chemical space for discovery of novel and potent inhibitors of β-lactamases.
PLoS ONE, 2012
The tremendous social and economic impact of thrombotic disorders, together with the considerable... more The tremendous social and economic impact of thrombotic disorders, together with the considerable risks associated to the currently available therapies, prompt for the development of more efficient and safer anticoagulants. Novel peptide-based thrombin inhibitors were identified using in silico structure-based design and further validated in vitro. The best candidate compounds contained both L- and D-amino acids, with the general sequence D-Phe(P3)-Pro(P2)-D-Arg(P1)-P1'-CONH₂. The P1' position was scanned with L- and D-isomers of natural or unnatural amino acids, covering the major chemical classes. The most potent non-covalent and proteolysis-resistant inhibitors contain small hydrophobic or polar amino acids (Gly, Ala, Ser, Cys, Thr) at the P1' position. The lead tetrapeptide, D-Phe-Pro-D-Arg-D-Thr-CONH₂, competitively inhibits α-thrombin's cleavage of the S2238 chromogenic substrate with a K(i) of 0.92 µM. In order to understand the molecular details of their inhibitory action, the three-dimensional structure of three peptides (with…
Cell Reports, 2012
A hallmark of aging is an imbalance between production and clearance of reactive oxygen species a... more A hallmark of aging is an imbalance between production and clearance of reactive oxygen species and increased levels of oxidatively damaged biomolecules. Herein, we demonstrate that splenic and nodal antigen-presenting cells purified from aging mice accumulate oxidatively modified proteins with sidechain carbonylation, advanced glycation end products, and lipid peroxidation. Furthermore, we show that the endosomal accumulation of oxidatively modified proteins interferes with the efficient processing of exogenous antigens and degradation of macroautophagy-delivered proteins. In support of a causative role for oxidized products in the inefficient immune response, a decrease in oxidative stress improved the adaptive immune response to immunizing antigens. These findings underscore a previously unrecognized negative effect of agedependent changes in cellular proteostasis on the immune response.
Peptides with enhanced resistance to proteolysis, based on the amino acid sequence of the F11 rec... more Peptides with enhanced resistance to proteolysis, based on the amino acid sequence of the F11 receptor molecule (F11R, aka JAM-A/Junctional adhesion molecule-A), were designed, prepared, and examined as potential candidates for the development of anti-atherosclerotic and anti-thrombotic therapeutic drugs. A sequence at the N-terminal of F11R together with another sequence located in the first Ig-loop
of this protein, were identified to form a steric active-site operating in the F11R-dependent adhesion between cells that express F11R molecules on their external surface. In silico modeling of the complex between two polypeptide chains with the sequences positioned
in the active-site was used to generate peptide candidates designed to inhibit homophilic interactions between surface-located F11R molecules. The two lead F11R peptides were modified with D-Arg and D-Lys at selective sites, for attaining higher stability to proteolysis in vivo. Using molecular docking experiments we tested different conformational states and the putative binding affinity between two selected D-Arg and D-Lys-modified F11R peptides and the proposed binding pocket. The inhibitory effects of the F11R peptide 2HN-(dK)-SVT-(dR)-EDTGTYTC-CONH2 on antibody-induced platelet aggregation and on the adhesion of platelets to cytokine inflammed
endothelial cells are reported in detail, and the results point out the significant potential utilization of F11R peptides for the prevention and treatment of atherosclerotic plaques and associated thrombotic events.
Peptides 2015, Proceedings of the 24th American Peptide Symposium, 2015
Advances in Experimental Medicine and Biology, 2009
Immunology of the Lymphatic System, 2013
Proceedings of the National Academy of Sciences, 2009
Triple-negative breast cancers (TNBCs) are defined by a lack of expression of estrogen, progester... more Triple-negative breast cancers (TNBCs) are defined by a lack of expression of estrogen, progesterone, and HER2 receptors. Because of the absence of identified targets and targeted therapies, and due to a heterogeneous molecular presentation, treatment guidelines for patients with TNBC include only conventional chemotherapy. Such treatment, while effective for some, leaves others with high rates of early relapse and is not curative for any patient with metastatic disease. Here, we demonstrate that these tumors are sensitive to the heat shock protein 90 (Hsp90) inhibitor PU-H71. Potent and durable anti-tumor effects in TNBC xenografts, including complete response and tumor regression, without toxicity to the host are achieved with this agent. Notably, TNBC tumors respond to retreatment with PU-H71 for several cycles extending for over 5 months without evidence of resistance or toxicity. Through a proteomics approach, we show that multiple oncoproteins involved in tumor proliferation, survival, and invasive potential are in complex with PU-H71-bound Hsp90 in TNBC. PU-H71 induces efficient and sustained downregulation and inactivation, both in vitro and in vivo, of these proteins. Among them, we identify downregulation of components of the Ras/Raf/MAPK pathway and G 2-M phase to contribute to its anti-proliferative effect, degradation of activated Akt and Bcl-xL to induce apoptosis, and inhibition of activated NF-B, Akt, ERK2, Tyk2, and PKC to reduce TNBC invasive potential. The results identify Hsp90 as a critical and multimodal target in this most difficult to treat breast cancer subtype and support the use of the Hsp90 inhibitor PU-H71 for clinical trials involving patients with TNBC.
Nature Chemical Biology, 2007
The heat shock protein 90 (Hsp90) has a critical role in malignant transformation. Whereas its ab... more The heat shock protein 90 (Hsp90) has a critical role in malignant transformation. Whereas its ability to maintain the functional conformations of mutant and aberrant oncoproteins is established, a transformation-specific regulation of the antiapoptotic phenotype by Hsp90 is poorly understood. By using selective compounds, we have discovered that small-cell lung carcinoma is a distinctive cellular system in which apoptosis is mainly regulated by Hsp90. Unlike the well-characterized antiapoptotic chaperone Hsp70, Hsp90 is not a general inhibitor of apoptosis, but it assumes this role in systems such as small-cell lung carcinoma, in which apoptosis is uniquely dependent on and effected through the intrinsic pathway, without involvement of caspase elements upstream of mitochondria or alternate pathways that are not apoptosome-channeled. These results provide important evidence for a transformation-specific interplay between chaperones in regulating apoptosis in malignant cells.
Journal of Medicinal Chemistry, 2014
The discovery and development of heat shock protein 70 (Hsp70) inhibitors is currently a hot topi... more The discovery and development of heat shock protein 70 (Hsp70) inhibitors is currently a hot topic in cancer. In the preceding paper in this issue ( 10.1021/jm401551n ), we have described structure-activity relationship studies in the first Hsp70 inhibitor class rationally designed to bind to a novel allosteric pocket located in the N-terminal domain of the protein. These ligands contained an acrylamide to take advantage of an active cysteine embedded in the allosteric pocket and acted as covalent protein modifiers upon binding. Here, we perform chemical modifications around the irreversible inhibitor scaffold to demonstrate that covalent modification is not a requirement for activity within this class of compounds. The study identifies derivative 27c, which mimics the biological effects of the irreversible inhibitors at comparable concentrations. Collectively, the back-to-back manuscripts describe the first pharmacophores that favorably and selectively interact with a never explored pocket in Hsp70 and provide a novel blueprint for a cancer-oriented development of Hsp70-directed ligands.
Journal of Medicinal Chemistry, 2014
Heat shock protein 70 (Hsp70) is an important emerging cancer target whose inhibition may affect ... more Heat shock protein 70 (Hsp70) is an important emerging cancer target whose inhibition may affect multiple cancer-associated signaling pathways and, moreover, result in significant cancer cell apoptosis. Despite considerable interest from both academia and pharmaceutical companies in the discovery and development of druglike Hsp70 inhibitors, little success has been reported so far. Here we describe structure-activity relationship studies in the first rationally designed Hsp70 inhibitor class that binds to a novel allosteric pocket located in the N-terminal domain of the protein. These 2,5'-thiodipyrimidine and 5-(phenylthio)pyrimidine acrylamides take advantage of an active cysteine embedded in the allosteric pocket to act as covalent protein modifiers upon binding. The study identifies derivatives 17a and 20a, which selectively bind to Hsp70 in cancer cells. Addition of high nanomolar to low micromolar concentrations of these inhibitors to cancer cells leads to a reduction in the steady-state levels of Hsp70-sheltered oncoproteins, an effect associated with inhibition of cancer cell growth and apoptosis. In summary, the described scaffolds represent a viable starting point for the development of druglike Hsp70 inhibitors as novel anticancer therapeutics.
FEBS Letters, 2008
Hsp110s are divergent relatives of Hsp70 chaperones that hydrolyze ATP. Hsp110s serve as Hsp70 nu... more Hsp110s are divergent relatives of Hsp70 chaperones that hydrolyze ATP. Hsp110s serve as Hsp70 nucleotide exchange factors and act directly to maintain polypeptide solubility. To date, the impact of peptide binding on Hsp110 ATPase activity is unknown and an Hsp110/peptide affinity has not been measured. We now report on a peptide that binds to the yeast Hsp110, Sse1p, with a K D of ~2 nM. Surprisingly, the binding of this peptide fails to stimulate Sse1p ATP hydrolysis. Moreover, an Hsp70-binding peptide is unable to associate with Sse1p, suggesting that Hsp70s and Hsp110s possess partially distinct peptide recognition motifs.
Cancer Cell, 2006
Although androgen receptor (AR)-mediated signaling is central to prostate cancer, the ability to ... more Although androgen receptor (AR)-mediated signaling is central to prostate cancer, the ability to modulate AR signaling states is limited. Here we establish a chemical genomic approach for discovery and target prediction of modulators of cancer phenotypes, as exemplified by AR signaling. We first identify AR activation inhibitors, including a group of structurally related compounds comprising celastrol, gedunin, and derivatives. To develop an in silico approach for target pathway identification, we apply a gene expression-based analysis that classifies HSP90 inhibitors as having similar activity to celastrol and gedunin. Validating this prediction, we demonstrate that celastrol and gedunin inhibit HSP90 activity and HSP90 clients, including AR. Broadly, this work identifies new modes of HSP90 modulation through a gene expression-based strategy.
Bioorganic & Medicinal Chemistry Letters, 2008
The 70 kDa heat shock proteins (Hsp70) are molecular chaperones that assist in folding of newly s... more The 70 kDa heat shock proteins (Hsp70) are molecular chaperones that assist in folding of newly synthesized polypeptides, refolding or denaturation of misfolded proteins, and translocation of proteins across biological membranes. In addition, Hsp70 play regulatory roles in signal transduction, cell cycle, and apoptosis. Here, we present a novel assay platform based on fluorescence polarization that is suitable for investigating the yet elusive molecular mechanics of human Hsp70 allosteric regulation.
Bioorganic & Medicinal Chemistry Letters, 2006
The synthesis of a red-shifted cy3B-GM ligand and its evaluation as a fluorescence polarization p... more The synthesis of a red-shifted cy3B-GM ligand and its evaluation as a fluorescence polarization probe for Hsp90 is presented.
ASSAY and Drug Development Technologies, 2011
Heat shock protein 70 (Hsp70) is a chaperone protein that helps protect against cellular stress, ... more Heat shock protein 70 (Hsp70) is a chaperone protein that helps protect against cellular stress, a function that may be co-opted to fight human diseases. In particular, the upregulation of Hsp70 can suppress the neurotoxicity of misfolded proteins, suggesting possible therapeutic strategies in neurodegenerative diseases. Alternatively, in cancer cells where high levels of Hsp70 inhibit both intrinsic and extrinsic apoptotic pathways, a reduction in Hsp70 levels may induce apoptosis. To evaluate and identify, in a single assay format, small molecules that induce or inhibit endogenous Hsp70, we have designed and optimized a microtiter assay that relies on whole-cell immunodetection of Hsp70. The assay utilizes a minimal number of neuronal or cancer cells, yet is sufficiently sensitive and reproducible to permit quantitative determinations. We further validated the assay using a panel of Hsp70 modulators. In conclusion, we have developed an assay that is fast, robust, and cost efficient. As such, it can be implemented in most research laboratories. The assay should greatly improve the speed at which novel Hsp70 inducers and inhibitors of expression can be identified and evaluated.
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Papers by Cristina C Clement
Mitomycin C (MC) is a well-known DNA alkylating agent. MC analog, 10-decarbamoyl mitomycin C (DMC), unlike MC, has stronger effects on cancer with p53 mutation. We previously demonstrated that MC/DMC could activate p21WAF1/CIP1 in MCF-7 (p53-proficient) and K562 (p53-deficient) cells in a p53-independent mode. This study aimed to elucidate the upstream signaling pathway of p21WAF1/CIP1 activation triggered by MC/DMC. Besides p53, Akt plays an important role on deactivating p21WAF1/CIP1 . The results showed that MC/DMC inhibited Akt in MCF-7 cells, but not in K562 cells. By knocking down p53, the Akt inhibition in MCF-7 cells was alleviated. This implied that the deactivated Akt caused by MC/DMC was p53-dependent. With Akt activator (SC79), p21WAF1/CIP1 activation triggered by MC/DMC in MCF-7 cells was not reduced. This indicated that Akt inhibition triggered by MC/DMC was not associated with MC/DMC-induced p21WAF1/CIP1 activation. Label-free quantitative proteomic profiling analysis revealed that DMC has a stronger effect on downregulating the PI3K/Akt signaling pathway in MCF-7 cells as compared with MC. No significant effect of MC/DMC on PI3K/Akt in K562 cells was observed. In summary, MC/DMC regulate Akt activation in a p53-dependent manner. This Akt deactivation is not associated with p21WAF1/CIP1 activation in response to MC/DMC. This article is protected by copyright. All rights reserved.
of this protein, were identified to form a steric active-site operating in the F11R-dependent adhesion between cells that express F11R molecules on their external surface. In silico modeling of the complex between two polypeptide chains with the sequences positioned
in the active-site was used to generate peptide candidates designed to inhibit homophilic interactions between surface-located F11R molecules. The two lead F11R peptides were modified with D-Arg and D-Lys at selective sites, for attaining higher stability to proteolysis in vivo. Using molecular docking experiments we tested different conformational states and the putative binding affinity between two selected D-Arg and D-Lys-modified F11R peptides and the proposed binding pocket. The inhibitory effects of the F11R peptide 2HN-(dK)-SVT-(dR)-EDTGTYTC-CONH2 on antibody-induced platelet aggregation and on the adhesion of platelets to cytokine inflammed
endothelial cells are reported in detail, and the results point out the significant potential utilization of F11R peptides for the prevention and treatment of atherosclerotic plaques and associated thrombotic events.
Mitomycin C (MC) is a well-known DNA alkylating agent. MC analog, 10-decarbamoyl mitomycin C (DMC), unlike MC, has stronger effects on cancer with p53 mutation. We previously demonstrated that MC/DMC could activate p21WAF1/CIP1 in MCF-7 (p53-proficient) and K562 (p53-deficient) cells in a p53-independent mode. This study aimed to elucidate the upstream signaling pathway of p21WAF1/CIP1 activation triggered by MC/DMC. Besides p53, Akt plays an important role on deactivating p21WAF1/CIP1 . The results showed that MC/DMC inhibited Akt in MCF-7 cells, but not in K562 cells. By knocking down p53, the Akt inhibition in MCF-7 cells was alleviated. This implied that the deactivated Akt caused by MC/DMC was p53-dependent. With Akt activator (SC79), p21WAF1/CIP1 activation triggered by MC/DMC in MCF-7 cells was not reduced. This indicated that Akt inhibition triggered by MC/DMC was not associated with MC/DMC-induced p21WAF1/CIP1 activation. Label-free quantitative proteomic profiling analysis revealed that DMC has a stronger effect on downregulating the PI3K/Akt signaling pathway in MCF-7 cells as compared with MC. No significant effect of MC/DMC on PI3K/Akt in K562 cells was observed. In summary, MC/DMC regulate Akt activation in a p53-dependent manner. This Akt deactivation is not associated with p21WAF1/CIP1 activation in response to MC/DMC. This article is protected by copyright. All rights reserved.
of this protein, were identified to form a steric active-site operating in the F11R-dependent adhesion between cells that express F11R molecules on their external surface. In silico modeling of the complex between two polypeptide chains with the sequences positioned
in the active-site was used to generate peptide candidates designed to inhibit homophilic interactions between surface-located F11R molecules. The two lead F11R peptides were modified with D-Arg and D-Lys at selective sites, for attaining higher stability to proteolysis in vivo. Using molecular docking experiments we tested different conformational states and the putative binding affinity between two selected D-Arg and D-Lys-modified F11R peptides and the proposed binding pocket. The inhibitory effects of the F11R peptide 2HN-(dK)-SVT-(dR)-EDTGTYTC-CONH2 on antibody-induced platelet aggregation and on the adhesion of platelets to cytokine inflammed
endothelial cells are reported in detail, and the results point out the significant potential utilization of F11R peptides for the prevention and treatment of atherosclerotic plaques and associated thrombotic events.