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1.
SLAS Discov ; 2023 Aug 06.
Article in English | MEDLINE | ID: mdl-37549772

ABSTRACT

Three series of compounds were prioritized from a high content screening campaign that identified molecules that blocked dihydrotestosterone (DHT) induced formation of Androgen Receptor (AR) protein-protein interactions (PPIs) with the Transcriptional Intermediary Factor 2 (TIF2) coactivator and also disrupted preformed AR-TIF2 PPI complexes; the hydrobenzo-oxazepins (S1), thiadiazol-5-piperidine-carboxamides (S2), and phenyl-methyl-indoles (S3). Compounds from these series inhibited AR PPIs with TIF2 and SRC-1, another p160 coactivator, in mammalian 2-hybrid assays and blocked transcriptional activation in reporter assays driven by full length AR or AR-V7 splice variants. Compounds inhibited the growth of five prostate cancer cell lines, with many exhibiting differential cytotoxicity towards AR positive cell lines. Representative compounds from the 3 series substantially reduced both endogenous and DHT-enhanced expression and secretion of the prostate specific antigen (PSA) cancer biomarker in the C4-2 castration resistant prostate cancer (CRPC) cell line. The comparatively weak activities of series compounds in the H3-DHT and/or TIF2 box 3 LXXLL-peptide binding assays to the recombinant ligand binding domain of AR suggest that direct antagonism at the orthosteric ligand binding site or AF-2 surface respectively are unlikely mechanisms of action. Cellular enhanced thermal stability assays (CETSA) indicated that compounds engaged AR and reduced the maximum efficacy and right shifted the EC50 of DHT-enhanced AR thermal stabilization consistent with the effects of negative allosteric modulators. Molecular docking of potent representative hits from each series to AR structures suggest that S1-1 and S2-6 engage a novel binding pocket (BP-1) adjacent to the orthosteric ligand binding site, while S3-11 occupies the AR binding function 3 (BF-3) allosteric pocket. Hit binding poses indicate spaces and residues adjacent to the BP-1 and BF-3 pockets that will be exploited in future medicinal chemistry optimization studies. Small molecule allosteric modulators that prevent/disrupt AR PPIs with coactivators like TIF2 to alter transcriptional activation in the presence of orthosteric agonists might evade the resistance mechanisms to existing prostate cancer drugs and provide novel starting points for medicinal chemistry lead optimization and future development into therapies for metastatic CRPC.

2.
J Biol Chem ; 298(2): 101535, 2022 02.
Article in English | MEDLINE | ID: mdl-34954143

ABSTRACT

Cancer cells frequently exhibit uncoupling of the glycolytic pathway from the TCA cycle (i.e., the "Warburg effect") and as a result, often become dependent on their ability to increase glutamine catabolism. The mitochondrial enzyme Glutaminase C (GAC) helps to satisfy this 'glutamine addiction' of cancer cells by catalyzing the hydrolysis of glutamine to glutamate, which is then converted to the TCA-cycle intermediate α-ketoglutarate. This makes GAC an intriguing drug target and spurred the molecules derived from bis-2-(5-phenylacetamido-1,3,4-thiadiazol-2-yl)ethyl sulfide (the so-called BPTES class of allosteric GAC inhibitors), including CB-839, which is currently in clinical trials. However, none of the drugs targeting GAC are yet approved for cancer treatment and their mechanism of action is not well understood. Here, we shed new light on the underlying basis for the differential potencies exhibited by members of the BPTES/CB-839 family of compounds, which could not previously be explained with standard cryo-cooled X-ray crystal structures of GAC bound to CB-839 or its analogs. Using an emerging technique known as serial room temperature crystallography, we were able to observe clear differences between the binding conformations of inhibitors with significantly different potencies. We also developed a computational model to further elucidate the molecular basis of differential inhibitor potency. We then corroborated the results from our modeling efforts using recently established fluorescence assays that directly read out inhibitor binding to GAC. Together, these findings should aid in future design of more potent GAC inhibitors with better clinical outlook.


Subject(s)
Enzyme Inhibitors , Glutaminase , Neoplasms , Sulfides , Thiadiazoles , Crystallography , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/pharmacology , Glutaminase/antagonists & inhibitors , Glutaminase/chemistry , Glutaminase/metabolism , Glutamine/metabolism , Neoplasms/drug therapy , Neoplasms/metabolism , Sulfides/chemistry , Sulfides/pharmacology , Temperature , Thiadiazoles/chemistry , Thiadiazoles/pharmacology
3.
J Neurotrauma ; 37(22): 2435-2444, 2020 11 15.
Article in English | MEDLINE | ID: mdl-30816062

ABSTRACT

The purpose of this review is to highlight the pharmacological barrier to drug development for traumatic brain injury (TBI) and to discuss best practice strategies to overcome such barriers. Specifically, this article will review the pharmacological considerations of moving from the disease target "hit" to the "lead" compound with drug-like and central nervous system (CNS) penetrant properties. In vitro assessment of drug-like properties will be detailed, followed by pre-clinical studies to ensure adequate pharmacokinetic and pharmacodynamic characteristics of response. The importance of biomarker development and utilization in both pre-clinical and clinical studies will be detailed, along with the importance of identifying diagnostic, pharmacodynamic/response, and prognostic biomarkers of injury type or severity, drug target engagement, and disease progression. This review will detail the important considerations in determining in vivo pre-clinical dose selection, as well as cross-species and human equivalent dose selection. Specific use of allometric scaling, pharmacokinetic and pharmacodynamic criteria, as well as incorporation of biomarker assessments in human dose selection for clinical trial design will also be discussed. The overarching goal of this review is to detail the pharmacological considerations in the drug development process as a method to improve both pre-clinical and clinical study design as we evaluate novel therapies to improve outcomes in patients with TBI.


Subject(s)
Brain Injuries, Traumatic/drug therapy , Drug Development/methods , Neuroprotective Agents/pharmacology , Animals , Biomarkers/metabolism , Humans
4.
Bioorg Med Chem Lett ; 29(19): 126632, 2019 10 01.
Article in English | MEDLINE | ID: mdl-31474484

ABSTRACT

Allosteric inhibitors of glutaminase (GAC), such as BPTES, CB-839 and UPGL00019, have great promise as inhibitors of cancer cell growth, but potent inhibitors with drug-like qualities have been difficult to achieve. Here, a small library of GAC inhibitors based on the UPGL00019 core is described. This set of derivatives was designed to assess if one or both of the phenylacetyl groups flanking the UPGL00019 core can be replaced by smaller simple aliphatic acyl groups without loss in potency. We found that one of the phenylacetyl moieties can be replaced by a set of small aliphatic moieties without loss in potency. We also found that enzymatic potency co-varies with the VDW volume or the maximum projection area of the groups used as replacements of the phenylacetyl moiety and used literature X-ray data to provide an explanation for this finding.


Subject(s)
Antineoplastic Agents/pharmacology , Breast Neoplasms/drug therapy , Enzyme Inhibitors/pharmacology , Glutaminase/antagonists & inhibitors , Piperidines/chemistry , Small Molecule Libraries/pharmacology , Antineoplastic Agents/chemistry , Breast Neoplasms/enzymology , Breast Neoplasms/pathology , Cell Proliferation , Enzyme Inhibitors/chemistry , Female , Humans , Models, Molecular , Molecular Structure , Small Molecule Libraries/chemistry , Tumor Cells, Cultured
5.
Arch Biochem Biophys ; 653: 71-79, 2018 09 01.
Article in English | MEDLINE | ID: mdl-29991441

ABSTRACT

Serine hydroxymethyltransferase (SHMT) is a pivotal enzyme in one-carbon metabolism that catalyses the reversible conversion of serine and tetrahydrofolate into glycine and methylenetetrahydrofolate. It exists in cytosolic (SHMT1) and mitochondrial (SHMT2) isoforms. Research on one-carbon metabolism in cancer cell lines has shown that SHMT1 preferentially catalyses serine synthesis, whereas in mitochondria SHMT2 is involved in serine breakdown. Recent research has focused on the identification of inhibitors that bind at the folate pocket. We have previously found that a representative derivative of the pyrazolopyran scaffold, namely 2.12, inhibits both SHMT isoforms, with a preference for SHMT1, causing apoptosis in lung cancer cell lines. Here we show that the affinity of 2.12 for SHMT depends on the identity of the amino acid substrate bound to the enzyme. The dissociation constant of 2.12 is 50-fold lower when it binds to SHMT1 enzyme-serine complex, as compared to the enzyme-glycine complex. Evidence is presented for a similar behaviour of compound 2.12 in the cellular environment. These findings suggest that the presence and identity of the amino acid substrate should be considered when designing SHMT inhibitors. Moreover, our data provide the proof-of-concept that SHMT inhibitors selectively targeting the directionality of one-carbon metabolism flux could be designed.


Subject(s)
Enzyme Inhibitors/pharmacology , Glycine Hydroxymethyltransferase/antagonists & inhibitors , Glycine Hydroxymethyltransferase/chemistry , Glycine/chemistry , Pyrans/pharmacology , Pyrazoles/pharmacology , Serine/chemistry , Apoptosis/drug effects , Cell Line, Tumor , Enzyme Inhibitors/metabolism , Humans , Hydrogen Bonding , Lung Neoplasms/pathology , Pyrans/chemistry , Pyrazoles/chemistry , Spectrometry, Fluorescence , Substrate Specificity
6.
J Biol Chem ; 293(10): 3535-3545, 2018 03 09.
Article in English | MEDLINE | ID: mdl-29317493

ABSTRACT

Altered glycolytic flux in cancer cells (the "Warburg effect") causes their proliferation to rely upon elevated glutamine metabolism ("glutamine addiction"). This requirement is met by the overexpression of glutaminase C (GAC), which catalyzes the first step in glutamine metabolism and therefore represents a potential therapeutic target. The small molecule CB-839 was reported to be more potent than other allosteric GAC inhibitors, including the parent compound bis-2-(5-phenylacetamido-1,2,4-thiadiazol-2-yl)ethyl (BPTES), and is in clinical trials. Recently, we described the synthesis of BPTES analogs having distinct saturated heterocyclic cores as a replacement for the flexible chain moiety, with improved microsomal stability relative to CB-839 and BPTES. Here, we show that one of these new compounds, UPGL00004, like CB-839, more potently inhibits the enzymatic activity of GAC, compared with BPTES. We also compare the abilities of UPGL00004, CB-839, and BPTES to directly bind to recombinant GAC and demonstrate that UPGL00004 has a similar binding affinity as CB-839 for GAC. We also show that UPGL00004 potently inhibits the growth of triple-negative breast cancer cells, as well as tumor growth when combined with the anti-vascular endothelial growth factor antibody bevacizumab. Finally, we compare the X-ray crystal structures for UPGL00004 and CB-839 bound to GAC, verifying that UPGL00004 occupies the same binding site as CB-839 or BPTES and that all three inhibitors regulate the enzymatic activity of GAC via a similar allosteric mechanism. These results provide insights regarding the potency of these inhibitors that will be useful in designing novel small-molecules that target a key enzyme in cancer cell metabolism.


Subject(s)
Antineoplastic Agents/pharmacology , Enzyme Inhibitors/pharmacology , Glutaminase/antagonists & inhibitors , Models, Molecular , Neoplasm Proteins/antagonists & inhibitors , Triple Negative Breast Neoplasms/drug therapy , Allosteric Site/drug effects , Amino Acid Substitution , Antineoplastic Agents/chemistry , Antineoplastic Agents/metabolism , Benzeneacetamides/chemistry , Benzeneacetamides/metabolism , Benzeneacetamides/pharmacology , Binding, Competitive , Cell Line, Tumor , Cell Proliferation/drug effects , Crystallography, X-Ray , Drug Resistance, Multiple , Drug Resistance, Neoplasm , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/metabolism , Glutaminase/chemistry , Glutaminase/genetics , Glutaminase/metabolism , Glutamine/antagonists & inhibitors , Glutamine/chemistry , Glutamine/metabolism , Humans , Hydrogen Bonding , Molecular Conformation , Mutation , Neoplasm Proteins/chemistry , Neoplasm Proteins/genetics , Neoplasm Proteins/metabolism , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism , Sulfides/chemistry , Sulfides/metabolism , Sulfides/pharmacology , Thiadiazoles/chemistry , Thiadiazoles/metabolism , Thiadiazoles/pharmacology , Triple Negative Breast Neoplasms/metabolism , Triple Negative Breast Neoplasms/pathology
7.
J Biol Chem ; 293(7): 2606-2616, 2018 02 16.
Article in English | MEDLINE | ID: mdl-29282288

ABSTRACT

Profilin 1 (Pfn1) is an important regulator of the actin cytoskeleton and plays a vital role in many actin-based cellular processes. Therefore, identification of a small-molecule intervention strategy targeted against the Pfn1-actin interaction could have broad utility in cytoskeletal research and further our understanding of the role of Pfn1 in actin-mediated biological processes. Based on an already resolved Pfn1-actin complex crystal structure, we performed structure-based virtual screening of small-molecule libraries to seek inhibitors of the Pfn1-actin interaction. We identified compounds that match the pharmacophore of the key actin residues of Pfn1-actin interaction and therefore have the potential to act as competitive inhibitors of this interaction. Subsequent biochemical assays identified two candidate compounds with nearly identical structures that can mitigate the effect of Pfn1 on actin polymerization in vitro As a further proof-of-concept test for cellular effects of these compounds, we performed proximity ligation assays in endothelial cells (ECs) to demonstrate compound-induced inhibition of Pfn1-actin interaction. Consistent with the important role of Pfn1 in regulating actin polymerization and various fundamental actin-based cellular activities (migration and proliferation), treatment of these compounds reduced the overall level of cellular filamentous (F) actin, slowed EC migration and proliferation, and inhibited the angiogenic ability of ECs both in vitro and ex vivo In summary, this study provides the first proof of principle of small-molecule-mediated interference with the Pfn1-actin interaction. Our findings may have potential general utility for perturbing actin-mediated cellular activities and biological processes.


Subject(s)
Actins/metabolism , Profilins/metabolism , Small Molecule Libraries/chemistry , Actin Cytoskeleton/genetics , Actin Cytoskeleton/metabolism , Actins/antagonists & inhibitors , Actins/genetics , Animals , Aorta, Thoracic/metabolism , Cell Movement/drug effects , Cell Proliferation/drug effects , Crystallography, X-Ray , Drug Evaluation, Preclinical , Endothelial Cells/cytology , Endothelial Cells/metabolism , Humans , Mice , Mice, Inbred C57BL , Polymerization/drug effects , Profilins/antagonists & inhibitors , Profilins/chemistry , Profilins/genetics , Protein Binding/drug effects , Small Molecule Libraries/pharmacology
8.
Sci Rep ; 7(1): 17803, 2017 12 19.
Article in English | MEDLINE | ID: mdl-29259176

ABSTRACT

Quantitative Systems Pharmacology (QSP) is a drug discovery approach that integrates computational and experimental methods in an iterative way to gain a comprehensive, unbiased understanding of disease processes to inform effective therapeutic strategies. We report the implementation of QSP to Huntington's Disease, with the application of a chemogenomics platform to identify strategies to protect neuronal cells from mutant huntingtin induced death. Using the STHdh Q111 cell model, we investigated the protective effects of small molecule probes having diverse canonical modes-of-action to infer pathways of neuronal cell protection connected to drug mechanism. Several mechanistically diverse protective probes were identified, most of which showed less than 50% efficacy. Specific combinations of these probes were synergistic in enhancing efficacy. Computational analysis of these probes revealed a convergence of pathways indicating activation of PKA. Analysis of phospho-PKA levels showed lower cytoplasmic levels in STHdh Q111 cells compared to wild type STHdh Q7 cells, and these levels were increased by several of the protective compounds. Pharmacological inhibition of PKA activity reduced protection supporting the hypothesis that protection may be working, in part, through activation of the PKA network. The systems-level studies described here can be broadly applied to any discovery strategy involving small molecule modulation of disease phenotype.


Subject(s)
Huntington Disease/drug therapy , Huntington Disease/metabolism , Neurons/drug effects , Neurons/metabolism , Protective Agents/pharmacology , Animals , Cyclic AMP-Dependent Protein Kinases/metabolism , Disease Models, Animal , Drug Combinations , Huntingtin Protein/metabolism , Mice , Mutation/drug effects , Phenotype , Signal Transduction/drug effects , Small Molecule Libraries/pharmacology
9.
Bioorg Med Chem ; 24(8): 1819-39, 2016 Apr 15.
Article in English | MEDLINE | ID: mdl-26988803

ABSTRACT

A novel set of GAC (kidney glutaminase isoform C) inhibitors able to inhibit the enzymatic activity of GAC and the growth of the triple negative MDA-MB-231 breast cancer cells with low nanomolar potency is described. Compounds in this series have a reduced number of rotatable bonds, improved ClogPs, microsomal stability and ligand efficiency when compared to the leading GAC inhibitors BPTES and CB-839. Property improvements were achieved by the replacement of the flexible n-diethylthio or the n-butyl moiety present in the leading inhibitors by heteroatom substituted heterocycloalkanes.


Subject(s)
Benzeneacetamides/pharmacology , Drug Design , Enzyme Inhibitors/pharmacology , Glutaminase/antagonists & inhibitors , Sulfides/pharmacology , Thiadiazoles/pharmacology , Benzeneacetamides/chemistry , Benzeneacetamides/metabolism , Cell Line, Tumor , Dose-Response Relationship, Drug , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/metabolism , Glutaminase/metabolism , Humans , Microsomes, Liver/chemistry , Microsomes, Liver/metabolism , Models, Molecular , Molecular Structure , Structure-Activity Relationship , Sulfides/chemistry , Sulfides/metabolism , Thiadiazoles/chemistry , Thiadiazoles/metabolism
10.
Am J Physiol Renal Physiol ; 310(8): F705-F716, 2016 04 15.
Article in English | MEDLINE | ID: mdl-26661656

ABSTRACT

No therapies have been shown to accelerate recovery or prevent fibrosis after acute kidney injury (AKI). In part, this is because most therapeutic candidates have to be given at the time of injury and the diagnosis of AKI is usually made too late for drugs to be efficacious. Strategies to enhance post-AKI repair represent an attractive approach to address this. Using a phenotypic screen in zebrafish, we identified 4-(phenylthio)butanoic acid (PTBA), which promotes proliferation of embryonic kidney progenitor cells (EKPCs), and the PTBA methyl ester UPHD25, which also increases postinjury repair in ischemia-reperfusion and aristolochic acid-induced AKI in mice. In these studies, a new panel of PTBA analogs was evaluated. Initial screening was performed in zebrafish EKPC assays followed by survival assays in a gentamicin-induced AKI larvae zebrafish model. Using this approach, we identified UPHD186, which in contrast to UPHD25, accelerates recovery and reduces fibrosis when administered several days after ischemia-reperfusion AKI and reduces fibrosis after unilateral ureteric obstruction in mice. UPHD25 and 186 are efficiently metabolized to the active analog PTBA in liver and kidney microsome assays, indicating both compounds may act as PTBA prodrugs in vivo. UPHD186 persists longer in the circulation than UPHD25, suggesting that sustained levels of UPHD186 may increase efficacy by acting as a reservoir for renal metabolism to PTBA. These findings validate use of zebrafish EKPC and AKI assays as a drug discovery strategy for molecules that reduce fibrosis in multiple AKI models and can be administered days after initiation of injury.


Subject(s)
Acute Kidney Injury/drug therapy , Butyrates/therapeutic use , Kidney/drug effects , Sulfides/therapeutic use , Acute Kidney Injury/pathology , Animals , Butyrates/pharmacology , Cell Proliferation/drug effects , Cell Survival/drug effects , Disease Models, Animal , Fibrosis/drug therapy , Fibrosis/pathology , Kidney/pathology , Male , Mice , Sulfides/pharmacology , Zebrafish
11.
Oncotarget ; 7(4): 4570-83, 2016 Jan 26.
Article in English | MEDLINE | ID: mdl-26717037

ABSTRACT

Serine hydroxymethyltransferase (SHMT) is a central enzyme in the metabolic reprogramming of cancer cells, providing activated one-carbon units in the serine-glycine one-carbon metabolism. Previous studies demonstrated that the cytoplasmic isoform of SHMT (SHMT1) plays a relevant role in lung cancer. SHMT1 is overexpressed in lung cancer patients and NSCLC cell lines. Moreover, SHMT1 is required to maintain DNA integrity. Depletion in lung cancer cell lines causes cell cycle arrest and uracil accumulation and ultimately leads to apoptosis. We found that a pyrazolopyran compound, namely 2.12, preferentially inhibits SHMT1 compared to the mitochondrial counterpart SHMT2. Computational and crystallographic approaches suggest binding at the active site of SHMT1 and a competitive inhibition mechanism. A radio isotopic activity assay shows that inhibition of SHMT by 2.12 also occurs in living cells. Moreover, administration of 2.12 in A549 and H1299 lung cancer cell lines causes apoptosis at LD50 34 µM and rescue experiments underlined selectivity towards SHMT1. These data not only further highlight the relevance of the cytoplasmic isoform SHMT1 in lung cancer but, more importantly, demonstrate that, at least in vitro, it is possible to find selective inhibitors against one specific isoform of SHMT, a key target in metabolic reprogramming of many cancer types.


Subject(s)
Antineoplastic Agents/pharmacology , Apoptosis/drug effects , Cytoplasm/enzymology , Enzyme Inhibitors/pharmacology , Glycine Hydroxymethyltransferase/metabolism , Lung Neoplasms/pathology , Pyrans/chemistry , Blotting, Western , Catalytic Domain , Cell Proliferation/drug effects , Glycine Hydroxymethyltransferase/chemistry , Glycine Hydroxymethyltransferase/genetics , Humans , Immunoenzyme Techniques , Lung Neoplasms/drug therapy , Lung Neoplasms/genetics , Lung Neoplasms/metabolism , Molecular Dynamics Simulation , Protein Conformation , Pyrazoles/chemistry , RNA, Messenger/genetics , Real-Time Polymerase Chain Reaction , Reverse Transcriptase Polymerase Chain Reaction , Tumor Cells, Cultured
12.
ChemMedChem ; 10(3): 490-7, 2015 Mar.
Article in English | MEDLINE | ID: mdl-25677305

ABSTRACT

Metabolic reprogramming of tumor cells toward serine catabolism is now recognized as a hallmark of cancer. Serine hydroxymethyltransferase (SHMT), the enzyme providing one-carbon units by converting serine and tetrahydrofolate (H4 PteGlu) to glycine and 5,10-CH2 -H4 PteGlu, therefore represents a target of interest in developing new chemotherapeutic drugs. In this study, 13 folate analogues under clinical evaluation or in therapeutic use were in silico screened against SHMT, ultimately identifying four antifolate agents worthy of closer evaluation. The interaction mode of SHMT with these four antifolate drugs (lometrexol, nolatrexed, raltitrexed, and methotrexate) was assessed. The mechanism of SHMT inhibition by the selected antifolate agents was investigated in vitro using the human cytosolic isozyme. The results of this study showed that lometrexol competitively inhibits SHMT with inhibition constant (Ki ) values in the low micromolar. The binding mode of lometrexol to SHMT was further investigated by molecular docking. These results thus provide insights into the mechanism of action of antifolate drugs and constitute the basis for the rational design of novel and more potent inhibitors of SHMT.


Subject(s)
Folic Acid Antagonists/chemistry , Folic Acid Antagonists/pharmacology , Glycine Hydroxymethyltransferase/antagonists & inhibitors , Glycine Hydroxymethyltransferase/metabolism , Humans , Methotrexate/chemistry , Methotrexate/pharmacology , Molecular Docking Simulation , Quinazolines/chemistry , Quinazolines/pharmacology , Tetrahydrofolates/chemistry , Tetrahydrofolates/pharmacology , Thiophenes/chemistry , Thiophenes/pharmacology
13.
Oncotarget ; 5(12): 4180-94, 2014 Jun 30.
Article in English | MEDLINE | ID: mdl-24952704

ABSTRACT

Overcoming platinum drug resistance represents a major clinical challenge in cancer treatment. We discovered a novel drug combination using cisplatin and a class of thioquinazolinone derivatives including mdivi-1 (mitochondrial division inhibitor-1), that induces synergistic apoptosis in platinum resistant tumor cells, including those from cisplatin-refractory endstage ovarian cancer patients. However, through study of the combination effect on Drp1 (the reported target of mdivi-1) knockout MEF cells and the functional analysis of mdivi-1 analogs, we revealed that the synergism between mdivi-1 and cisplatin is Drp1-independent. Mdivi-1 impairs DNA replication and its combination with cisplatin induces a synergistic increase of replication stress and DNA damage, causing a preferential upregulation of a BH3-only protein Noxa. Mdivi-1 also represses mitochondrial respiration independent of Drp1, and the combination of mdivi-1 and cisplatin triggers substantial mitochondrial uncoupling and swelling. Upregulation of Noxa and simultaneous mitochondrial swelling causes synergistic induction of mitochondrial outer membrane permeabilization (MOMP), proceeding robust mitochondrial apoptotic signaling independent of Bax/Bak. Thus, the novel mode of MOMP induction by the combination through the "dual-targeting" potential of mdivi-1 on DNA replication and mitochondrial respiration suggests a novel class of compounds for platinum-based combination option in the treatment of platinum as well as multidrug resistant tumors.


Subject(s)
Cisplatin/therapeutic use , Neoplasms/metabolism , Platinum/therapeutic use , Quinazolinones/therapeutic use , Apoptosis , Cell Line, Tumor , Cisplatin/metabolism , Drug Resistance, Neoplasm , Drug Synergism , Humans , Mitochondrial Dynamics , Platinum/metabolism , Quinazolinones/metabolism
14.
Am J Physiol Renal Physiol ; 306(5): F496-504, 2014 Mar 01.
Article in English | MEDLINE | ID: mdl-24370591

ABSTRACT

Phenylthiobutanoic acids (PTBAs) are a new class of histone deacetylase (HDAC) inhibitors that accelerate recovery and reduce postinjury fibrosis after ischemia-reperfusion-induced acute kidney injury. However, unlike the more common scenario in which patients present with protracted and less clearly defined onset of renal injury, this model of acute kidney injury gives rise to a clearly defined injury that begins to resolve over a short period of time. In these studies, we show for the first time that treatment with the PTBA analog methyl-4-(phenylthio)butanoate (M4PTB) accelerates recovery and reduces postinjury fibrosis in a progressive model of acute kidney injury and renal fibrosis that occurs after aristolochic acid injection in mice. These effects are apparent when M4PTB treatment is delayed 4 days after the initiating injury and are associated with increased proliferation and decreased G2/M arrest of regenerating renal tubular epithelial cells. In addition, there is reduced peritubular macrophage infiltration and decreased expression of the macrophage chemokines CX3Cl1 and CCL2. Since macrophage infiltration plays a role in promoting kidney injury, and since renal tubular epithelial cells show defective repair and a marked increase in maladaptive G2/M arrest after aristolochic acid injury, these findings suggest M4PTB may be particularly beneficial in reducing injury and enhancing intrinsic cellular repair even when administered days after aristolochic acid ingestion.


Subject(s)
Acute Kidney Injury/drug therapy , Butyrates/pharmacology , Sulfides/pharmacology , Acute Kidney Injury/chemically induced , Animals , Aristolochic Acids/pharmacology , Butyrates/analysis , Disease Models, Animal , Fibrosis/drug therapy , Fibrosis/prevention & control , Histone Deacetylase Inhibitors/pharmacology , Kidney Tubules/drug effects , Kidney Tubules/metabolism , Mice , Mice, Biozzi , Reperfusion Injury/chemically induced , Reperfusion Injury/drug therapy , Reperfusion Injury/metabolism , Sulfides/analysis
15.
J Biomol Screen ; 18(10): 1193-202, 2013 Dec.
Article in English | MEDLINE | ID: mdl-23832868

ABSTRACT

Reactivation of genes normally expressed during organogenesis is a characteristic of kidney regeneration. Enhancing this reactivation could potentially be a therapeutic target to augment kidney regeneration. The inductive events that drive kidney organogenesis in zebrafish are similar to the initial steps in mammalian kidney organogenesis. Therefore, quantifying embryonic signals that drive zebrafish kidney development is an attractive strategy for the discovery of potential novel therapeutic modalities that accelerate kidney regeneration. The Lim1 homeobox protein, Lhx1, is a marker of kidney development that is also expressed in the regenerating kidneys after injury. Using a fluorescent Lhx1a-EGFP transgene whose phenotype faithfully recapitulates that of the endogenous protein, we developed a high-content assay for Lhx1a-EGFP expression in transgenic zebrafish embryos employing an artificial intelligence-based image analysis method termed cognition network technology (CNT). Implementation of the CNT assay on high-content readers enabled automated real-time in vivo time-course, dose-response, and variability studies in the developing embryo. The Lhx1a assay was complemented with a kidney-specific secondary CNT assay that enables direct measurements of the embryonic renal tubule cell population. The integration of fluorescent transgenic zebrafish embryos with automated imaging and artificial intelligence-based image analysis provides an in vivo analysis system for structure-activity relationship studies and de novo discovery of novel agents that augment innate regenerative processes.


Subject(s)
Drug Evaluation, Preclinical/methods , Kidney/physiology , Stem Cells/physiology , Animals , Biological Assay , Cell Proliferation/drug effects , Embryo, Nonmammalian/drug effects , Embryo, Nonmammalian/metabolism , Gene Expression/drug effects , Green Fluorescent Proteins/biosynthesis , Green Fluorescent Proteins/genetics , Histone Deacetylase Inhibitors/pharmacology , Kidney/cytology , LIM-Homeodomain Proteins/genetics , LIM-Homeodomain Proteins/metabolism , Phenylbutyrates/pharmacology , Recombinant Fusion Proteins/biosynthesis , Recombinant Fusion Proteins/genetics , Regeneration , Stem Cells/drug effects , Transcription Factors/genetics , Transcription Factors/metabolism , Zebrafish , Zebrafish Proteins/genetics , Zebrafish Proteins/metabolism
16.
J Am Soc Nephrol ; 24(6): 943-53, 2013 May.
Article in English | MEDLINE | ID: mdl-23620402

ABSTRACT

At present, there are no effective therapies to ameliorate injury, accelerate recovery, or prevent postinjury fibrosis after AKI. Here, we sought to identify candidate compounds that accelerate recovery after AKI by screening for small molecules that increase proliferation of renal progenitor cells in zebrafish embryos. One compound identified from this screen was the histone deacetylase inhibitor methyl-4-(phenylthio)butanoate, which we subsequently administered to zebrafish larvae and mice 24-48 hours after inducing AKI. In zebrafish, treatment with the compound increased larval survival and proliferation of renal tubular epithelial cells. In mice, treatment accelerated recovery, reduced postinjury tubular atrophy and interstitial fibrosis, and increased the regenerative capacity of actively cycling renal tubular cells by decreasing the number of cells in G2/M arrest. These data suggest that accelerating recovery may be a viable approach to treating AKI and provide proof of concept that a screen in zebrafish embryos can identify therapeutic candidates for kidney injury.


Subject(s)
Acute Kidney Injury/drug therapy , Acute Kidney Injury/enzymology , Histone Deacetylase 1/antagonists & inhibitors , Histone Deacetylase Inhibitors/pharmacology , Phenylbutyrates/pharmacology , Zebrafish Proteins/antagonists & inhibitors , Acute Kidney Injury/pathology , Animals , Disease Models, Animal , Fibrosis , Gentamicins/toxicity , Histone Deacetylase 1/metabolism , Ischemia/drug therapy , Ischemia/enzymology , Ischemia/pathology , Kidney/drug effects , Kidney/enzymology , Kidney/pathology , Male , Mice , Mice, Inbred BALB C , Protein Synthesis Inhibitors/toxicity , Recovery of Function/drug effects , Zebrafish , Zebrafish Proteins/metabolism
17.
J Med Chem ; 54(7): 2433-46, 2011 Apr 14.
Article in English | MEDLINE | ID: mdl-21413799

ABSTRACT

Diacylglycerol acyltransferase-1 (DGAT-1) is the enzyme that catalyzes the final and committed step of triglyceride formation, namely, the acylation of diacylglycerol with acyl coenzyme A. DGAT-1 deficient mice demonstrate resistance to weight gain on high fat diet, improved insulin sensitivity, and reduced liver triglyceride content. Inhibition of DGAT-1 thus represents a potential novel approach for the treatment of obesity, dyslipidemia, and metabolic syndrome. In this communication, we report the identification of the lead structure 6 and our lead optimization efforts culminating in the discovery of potent, selective, and orally efficacious carboxylic acid derivatives of 2-phenyl-5-trifluoromethyloxazole-4-carboxamides. In particular, compound 29 (DGAT-1 enzyme assay, IC(50) = 57 nM; CHO-K1 cell triglyceride formation assay, EC(50) = 0.5 µM) demonstrated dose dependent inhibition of weight gain in diet induced obese (DIO) rats (0.3, 1, and 3 mg/kg, p.o., qd) during a 21-day efficacy study. Furthermore, compound 29 demonstrated improved glucose tolerance determined by an oral glucose tolerance test (OGTT).


Subject(s)
Amides/chemistry , Amides/pharmacology , Carboxylic Acids/chemistry , Diabetes Mellitus/drug therapy , Diacylglycerol O-Acyltransferase/antagonists & inhibitors , Drug Discovery , Obesity/drug therapy , Oxazoles/chemistry , Oxazoles/pharmacology , Administration, Oral , Amides/administration & dosage , Amides/pharmacokinetics , Animals , Cell Line , Diabetes Mellitus/enzymology , Dogs , Enzyme Inhibitors/administration & dosage , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/pharmacokinetics , Enzyme Inhibitors/pharmacology , Ether-A-Go-Go Potassium Channels/antagonists & inhibitors , Humans , Inhibitory Concentration 50 , Male , Mice , Obesity/enzymology , Oxazoles/administration & dosage , Oxazoles/pharmacokinetics , Rats
18.
Mol Pharmacol ; 75(1): 196-207, 2009 Jan.
Article in English | MEDLINE | ID: mdl-18849352

ABSTRACT

Despite widespread expression of epidermal growth factor (EGF) receptors (EGFRs) and EGF family ligands in non-small-cell lung cancer (NSCLC), EGFR-specific tyrosine kinase inhibitors (TKIs) such as gefitinib exhibit limited activity in this cancer. We propose that autocrine growth signaling pathways distinct from EGFR are active in NSCLC cells. To this end, gene expression profiling revealed frequent coexpression of specific fibroblast growth factors (FGFs) and FGF receptors (FGFRs) in NSCLC cell lines. It is noteworthy that FGF2 and FGF9 as well as FGFR1 IIIc and/or FGFR2 IIIc mRNA and protein are frequently coexpressed in NSCLC cell lines, especially those that are insensitive to gefitinib. Specific silencing of FGF2 reduced anchorage-independent growth of two independent NSCLC cell lines that secrete FGF2 and coexpress FGFR1 IIIc and/or FGFR2 IIIc. Moreover, a TKI [(+/-)-1-(anti-3-hydroxy-cyclopentyl)-3-(4-methoxy-phenyl)-7-phenylamino-3,4-dihydro-1H-pyrimido-[4,5-d]pyrimidin-2-one (RO4383596)] that targets FGFRs inhibited basal FRS2 and extracellular signal-regulated kinase phosphorylation, two measures of FGFR activity, as well as proliferation and anchorage-independent growth of NSCLC cell lines that coexpress FGF2 or FGF9 and FGFRs. By contrast, RO4383596 influenced neither signal transduction nor growth of NSCLC cell lines lacking FGF2, FGF9, FGFR1, or FGFR2 expression. Thus, FGF2, FGF9 and their respective high-affinity FGFRs comprise a growth factor autocrine loop that is active in a subset of gefitinib-insensitive NSCLC cell lines.


Subject(s)
Carcinoma, Non-Small-Cell Lung/genetics , Fibroblast Growth Factors/metabolism , Lung Neoplasms/genetics , Receptors, Fibroblast Growth Factor/metabolism , Signal Transduction/genetics , Cell Line, Tumor , Fibroblast Growth Factors/genetics , Humans , RNA, Small Interfering/metabolism , Receptors, Fibroblast Growth Factor/genetics
20.
Bioorg Med Chem ; 13(16): 4835-41, 2005 Aug 15.
Article in English | MEDLINE | ID: mdl-15953730
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