Jing Yuan Liu

A small molecule compound targeting STAT3 DNA-binding domain inhibits cancer cell proliferation, migration, and invasion.

Signal transducer and activator of transcription 3 (STAT3) plays important roles in multiple aspects of cancer aggressiveness including migration, invasion, survival, self-renewal, angiogenesis, and tumor cell immune evasion by regulating the expression of multiple downstream target genes. STAT3 is constitutively activated in many malignant tumors and its activation is associated with high histological grade and advanced cancer stages. Thus, inhibiting STAT3 promises an attracting strategy for treatment of advanced and metastatic cancers. Herein, we identified a STAT3 inhibitor, inS3-54, by targeting the DNA-binding domain of STAT3 using an improved virtual screening strategy. InS3-54 preferentially suppresses proliferation of cancer over non-cancer cells and inhibits migration and invasion of malignant cells. Biochemical analyses show that inS3-54 selectively inhibits STAT3 binding to DNA without affecting the activation and dimerization of STAT3. Furthermore, inS3-54 inhibits expression of STAT3 downstream target genes and STAT3 binding to chromatin in situ. Thus, inS3-54 represents a novel probe for development of specific inhibitors targeting the DNA-binding domain of STAT3 and a potential therapeutic for cancer treatments.

Pyrithiamine as a Substrate for Thiamine Pyrophosphokinase

Thiamine pyrophosphokinase transfers a pyrophosphate group from a nucleoside triphosphate, such as ATP, to the hydroxyl group of thiamine to produce thiamine pyrophosphate. Deficiencies in thiamine can result in the development of the neurological disorder Wernicke-Korsakoff Syndrome as well as the potentially fatal cardiovascular disease wet beriberi. Pyrithiamine is an inhibitor of thiamine metabolism that induces neurological symptoms similar to that of Wernicke-Korsakoff Syndrome in animals. However, the mechanism by which pyrithiamine interferes with cellular thiamine phosphoester homeostasis is not entirely clear. We used kinetic assays coupled with mass spectrometry of the reaction products and x-ray crystallography of an equilibrium reaction mixture of thiamine pyrophosphokinase, pyrithiamine, and Mg2+/ATP to elucidate the mechanism by which pyrithiamine inhibits the enzymatic production of thiamine pyrophosphate. Three lines of evidence support the ability of thiamine pyrophosphokinase to form pyrithiamine pyrophosphate. First, a coupled enzyme assay clearly demonstrated the ability of thiamine pyrophosphokinase to produce AMP when pyrithiamine was used as substrate. Second, an analysis of the reaction mixture by mass spectrometry directly identified pyrithiamine pyrophosphate in the reaction mixture. Last, the structure of thiamine pyrophosphokinase crystallized from an equilibrium substrate/product mixture shows clear electron density for pyrithiamine pyrophosphate bound in the enzyme active site. This structure also provides the first clear picture of the binding pocket for the nucleoside triphosphate and permits the first detailed understanding of the catalytic requirements for catalysis in this enzyme.

Small-molecule inhibitors targeting the DNA-binding domain of STAT3 suppress tumor growth, metastasis and STAT3 target gene expression in vivo.

Signal transducer and activator of transcription 3 (STAT3) is constitutively activated in malignant tumors and has important roles in multiple aspects of cancer aggressiveness. Thus targeting STAT3 promises to be an attractive strategy for treatment of advanced metastatic tumors. Although many STAT3 inhibitors targeting the SH2 domain have been reported, few have moved into clinical trials. Targeting the DNA-binding domain (DBD) of STAT3, however, has been avoided due to its ‘undruggable‘ nature and potentially limited selectivity. In a previous study, we reported an improved in silico approach targeting the DBD of STAT3 that resulted in a small-molecule STAT3 inhibitor (inS3-54). Further studies, however, showed that inS3-54 has off-target effect although it is selective to STAT3 over STAT1. In this study, we describe an extensive structure and activity-guided hit optimization and mechanistic characterization effort, which led to identification of an improved lead compound (inS3-54A18) with increased specificity and pharmacological properties. InS3-54A18 not only binds directly to the DBD and inhibits the DNA-binding activity of STAT3 both in vitro and in situ but also effectively inhibits the constitutive and interleukin-6-stimulated expression of STAT3 downstream target genes. InS3-54A18 is completely soluble in an oral formulation and effectively inhibits lung xenograft tumor growth and metastasis with little adverse effect on animals. Thus inS3-54A18 may serve as a potential candidate for further development as anticancer therapeutics targeting the DBD of human STAT3 and DBD of transcription factors may not be ‘undruggable‘ as previously thought.

Identification of novel small molecule inhibitors of the XPA protein using in silico based screening

The nucleotide excision repair pathway catalyzes the removal of bulky adduct damage from DNA and requires the activity of more than 30 individual proteins and complexes. A diverse array of damage can be recognized and removed by the NER pathway including UV-induced adducts and intrastrand adducts induced by the chemotherapeutic compound cisplatin. The recognition of DNA damage is complex and involves a series of proteins including the xeroderma pigmentosum group A and C proteins and the UV-damage DNA binding protein. The xeroderma pigmentosum group A protein is unique in the sense that it is required for both transcription coupled and global genomic nucleotide excision repair. In addition, xeroderma pigmentosum group A protein is required for the removal of all types of DNA lesions repaired by nucleotide excision repair. Considering its importance in the damage recognition process, the minimal information available on the mechanism of DNA binding, and the potential that inhibition of xeroderma pigmentosum group A protein could enhance the therapeutic efficacy of platinum based anticancer drugs, we sought to identify and characterize small molecule inhibitors of the DNA binding activity of the xeroderma pigmentosum group A protein. In silico screening of a virtual small molecule library resulted in the identification of a class of molecules confirmed to inhibit the xeroderma pigmentosum group A protein-DNA interaction. Biochemical analysis of inhibition with varying DNA substrates revealed a common mechanism of xeroderma pigmentosum group A protein DNA binding to single-stranded DNA and cisplatin-damaged DNA.

Effective Targeting of the Survivin Dimerization Interface with Small-Molecule Inhibitors

Many oncoproteins are considered undruggable because they lack enzymatic activities. In this study, we present a small-molecule-based anticancer agent that acts by inhibiting dimerization of the oncoprotein survivin, thereby promoting its degradation along with spontaneous apoptosis in cancer cells. Through a combination of computational analysis of the dimerization interface and in silico screening, we identified one compound that induced proteasome-dependent survivin degradation. Analysis of a set of structural analogues led us to identify a lead compound (LQZ-7F), which was effective in blocking the survival of multiple cancer cell lines in a low micromolar concentration range. LQZ-7F induced proteasome-dependent survivin degradation, mitotic arrest, and apoptosis, and it blocked the growth of human tumors in mouse xenograft assays. In addition to providing preclinical proof of concept for a survivin-targeting anticancer agent, our work offers novel in silico screening strategies to therapeutically target homodimeric oncogenic proteins considered undruggable.

Repositioning proton pump inhibitors as anticancer drugs by targeting the thioesterase domain of human fatty acid synthase.

Fatty acid synthase (FASN), the enzyme responsible for de novo synthesis of free fatty acids, is up-regulated in many cancers. FASN is essential for cancer cell survival and contributes to drug resistance and poor prognosis. However, it is not expressed in most nonlipogenic normal tissues. Thus, FASN is a desirable target for drug discovery. Although different FASN inhibitors have been identified, none has successfully moved into clinical use. In this study, using in silico screening of an FDA-approved drug database, we identified proton pump inhibitors (PPIs) as effective inhibitors of the thioesterase activity of human FASN. Further investigation showed that PPIs inhibited proliferation and induced apoptosis of cancer cells. Supplementation of palmitate, the end product of FASN catalysis, rescued cancer cells from PPI-induced cell death. These findings provide new evidence for the mechanism by which this FDA-approved class of compounds may be acting on cancer cells.

Targeting survivin for therapeutic discovery: past, present, and future promises

Survivin, the smallest member of the inhibitor of apoptosis protein (IAP) family, is overexpressed in cells of almost all cancers but not in most normal tissues in adults. Survivin expression is required for cancer cell survival and knocking down its expression or inhibiting its function using molecular approaches results in spontaneous apoptosis. Thus, survivin is an attractive and perhaps ideal target for cancer drug discovery. However, a US Food and Drug Administration (FDA)-approved drug targeting survivin has yet to emerge. In this Foundation Review, we examine and evaluate various strategies that have been used to target survivin and the stages of each survivin inhibitor to help understand this lack of success. We also provide future perspectives moving forward in targeting survivin for drug discovery.

FASN regulates cellular response to genotoxic treatments by increasing PARP-1 expression and DNA repair activity via NF-κB and SP1

Significance The findings of this study have revealed a potential molecular pathway for how fatty acid synthase (FASN) overexpression causes drug and radiation resistance and contributes to poor clinical prognosis of cancer diseases. FASN is the sole cytosolic enzyme responsible for de novo lipid synthesis, required for cancer cell survival but not for most normal nonadipose tissues. The finding that FASN regulates DNA repair by regulating specificity protein 1 and NF-κB in cancer cell responses to anticancer treatments will have a profound impact on designing future treatment strategies. It will also help establish FASN as a target for therapeutic discovery to sensitize drug and radiation resistance. Fatty acid synthase (FASN), the sole cytosolic mammalian enzyme for de novo lipid synthesis, is crucial for cancer cell survival and associates with poor prognosis. FASN overexpression has been found to cause resistance to genotoxic insults. Here we tested the hypothesis that FASN regulates DNA repair to facilitate survival against genotoxic insults and found that FASN suppresses NF-κB but increases specificity protein 1 (SP1) expression. NF-κB and SP1 bind to a composite element in the poly(ADP-ribose) polymerase 1 (PARP-1) promoter in a mutually exclusive manner and regulate PARP-1 expression. Up-regulation of PARP-1 by FASN in turn increases Ku protein recruitment and DNA repair. Furthermore, lipid deprivation suppresses SP1 expression, which is able to be rescued by palmitate supplementation. However, lipid deprivation or palmitate supplementation has no effect on NF-κB expression. Thus, FASN may regulate NF-κB and SP1 expression using different mechanisms. Altogether, we conclude that FASN regulates cellular response against genotoxic insults by up-regulating PARP-1 and DNA repair via NF-κB and SP1.

Mechanism of Orlistat Hydrolysis by the Thioesterase of Human Fatty Acid Synthase

Fatty acid synthase (FASN), the sole protein capable of de novo synthesis of free fatty acids, is overexpressed in a wide variety of human cancers and is associated with poor prognosis and aggressiveness of these cancers. Orlistat, an FDA-approved drug for obesity treatment that inhibits pancreatic lipases in the GI tract, also inhibits the thioesterase (TE) of human FASN. The cocrystal structure of TE with orlistat shows a pseudo TE dimer containing two different forms of orlistat in the active site, an intermediate that is covalently bound to a serine residue (Ser2308) and a hydrolyzed and inactivated product. In this study, we attempted to understand the mechanism of TE-catalyzed orlistat hydrolysis by examining the role of the hexyl tail of the covalently bound orlistat in water activation for hydrolysis using molecular dynamics simulations. We found that the hexyl tail of the covalently bound orlistat undergoes a conformational transition, which is accompanied by destabilization of a hydrogen bond between a hydroxyl moiety of orlistat and the catalytic His2481 of TE that in turn leads to an increased hydrogen bonding between water molecules and His2481 and increased chance for water activation to hydrolyze the covalent bond between orlistat and Ser2308. Thus, the conformation of the hexyl tail of orlistat plays an important role in orlistat hydrolysis. Strategies that stabilize the hexyl tail may lead to the design of more potent irreversible inhibitors that target FASN and block TE activity with greater endurance.

Biochemistry and Pharmacology of Human ABCC1/MRP1 and its Role in Detoxification and in Multidrug Resistance of Cancer Chemotherapy

Human ABCC1 is a member of the ATP-binding cassette (ABC) transporter superfamily and serves as an efflux pump to a wide variety of substrates. Similar to most other ABC transporters, ABCC1 consists of both membrane-spanning and nucleotide-binding domains which function coordinately to actively transport substrates with the energy provided by ATP hydrolysis. Different from many other ABC transporters, ABCC1 has an additional membrane-spanning domain which may be responsible for its dimerization as well as a putative gating function. ABCC1 is ubiquitously expressed and its substrates vary from anticancer drugs to conjugated organic anions to heavy metals. Thus, it likely protects normal tissues where it is expressed from environmental cytotoxins and also protects cancer cells from cytotoxicities of anticancer drugs. Inhibitors of ABCC1 have been discovered and many are in process of further development. In this chapter, we review the current knowledge and recent progress in the studies of ABCC1, its structure and mechanism of action, its role in physiologic protection and drug resistance, as well as the discovery of ABCC1 inhibitors for development as chemosensitizers for combinational therapy of human cancers.