Phenotype plasticity and altered sensitivity to chemotherapeutic agents in aggressive prostate cancer cells.

In 2023, approximately 288,300 new diagnoses of prostate cancer will occur, with 34,700 disease-related deaths. Death from prostate cancer is associated with metastasis, enabled by progression of tumor phenotypes and successful extracapsular extension to reach Batson's venous plexus, a specific route to the spine and brain. Using a mouse-human tumor xenograft model, we isolated an aggressive muscle invasive cell population of prostate cancer, called DU145J7 with a distinct biophysical phenotype, elevated histone H3K27, and increased matrix metalloproteinase 14 expression as compared to the non-aggressive parent cell population called DU145WT. Our goal was to determine the sensitivities to known chemotherapeutic agents of the aggressive cells as compared to the parent population. High-throughput screening was performed with 5,578 compounds, comprising of approved and investigational drugs for oncology. Eleven compounds were selected for additional testing, which revealed that vorinostat, 5-azacitidine, and fimepinostat (epigenetic inhibitors) showed 2.6-to-7.5-fold increases in lethality for the aggressive prostate cancer cell population as compared to the parent, as judged by the concentration of drug to inhibit 50% cell growth (IC50). On the other hand, the DU145J7 cells were 2.2-to-4.0-fold resistant to mitoxantrone, daunorubicin, and gimatecan (topoisomerase inhibitors) as compared to DU145WT. No differences in sensitivities between cell populations were found for docetaxel or pirarubicin. The increased sensitivity of DU145J7 prostate cancer cells to chromatin modifying agents suggests a therapeutic vulnerability occurs after tumor cells invade into and through muscle. Future work will determine which epigenetic modifiers and what combinations will be most effective to eradicate early aggressive tumor populations.

Real-Time Cellular Thermal Shift Assay to Monitor Target Engagement.

Determining a molecule's mechanism of action is paramount during chemical probe development and drug discovery. The cellular thermal shift assay (CETSA) is a valuable tool to confirm target engagement in cells for a small molecule that demonstrates a pharmacological effect. CETSA directly detects biophysical interactions between ligands and protein targets, which can alter a protein's unfolding and aggregation properties in response to thermal challenge. In traditional CETSA experiments, each temperature requires an individual sample, which restricts throughput and requires substantial optimization. To capture the full aggregation profile of a protein from a single sample, we developed a prototype real-time CETSA (RT-CETSA) platform by coupling a real-time PCR instrument with a CCD camera to detect luminescence. A thermally stable Nanoluciferase variant (ThermLuc) was bioengineered to withstand unfolding at temperatures greater than 90 °C and was compatible with monitoring target engagement events when fused to diverse targets. Utilizing well-characterized inhibitors of lactate dehydrogenase alpha, RT-CETSA showed significant correlation with enzymatic, biophysical, and other cell-based assays. A data analysis pipeline was developed to enhance the sensitivity of RT-CETSA to detect on-target binding. RT-CETSA technology advances capabilities of the CETSA method and facilitates the identification of ligand-target engagement in cells, a critical step in assessing the mechanism of action of a small molecule.

High-Throughput Cellular Thermal Shift Assay Using Acoustic Transfer of Protein Lysates.

Cellular thermal shift assay (CETSA) is a valuable method to confirm target engagement within a complex cellular environment, by detecting changes in a protein's thermal stability upon ligand binding. The classical CETSA method measures changes in the thermal stability of endogenous proteins using immunoblotting, which is low-throughput and laborious. Reverse-phase protein arrays (RPPAs) have been demonstrated as a detection modality for CETSA; however, the reported procedure requires manual processing steps that limit throughput and preclude screening applications. We developed a high-throughput CETSA using an acoustic RPPA (HT-CETSA-aRPPA) protocol that is compatible with 96- and 384-well microplates from start-to-finish, using low speed centrifugation to remove thermally destabilized proteins. The utility of HT-CETSA-aRPPA for guiding structure-activity relationship studies was demonstrated for inhibitors of lactate dehydrogenase A. Additionally, a collection of kinase inhibitors was screened to identify compounds that engage MEK1, a clinically relevant kinase target.

High-Throughput Detection of Ligand-Protein Binding Using a SplitLuc Cellular Thermal Shift Assay.

The confirmation of a small molecule binding to a protein target can be challenging when switching from biochemical assays to physiologically relevant cellular models. The cellular thermal shift assay (CETSA) is an approach to validate ligand-protein binding in a cellular environment by examining a protein's melting profile which can shift to a higher or lower temperature when bound by a small molecule. Traditional CETSA uses SDS-PAGE and Western blotting to quantify protein levels, a process that is both time consuming and low-throughput when screening multiple compounds and concentrations. Herein, we outline the reagents and methods to implement split Nano Luciferase (SplitLuc) CETSA, which is a reporter-based target engagement assay designed for high-throughput screening in 384- or 1536-well plate formats.

Inhibition of Human Immunodeficiency Virus-1 Integrase by ß-Diketo Acid Coated Gold Nanoparticles.

Gold nanoparticles (GNPs) have been proposed as carriers for drugs to improve their intrinsic therapeutic activities and to overcome pharmacokinetic problems. In this study, novel nanosystems constituted by a model ß-diketo acid (DKA) grafted to the surface of GNPs were designed and synthesized following the "multivalent high-affinity" binding strategy. These first nanoscale DKA prototypes showed improved inhibition of HIV-1 integrase (HIV-1 IN) catalytic activities as compared with free DKA ligands.

Alpha-Enolase: Emerging Tumor-Associated Antigen, Cancer Biomarker, and Oncotherapeutic Target.

Alpha-enolase, also known as enolase-1 (ENO1), is a glycolytic enzyme that "moonlights" as a plasminogen receptor in the cell surface, particularly in tumors, contributing to cancer cell proliferation, migration, invasion, and metastasis. ENO1 also promotes other oncogenic events, including protein-protein interactions that regulate glycolysis, activation of signaling pathways, and resistance to chemotherapy. ENO1 overexpression has been established in a broad range of human cancers and is often associated with poor prognosis. This increased expression is usually accompanied by the generation of anti-ENO1 autoantibodies in some cancer patients, making this protein a tumor associated antigen. These autoantibodies are common in patients with cancer associated retinopathy, where they exert pathogenic effects, and may be triggered by immunodominant peptides within the ENO1 sequence or by posttranslational modifications. ENO1 overexpression in multiple cancer types, localization in the tumor cell surface, and demonstrated targetability make this protein a promising cancer biomarker and therapeutic target. This mini-review summarizes our current knowledge of ENO1 functions in cancer and its growing potential as a cancer biomarker and guide for the development of novel anti-tumor treatments.

Maternal plasma proteomics in a rat model of pregnancy complications reveals immune and pro-coagulant gene pathway activation.

PROBLEM: The Brown Norway (BN) rat is a model of T-helper 2 immune diseases, and also a model of pregnancy disorders that include placental insufficiency, fetal loss, and pre-eclampsia-like symptoms. The aim of this study was to investigate the plasma proteomic/cytokine profile of pregnant BN rats in comparison to that of the Lewis (LEW) rat strain. METHOD OF STUDY: Plasma proteomics differences were studied at day 13 of pregnancy in pooled plasma samples by differential in-gel electrophoresis, and protein identification was performed by mass spectrometry. Key protein findings and predicted cytokine differences were validated by ELISA using plasma from rats at various pregnancy stages. Proteomics data were used for ingenuity pathway analysis (IPA). RESULTS: In-gel analysis revealed 74 proteins with differential expression between BN and LEW pregnant dams. ELISA studies confirmed increased maternal plasma levels of complement 4, prothrombin, and C-reactive protein in BN compared to LEW pregnancies. LEW pregnancies showed higher maternal plasma levels of transthyretin and haptoglobin than BN pregnancies. Ingenuity pathway analysis revealed that BN pregnancies are characterized by activation of pro-coagulant, reactive oxygen species, and immune-mediated chronic inflammation pathways, and suggested increased interleukin 6 and decreased transforming growth factor-ß1 as potential upstream events. Plasma cytokine analysis revealed that pregnant BN dams have a switch from anti- to pro-inflammatory cytokines with the opposite switch observed in pregnant LEW dams. CONCLUSION: Brown Norway rats show a maternal pro-inflammatory response to pregnancy that likely contributes to the reproductive outcomes observed in this rat strain.

Racial differences in the expression of inhibitors of apoptosis (IAP) proteins in extracellular vesicles (EV) from prostate cancer patients.

African-American men with prostate cancer typically develop more aggressive tumors than men from other racial/ethnic groups, resulting in a disproportionately high mortality from this malignancy. This study evaluated differences in the expression of inhibitors of apoptosis proteins (IAPs), a known family of oncoproteins, in blood-derived exosomal vesicles (EV) between African-American and European-American men with prostate cancer. The ExoQuick™ method was used to isolate EV from both plasma and sera of African-American (n = 41) and European-American (n = 31) men with prostate cancer, as well as from controls with no cancer diagnosis (n = 10). EV preparations were quantified by acetylcholinesterase activity assays, and assessed for their IAP content by Western blotting and densitometric analysis. Circulating levels of the IAP Survivin were evaluated by ELISA. We detected a significant increase in the levels of circulating Survivin in prostate cancer patients compared to controls (P<0.01), with the highest levels in African-American patients (P<0.01). African-American patients with prostate cancer also contained significantly higher amounts of EVs in their plasma (P<0.01) and sera (P<0.05) than European-American patients. In addition, EVs from African-American patients with prostate cancer contained significantly higher amounts of the IAPs Survivin (P<0.05), XIAP (P<0.001), and cIAP-2 (P<0.01) than EVs from European-American patients. There was no significant correlation between expression of IAPs and clinicopathological parameters in the two patient groups. Increased expression of IAPs in EVs from African-American patients with prostate cancer may influence tumor aggressiveness and contribute to the mortality disparity observed in this patient population. EVs could serve as reservoirs of novel biomarkers and therapeutic targets that may have clinical utility in reducing prostate cancer health disparities.

Targeting the stress oncoprotein LEDGF/p75 to sensitize chemoresistant prostate cancer cells to taxanes.

Prostate cancer (PCa) is associated with chronic prostate inflammation resulting in activation of stress and pro-survival pathways that contribute to disease progression and chemoresistance. The stress oncoprotein lens epithelium-derived growth factor p75 (LEDGF/p75), also known as DFS70 autoantigen, promotes cellular survival against environmental stressors, including oxidative stress, radiation, and cytotoxic drugs. Furthermore, LEDGF/p75 overexpression in PCa and other cancers has been associated with features of tumor aggressiveness, including resistance to cell death and chemotherapy. We report here that the endogenous levels of LEDGF/p75 are upregulated in metastatic castration resistant prostate cancer (mCRPC) cells selected for resistance to the taxane drug docetaxel (DTX). These cells also showed resistance to the taxanes cabazitaxel (CBZ) and paclitaxel (PTX), but not to the classical inducer of apoptosis TRAIL. Silencing LEDGF/p75 effectively sensitized taxane-resistant PC3 and DU145 cells to DTX and CBZ, as evidenced by a significant decrease in their clonogenic potential. While TRAIL induced apoptotic blebbing, caspase-3 processing, and apoptotic LEDGF/p75 cleavage, which leads to its inactivation, in both taxane-resistant and -sensitive PC3 and DU145 cells, treatment with DTX and CBZ failed to robustly induce these signature apoptotic events. These observations suggested that taxanes induce both caspase-dependent and -independent cell death in mCRPC cells, and that maintaining the structural integrity of LEDGF/p75 is critical for its role in promoting taxane-resistance. Our results further establish LEDGF/p75 as a stress oncoprotein that plays an important role in taxane-resistance in mCRPC cells, possibly by antagonizing drug-induced caspase-independent cell death.

Immunoseroproteomic Profiling in African American Men with Prostate Cancer: Evidence for an Autoantibody Response to Glycolysis and Plasminogen-Associated Proteins.

African American (AA) men suffer from a disproportionately high incidence and mortality of prostate cancer (PCa) compared with other racial/ethnic groups. Despite these disparities, African American men are underrepresented in clinical trials and in studies on PCa biology and biomarker discovery. We used immunoseroproteomics to profile antitumor autoantibody responses in AA and European American (EA) men with PCa, and explored differences in these responses. This minimally invasive approach detects autoantibodies to tumor-associated antigens that could serve as clinical biomarkers and immunotherapeutic agents. Sera from AA and EA men with PCa were probed by immunoblotting against PC3 cell proteins, with AA sera showing stronger immunoreactivity. Mass spectrometry analysis of immunoreactive protein spots revealed that several AA sera contained autoantibodies to a number of proteins associated with both the glycolysis and plasminogen pathways, particularly to alpha-enolase (ENO1). The proteomic data is deposited in ProteomeXchange with identifier PXD003968. Analysis of sera from 340 racially diverse men by enzyme-linked immunosorbent assays (ELISA) showed higher frequency of anti-ENO1 autoantibodies in PCa sera compared with control sera. We observed differences between AA-PCa and EA-PCa patients in their immunoreactivity against ENO1. Although EA-PCa sera reacted with higher frequency against purified ENO1 in ELISA and recognized by immunoblotting the endogenous cellular ENO1 across a panel of prostate cell lines, AA-PCa sera reacted weakly against this protein by ELISA but recognized it by immunoblotting preferentially in metastatic cell lines. These race-related differences in immunoreactivity to ENO1 could not be accounted by differential autoantibody recognition of phosphoepitopes within this antigen. Proteomic analysis revealed differences in the posttranslational modification profiles of ENO1 variants differentially recognized by AA-PCa and EA-PCa sera. These intriguing results suggest the possibility of race-related differences in the antitumor autoantibody response in PCa, and have implications for defining novel biological determinants of PCa health disparities.

Using Serological Proteome Analysis to Identify Serum Anti-Nucleophosmin 1 Autoantibody as a Potential Biomarker in European-American and African-American Patients With Prostate Cancer.

BACKGROUND: The prostate-specific antigen (PSA) testing has been widely implemented for the early detection and management of prostate cancer (PCa). However, the lack of specificity has led to overdiagnosis, resulting in many possibly unnecessary biopsies and overtreatment. Therefore, novel serological biomarkers with high sensitivity and specificity are of vital importance needed to complement PSA testing in the early diagnosis and effective management of PCa. This is particularly critical in the context of PCa health disparities, where early detection and management could help reduce the disproportionately high PCa mortality observed in African-American men. Previous studies have demonstrated that sera from patients with PCa contain autoantibodies that react with tumor-associated antigens (TAAs). METHODS: The serological proteome analysis (SERPA) approach was used to identify tumor-associated antigens (TAAs) of PCa. In evaluation study, the level of anti-NPM1 antibody was examined in sera from test cohort, validation cohort, as well as European-American (EA) and African-American (AA) men with PCa by using immunoassay. RESULTS: Nucleophosmin 1 (NPM1) as a 33 kDa TAA in PCa was identified and characterized by SERPA approach. Anti-NPM1 antibody level in PCa was higher than in benign prostatic hyperplasia (BPH) patients and healthy individuals. Receiver operating characteristic (ROC) curve analysis showed similar high diagnostic value for PCa in the test cohort (area under the curve (AUC):0.860) and validation cohort (AUC: 0.822) to differentiate from normal individuals and BPH. Interestingly, AUC values were significantly higher for AA PCa patients. When considering concurrent serum measurements of anti-NPM1 antibody and PSA, 97.1% PCa patients at early stage were identified correctly, while 69.2% BPH patients who had elevated PSA levels were found to be anti-NPM1 negative. Additionally, anti-NPM1 antibody levels in PCa patients at early stage significantly increased after surgery treatment. CONCLUSION: This intriguing data suggested that NPM1 can elicit autoantibody response in PCa and might be a potential biomarker for the immunodiagnosis and prognosis of PCa, and for supplementing PSA testing in distinguishing PCa from BPH. Prostate 76:1375-1386, 2016. © 2016 Wiley Periodicals, Inc.

The significance of autoantibodies to DFS70/LEDGFp75 in health and disease: integrating basic science with clinical understanding.

Antinuclear autoantibodies (ANAs) displaying the nuclear dense fine speckled immunofluorescence (DFS-IIF) pattern in HEp-2 substrates are commonly observed in clinical laboratory referrals. They target the dense fine speckled autoantigen of 70 kD (DFS70), most commonly known as lens epithelium-derived growth factor p75 (LEDGFp75). Interesting features of these ANAs include their low frequency in patients with systemic autoimmune rheumatic diseases (SARD), elevated prevalence in apparently healthy individuals, IgG isotype, strong trend to occur as the only ANA specificity in serum, and occurrence in moderate to high titers. These autoantibodies have also been detected at varied frequencies in patients with diverse non-SARD inflammatory and malignant conditions such as atopic diseases, asthma, eye diseases, and prostate cancer. These observations have recently stimulated vigorous research on their clinical and biological significance. Some studies have suggested that they are natural, protective antibodies that could serve as biomarkers to exclude a SARD diagnosis. Other studies suggest that they might be pathogenic in certain contexts. The emerging role of DFS70/LEDGFp75 as a stress protein relevant to human acquired immunodeficiency syndrome, cancer, and inflammation also points to the possibility that these autoantibodies could be sensors of cellular stress and inflammation associated with environmental factors. In this comprehensive review, we integrate our current knowledge of the biology of DFS70/LEDGFp75 with the clinical understanding of its autoantibodies in the contexts of health and disease.

DFS70/LEDGFp75: An Enigmatic Autoantigen at the Interface between Autoimmunity, AIDS, and Cancer.

Clinical and diagnostic laboratories often encounter patient sera containing antinuclear antibodies (ANAs) that produce a nuclear dense fine speckled immunofluorescence pattern on HEp-2 cells. These autoantibodies usually target the dense fine speckled protein of 70 kDa (DFS70), commonly known as lens epithelium-derived growth factor p75 (LEDGFp75). Anti-DFS70/LEDGFp75 autoantibodies have recently attracted much interest because of their relatively common occurrence in sera from patients with positive ANA tests with no clinical evidence of systemic autoimmune rheumatic disease (SARD). Their presence has been documented primarily in patients with diverse non-SARD inflammatory conditions and "apparently healthy" individuals. While there is circumstantial evidence that depending on the context these autoantibodies could play protective, pathogenic, or sensor roles, their significance remains elusive. DFS70/LEDGFp75 has emerged during the past decade as a stress transcription co-activator relevant to HIV integration, cancer, and inflammation. It is not clear, however, what makes this protein the target of such a common autoantibody response. We suggest that a better understanding of DFS70/LEDGFp75 biology is key to elucidating the significance of its associated autoantibodies. Here, we discuss briefly our current understanding of this enigmatic autoantigen and potential scenarios leading to its targeting by the immune system.

Design and discovery of 5-hydroxy-6-oxo-1,6-dihydropyrimidine-4-carboxamide inhibitors of HIV-1 integrase.

Raltegravir (RAL) is a first clinically approved integrase (IN) inhibitor for the treatment of HIV but rapid mutation of the virus has led to chemo-resistant strains. Therefore, there is a medical need to develop new IN inhibitors to overcome drug resistance. At present, several IN inhibitors are in different phases of clinical trials and few have been discontinued due to toxicity and lack of efficacy. The development of potent second-generation IN inhibitors with improved safety profiles is key for selecting new clinical candidates. In this article, we report the design and synthesis of potent 5-hydroxy-6-oxo-1,6-dihydropyrimidine-4-carboxamide analogues as second-generation IN inhibitors. These compounds satisfy two structural requirements known for potent inhibition of HIV-1 IN catalysis: a metal chelating moiety and a hydrophobic functionality necessary for selectivity against the strand transfer reaction. Most of the new compounds described herein are potent and selective for the strand transfer reaction and show antiviral activity in cell-based assays. Furthermore, this class of compounds are drug-like and suitable for further optimization and preclinical studies.

Design and discovery of flavonoid-based HIV-1 integrase inhibitors targeting both the active site and the interaction with LEDGF/p75.

HIV integrase (IN) is an essential enzyme for the viral replication. Currently, three IN inhibitors have been approved for treating HIV-1 infection. All three drugs selectively inhibit the strand transfer reaction by chelating a divalent metal ion in the enzyme active site. Flavonoids are a well-known class of natural products endowed with versatile biological activities. Their ß-ketoenol or catechol structures can serve as a metal chelation motif and be exploited for the design of novel IN inhibitors. Using the metal chelation as a common pharmacophore, we introduced appropriate hydrophobic moieties into the flavonol core to design natural product-based novel IN inhibitors. We developed selective and efficient syntheses to generate a series of mono 3/5/7/3'/4'-substituted flavonoid derivatives. Most of these new compounds showed excellent HIV-1 IN inhibitory activity in enzyme-based assays and protected against HIV-1 infection in cell-based assays. The 7-morpholino substituted 7c showed effective antiviral activity (EC50=0.826 µg/mL) and high therapeutic index (TI>242). More significantly, these hydroxyflavones block the IN-LEDGF/p75 interaction with low- to sub-micromolar IC50 values and represent a novel scaffold to design new generation of drugs simultaneously targeting the catalytic site as well as protein-protein interaction domains.

Design and synthesis of novel pyrimidone analogues as HIV-1 integrase inhibitors.

A series of novel pyrimidone analogues have been designed and synthesized as HIV-1 integrase (IN) inhibitors. This study demonstrated that introducing a substituent in the N1-position of the pyrimidone scaffold does not significantly influence IN inhibitory activity. Molecular docking studies showed these compounds could occupy the IN active site and form pi-pi interactions with viral DNA nucleotides DC16 and DA17 to displace reactive viral DNA 3'OH and block intasome activity.

Synthesis, docking, and biological studies of phenanthrene ß-diketo acids as novel HIV-1 integrase inhibitors.

In the present study we report the synthesis of halogen-substituted phenanthrene ß-diketo acids as new HIV-1 integrase inhibitors. The target phenanthrenes were obtained using both standard thermal- and microwave-assisted synthesis. 4-(6-Chlorophenanthren-2-yl)-2,4-dioxobutanoic acid (18) was the most active compound of the series, inhibiting both 3'-end processing (3'-P) and strand transfer (ST) with IC50 values of 5 and 1.3 µM, respectively. Docking studies revealed two predominant binding modes that were distinct from the binding modes of raltegravir and elvitegravir, and suggest a novel binding region in the IN active site. Moreover, these compounds are predicted not to interact significantly with some of the key amino acids (Q148 and N155) implicated in viral resistance. Therefore, this series of compounds can further be investigated for a possible chemotype to circumvent resistance to clinical HIV-1 IN inhibitors.

Design of cell-permeable stapled peptides as HIV-1 integrase inhibitors.

HIV-1 integrase (IN) catalyzes the integration of viral DNA into the host genome, involving several interactions with the viral and cellular proteins. We have previously identified peptide IN inhibitors derived from the α-helical regions along the dimeric interface of HIV-1 IN. Herein, we show that appropriate hydrocarbon stapling of these peptides to stabilize their helical structure remarkably improves the cell permeability, thus allowing inhibition of the HIV-1 replication in cell culture. Furthermore, the stabilized peptides inhibit the interaction of IN with the cellular cofactor LEDGF/p75. Cellular uptake of the stapled peptide was confirmed in four different cell lines using a fluorescein-labeled analogue. Given their enhanced potency and cell permeability, these stapled peptides can serve as not only lead IN inhibitors but also prototypical biochemical probes or "nanoneedles" for the elucidation of HIV-1 IN dimerization and host cofactor interactions within their native cellular environment.

Discovery of novel inhibitors of LEDGF/p75-IN protein-protein interactions.

Human lens epithelium-derived growth factor (LEDGF)/p75 plays an important role in the HIV life cycle by stimulating integrase (IN)-led viral DNA integration into cellular chromosomes. Mechanistic studies show the majority of IN inhibitors chelate magnesium ions in the catalytic active site, a region topologically distant from the LEDGF/p75 binding site. Compounds disrupting the formation of LEDGF/p75 and IN complexes serve as a novel mechanistic approach different from current antiretroviral therapies. We previously built pharmacophore models mimicking LEDGF/p75 residues and identified four classes of LEDGF/p75-IN inhibitors. Substructure and similarity searches yielded additional LEDGF/p75-IN inhibitors containing an acylhydrazone moiety. The most potent of the acylhydrazones inhibited LEDGF/p75-IN interaction with an IC(50) value of 400nM. We explored structure-activity relationships (SAR) and identified new acylhydrazones, hydrazines, and diazenes as lead molecules for further optimization. Two lead LEDGF/p75-IN inhibitors showed antiviral activity.

Ruthenium arene complexes as HIV-1 integrase strand transfer inhibitors.

The quinolone HL(1) and the hydroxypyrimidine-carboxamide HL(2) were designed and synthesized as models of the HIV integrase strand transfer inhibitors Elvitegravir and Raltegravir (brand name Isentress), with the aim to study their complexing behavior and their biological activity. The Ru(arene) complexes [RuCl(η(6)-p-cym)L(1)], [RuCl(η(6)-p-cym)L(2)] and [RuCl(hexamethylbenzene)L(2)] were also synthesized and spectroscopically characterized and their X-ray diffraction structures were discussed. The ligands and the complexes showed inhibition potency in the sub/low-micromolar concentration range in anti-HIV-1 integrase enzymatic assays, with selectivity toward strand transfer catalytic process, without any significant cytotoxicity on cancer cells.