Selective Agents Research Articles

In vitro and in silico analysis of the Anopheles anticholinesterase activity of terpenoids.

Anopheles gambiae, An. coluzzii, An. arabiensis, and An. funestus are major vectors in high malaria endemic African regions. Various terpenoid classes form the main chemical constituent repository of essential oils, many of which have been shown to possess insecticidal effects against Anopheles species. The current study aimed to assess the bioactivity of terpenoids including four sesquiterpene alcohols, farnesol, (-)-α-bisabolol, cis-nerolidol, and trans-nerolidol; a phenylpropanoid, methyleugenol, and a monoterpene, (R)-(+)-limonene, using the larvicidal screening assay against the four Anopheles species. The mechanism of action was investigated through in vitro acetylcholinesterase inhibition assay and in silico molecular modelling. All six terpenoids showed potent larvicidal activity against the four Anopheles species. Insights into the mechanism of action revealed that the six terpenoids are strong AChE inhibitors against An. funestus and An. arabiensis, while there was a moderate inhibitory activity against An. gambiae AChE, but very weak activity against An. coluzzii. Interestingly, in the in silico study, farnesol established a favourable hydrogen bonding interaction with a conserved amino acid residue, Cys447, at the entrance to the active site gorge. While (-)-α-bisabolol and methyleugenol displayed a strong interaction with the catalytic Ser360 and adjacent amino acid residues; but sparing the mutable Gly280 residue that confers resistance to the current anticholinesterase insecticides. As a result, this study identified farnesol, (-)-α-bisabolol, and methyleugenol as selective bioinsecticidal agents with potent Anopheles AChE inhibition. These terpenoids present as natural compounds for further development as anticholinesterase bioinsecticides.

Temporal stability and directional change in a color cline of a marine snail from NW Spain.

The evolution and maintenance of color clines is a classic topic of research in evolutionary ecology. However, studies analyzing the temporal dynamics of such clines are much less frequent, due to the difficulty of obtaining reliable data about past color distributions along environmental gradients. In this article, we describe a case of decades-long temporal stability and directional change in a color cline of the marine snail Littorina saxatilis along the coastal inlet of the Ría de Vigo (NW Spain). L. saxatilis from this area shows a clear color cline with 3 distinct areas from the innermost to the more wave-exposed localities of the Ría: the inner, protected localities show an abundance of fawn-like individuals; the intermediate localities show a high diversity of colors; and the outer, wave-exposed localities show populations with a high frequency of a black and lineated morph. We compare data from the 1970s and 2022 in the same localities, showing that the cline has kept relatively stable for at least over half a century, except for some directional change and local variability in the frequency of certain morphs. Multiple regression analyses and biodiversity measures are presented to provide clues into the selective pressures that might be involved in the maintenance of this color cline. Future research avenues to properly test the explanatory power of these selective agents as well as the possible origins of the cline are discussed.

Targeting the Dark Lipid Kinase PIP4K2C with a Potent and Selective Binder and Degrader.

Phosphatidylinositol 5-phosphate 4-kinase, type II, gamma (PIP4K2C) remains a poorly understood lipid kinase with minimal enzymatic activity but potential scaffolding roles in immune modulation and autophagy-dependent catabolism. Achieving potent and selective agents for PIP4K2C while sparing other lipid and non-lipid kinases has been challenging. Here, we report the discovery of the highly potent PIP4K2C binder TMX-4102, which shows exclusive binding selectivity for PIP4K2C. Furthermore, we elaborated the PIP4K2C binder into TMX-4153, a bivalent degrader capable of rapidly and selectively degrading endogenous PIP4K2C. Collectively, our work demonstrates that PIP4K2C is a tractable and degradable target, and that TMX-4102 and TMX-4153 are useful leads to further interrogate the biological roles and therapeutic potential of PIP4K2C.

Targeting the Dark Lipid Kinase PIP4K2C with a Potent and Selective Binder and Degrader

AbstractPhosphatidylinositol 5‐phosphate 4‐kinase, type II, gamma (PIP4K2C) remains a poorly understood lipid kinase with minimal enzymatic activity but potential scaffolding roles in immune modulation and autophagy‐dependent catabolism. Achieving potent and selective agents for PIP4K2C while sparing other lipid and non‐lipid kinases has been challenging. Here, we report the discovery of the highly potent PIP4K2C binder TMX‐4102, which shows exclusive binding selectivity for PIP4K2C. Furthermore, we elaborated the PIP4K2C binder into TMX‐4153, a bivalent degrader capable of rapidly and selectively degrading endogenous PIP4K2C. Collectively, our work demonstrates that PIP4K2C is a tractable and degradable target, and that TMX‐4102 and TMX‐4153 are useful leads to further interrogate the biological roles and therapeutic potential of PIP4K2C.

Efflux Pump Inhibitors in Controlling Antibiotic Resistance: Outlook under a Heavy Metal Contamination Context

Multi-drug resistance to antibiotics represents a growing challenge in treating infectious diseases. Outside the hospital, bacteria with the multi-drug resistance (MDR) phenotype have an increased prevalence in anthropized environments, thus implying that chemical stresses, such as metals, hydrocarbons, organic compounds, etc., are the source of such resistance. There is a developing hypothesis regarding the role of metal contamination in terrestrial and aquatic environments as a selective agent in the proliferation of antibiotic resistance caused by the co-selection of antibiotic and metal resistance genes carried by transmissible plasmids and/or associated with transposons. Efflux pumps are also known to be involved in either antibiotic or metal resistance. In order to deal with these situations, microorganisms use an effective strategy that includes a range of expressions based on biochemical and genetic mechanisms. The data from numerous studies suggest that heavy metal contamination could affect the dissemination of antibiotic-resistant genes. Environmental pollution caused by anthropogenic activities could lead to mutagenesis based on the synergy between antibiotic efficacy and the acquired resistance mechanism under stressors. Moreover, the acquired resistance includes plasmid-encoded specific efflux pumps. Soil microbiomes have been reported as reservoirs of resistance genes that are available for exchange with pathogenic bacteria. Importantly, metal-contaminated soil is a selective agent that proliferates antibiotic resistance through efflux pumps. Thus, the use of multi-drug efflux pump inhibitors (EPIs) originating from natural plants or synthetic compounds is a promising approach for restoring the efficacy of existing antibiotics, even though they face a lot of challenges.

Phenylpyrazolone-1,2,3-triazole Hybrids as Potent Antiviral Agents with Promising SARS-CoV-2 Main Protease Inhibition Potential

COVID-19 infection is now considered one of the leading causes of human death. As an attempt towards the discovery of novel medications for the COVID-19 pandemic, nineteen novel compounds containing 1,2,3-triazole side chains linked to phenylpyrazolone scaffold and terminal lipophilic aryl parts with prominent substituent functionalities were designed and synthesized via a click reaction based on our previous work. The novel compounds were assessed using an in vitro effect on the growth of SARS-CoV-2 virus-infested Vero cells with different compound concentrations: 1 and 10 μM. The data revealed that most of these derivatives showed potent cellular anti-COVID-19 activity and inhibited viral replication by more than 50% with no or weak cytotoxic effect on harboring cells. In addition, in vitro assay employing the SARS-CoV-2-Main protease inhibition assay was done to test the inhibitors' ability to block the common primary protease of the SARS-CoV-2 virus as a mode of action. The obtained results show that the one non-linker analog 6h and two amide-based linkers 6i and 6q were the most active compounds with IC50 values of 5.08, 3.16, and 7.55 μM, respectively, against the viral protease in comparison to data of the selective antiviral agent GC-376. Molecular modeling studies were done for compound placement within the binding pocket of protease which reveal conserved residues hydrogen bonding and non-hydrogen interactions of 6i analog fragments: triazole scaffold, aryl part, and linker. Moreover, the stability of compounds and their interactions with the target pocket were also studied and analyzed by molecular dynamic simulations. The physicochemical and toxicity profiles were predicted, and the results show that compounds behave as an antiviral activity with low or no cellular or organ toxicity. All research results point to the potential usage of new chemotype potent derivatives as promising leads to be explored in vivo that might open the door to rational drug development of SARS-CoV-2 Main protease potent medicines.

Laboratory evaluation on oil-soluble resin as selective water shut-off agent in water control fracturing for low-permeability hydrocarbon reservoirs with bottom aquifer

During the development of low-permeability hydrocarbon reservoirs with bottom aquifer, hydraulic fracturing is essentially employed for commercial oil flow while increasing the potential risk of serious water production, thus water control hydraulic fracturing technology plays important role on addressing this challenge. In this work, a water control fracturing technique is developed to integrate fracture height containment with selective water shut-off. Specifically, oil-soluble resin is used as selective water shut-off agent and is injected with sinking agent simultaneously, carried by fracturing fluid at the fracture generation stage, to establish an improved artificial barrier. Afterwards, a modified fracture conductivity test equipment is designed and manufactured to measure the selective conductivity of the artificial barrier, its sensitivity to temperature, ratio of oil-soluble resin to sinking agent, two-phase flow are studied to evaluate the capability of selective water shut-off.The results demonstrated that 1) the occupation in porous space of sinking agent (0.212–0.425 mm) and the status change from glassy state to high elastic state of oil-soluble resin are mainly responsible for water shut-off; 2) The ratio of oil-soluble resin to sinking agent in 10 g: 70 g and temperature at 75 °C are two critical value to significantly reduce water conductivity in our experiments; 3) Fast dissolution of resin in oil results in oil conductivity over 5 to 100 times higher than water conductivity to achieve selective water shut-off. The results presented herein will provide important guidance on water control in low permeability reservoirs with active bottom aquifer and will promote the unconventional hydrocarbon recovery.

An Efficient Agrobacterium-Mediated Genetic Transformation Method for Solanum betaceum Cav. Embryogenic Callus

Somatic embryogenesis in Solanum betaceum (tamarillo) has proven to be an effective model system for studying morphogenesis, since optimized plant regeneration protocols are available, and embryogenic competent cell lines can be induced from different explants. Nevertheless, an efficient genetic transformation system for embryogenic callus (EC) has not yet been implemented for this species. Here, an optimized faster protocol of genetic transformation using Agrobacterium tumefaciens is described for EC. The sensitivity of EC to three antibiotics was determined, and kanamycin proved to be the best selective agent for tamarillo callus. Two Agrobacterium strains, EHA105 and LBA4404, both harboring the p35SGUSINT plasmid, carrying the reporter gene for β-glucuronidase (gus) and the marker gene neomycin phosphotransferase (nptII), were used to test the efficiency of the process. To increase the success of the genetic transformation, a cold-shock treatment, coconut water, polyvinylpyrrolidone and an appropriate selection schedule based on antibiotic resistance were employed. The genetic transformation was evaluated by GUS assay and PCR-based techniques, and a 100% efficiency rate was confirmed in the kanamycin-resistant EC clumps. Genetic transformation with the EHA105 strain resulted in higher values for gus insertion in the genome. The protocol presented provides a useful tool for functional gene analysis and biotechnology approaches.

Electrochemical-Based Sensing Platforms for Detection of Glucose and H2O2 by Porous Metal–Organic Frameworks: A Review of Status and Prospects

Establishing enzyme-free sensing assays with great selectivity and sensitivity for glucose and H2O2 detection has been highly required in biological science. In particular, the exploitation of nanomaterials by using noble metals of high conductivity and surface area has been widely investigated to act as selective catalytic agents for molecular recognition in sensing platforms. Several approaches for a straightforward, speedy, selective, and sensitive recognition of glucose and H2O2 were requested. This paper reviews the current progress in electrochemical detection using metal-organic frameworks (MOFs) for H2O2 and glucose recognition. We have reviewed the latest electrochemical sensing assays for in-place detection with priorities including straightforward procedure and manipulation, high sensitivity, varied linear range, and economic prospects. The mentioned sensing assays apply electrochemical systems through a rapid detection time that enables real-time recognition. In profitable fields, the obstacles that have been associated with sample preparation and tool expense can be solved by applying these sensing means. Some parameters, including the impedance, intensity, and potential difference measurement methods have permitted low limit of detections (LODs) and noticeable durations in agricultural, water, and foodstuff samples with high levels of glucose and H2O2.

The Use of Phage Antibodies for Microbial Cells Detection (Review)

Phage antibody display technology has revolutionized the field of bacterial immunodetection. This technology allows the expression of an antibody fused to the coat protein of a filamentous bacteriophage. The use of phage display makes it possible to obtain high-affinity antibodies by passing the stage of animal immunization, reducing the time for obtaining stable antibody-producing clones from several months to several weeks, significantly reducing the cost of the process. These advantages make phage antibodies an important tool for bacterial detection. The paper presents a brief description of the technological methods for obtaining phage antibodies to microbial cells. The possibilities and prospects for using phage antibodies as a selective agent in analytical systems, including biosensors, are discussed.

Study on Selective Water-blocking Agent for Temperature and Salt Resistance, Water Blocking, Air Permeability

Aiming at the high temperature and high salinity of Keshen 8 gas reservoir, the selective water plugging agent system with high temperature resistance, salt resistance and water permeability was studied. The effect of the plugging system components on the performance of plugging agent was investigated by single factor experiment, and the best formula was determined. The compressive strength of the material was stable at about 4.6 MPa, and the plugging agent had good temperature and salt resistance. Through the physical simulation experiment of fracture core flow, the plugging effect of different plugging agent injection volume on plugging agent is discussed. When the injection volume is 0.3 PV, the liquid phase plugging rate is 86.69 %, and the gas phase plugging rate is 26.18 %, which has good water resistance and gas permeability.

A Systematic Review of Deucravacitinib as a Novel Systemic Therapy for Moderate to Severe Plaque Psoriasis

Psoriasis is a chronic skin disease which to date, available treatments can only prevent symptoms remission. Plaque psoriasis is the most common type. Moderate to severe plaque psoriasis are commonly treated with systemic agents which bring systemic side effects that are unfavourable to patients. Researchers and clinicians are now looking at a novel highly selective TYK2 inhibitor agent, Deucravacitinib, which carries a lot less systemic side effects. We aim to review its efficacy on current studies available. We performed systematic review conducted from MEDLINE, ScienceDirect, and ProQuest library. The authors screened the articles based on inclusion criteria: (1) Trials including patients with moderate-to-severe plaque psoriasis for more than six months (2) Deucravacitinib as interventional therapy (3) Human studies that have PASI score as an outcome (4) Written in English. PASI, sPGA, and DLQI score improvements are outcomes measured in this review. Three randomized control trials (RCT) studies involving 1,943 patients were included. PASI75, PASI90, sPGA 0/1, and DLQI 0/1 achievements in all studies were significantly higher in subjects receiving Deucravacitinib compared to placebo and Apremilast—other active comparator. Deucravacitinib has been proven to be more efficacious in treating moderate to severe plaque psoriasis, even compared to another active biologic agent, Apremilast.

Abstract ED12-3: ATR inhibitors and PARP1 selective PARP inhibitors

Abstract The targeting of Homologous Recombination deficient (HRD) malignancy using a synthetic lethal strategy based on the inhibition and trapping of PARP1 on DNA in manner that leads to tumour selective effects via dependency on the function of HR gene products such as BRCA1, BRCA2 and PALB2 for DNA replication fitness is now well established. However, there is a need to improve both the frequency and duration of response in the licensed indications and to explore combination PARP inhibitor (PARPi) strategies that may be effective a broader range of breast cancers with functional deficiencies in HR and the wider DNA damage response. ATR inhibitors exacerbate replication stress that is toxic to HR deficient cells. Combinations of an ATR inhibition (ATRi) and PARPi have been shown to be synergistic and to be active in PARPi resistant pre-clinical model contexts. The development of combination strategies of these agents and of platinums and ATRi have been limited by combinatorial toxicity but have recently reported results in breast cancer. PARP1 is part of a family of PARP enzymes and currently licensed PARPi inhibit several family members that underpin some of their toxicity. New PARP1 selective agents have recently reported results in early phase trials. I will review some of the mechanistic rationale, preclinical data and relevant clinical trial data in my lecture. Citation Format: Andrew Tutt. ATR inhibitors and PARP1 selective PARP inhibitors [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr ED12-3.

Improving Agrobacterium tumefaciens-Mediated Genetic Transformation for Gene Function Studies and Mutagenesis in Cucumber (Cucumis sativus L.).

In the post-genomics era, Agrobacterium tumefaciens-mediated genetic transformation is becoming an increasingly indispensable tool for characterization of gene functions and crop improvement in cucumber (Cucumis sativus L.). However, cucumber transformation efficiency is still low. In this study, we evaluated the effects of several key factors affecting the shoot-regeneration rate and overall transformation efficiency in cucumber including genotypes, the age and sources of explants, Agrobacterium strains, infection/co-cultivation conditions, and selective agents. We showed that in general, North China cucumbers exhibited higher shoot-regeneration rate than US pickling or slicing cucumbers. The subapical ground meristematic regions from cotyledons or the hypocotyl had a similar shoot-regeneration efficiency that was also affected by the age of the explants. Transformation with the Agrobacterium strain AGL1 yielded a higher frequency of positive transformants than with GV3101. The antibiotic kanamycin was effective in selection against non-transformants or chimeras. Optimization of various factors was exemplified with the development of transgenic plants overexpressing the LittleLeaf (LL) gene or RNAi of the APRR2 gene in three cucumber lines. The streamlined protocol was also tested in transgenic studies in three additional genes. The overall transformation efficiency defined by the number of verified transgenic plants out of the number of seeds across multiple experiments was 0.2-1.7%. Screening among T1 OE transgenic plants identified novel, inheritable mutants for leaf or fruit color or size/shape, suggesting T-DNA insertion as a potential source of mutagenesis. The Agrobacterium-mediated transformation protocol from this study could be used as the baseline for further improvements in cucumber transformation.

Rapid Single-Shot Synthesis of the 214 Amino Acid-Long N-Terminal Domain of Pyocin S2.

The impermeable outer membrane of Pseudomonas aeruginosa is bypassed by antibacterial proteins known as S-type pyocins. Because of their properties, pyocins are investigated as a potential new class of antimicrobials against Pseudomonas infections. Their production and modification, however, remain challenging. To address this limitation, we employed automated fast-flow peptide synthesis for the rapid production of a pyocin S2 import domain. The N-terminal domain sequence (PyS2NTD) was synthesized in under 10 h and purified to yield milligram quantities of the desired product. To our knowledge, the 214 amino acid sequence of PyS2NTD is among the longest peptides produced from a "single-shot" synthesis, i.e., made in a single stepwise route without the use of ligation techniques. Biophysical characterization of the PyS2NTD with circular dichroism was consistent with the literature reports. Fluorescently labeled PyS2NTD binds to P. aeruginosa expressing the cognate ferripyoverdine receptor and is taken up into the periplasm. This selective uptake was validated with confocal and super resolution microscopy, flow cytometry, and fluorescence recovery after photobleaching. These modified, synthetic S-type pyocin domains can be used to probe import mechanisms of P. aeruginosa and leveraged to develop selective antimicrobial agents that bypass the outer membrane.

Phytochemicals and quaternary phosphonium ionic liquids: Connecting the dots to develop a new class of antimicrobial agents

IntroductionThe infections by multidrug-resistant bacteria are a growing threat to human health, and the efficacy of the available antibiotics is gradually decreasing. As such, new antibiotic classes are urgently needed. ObjectivesThis study aims to evaluate the antimicrobial activity, safety and mechanism of action of phytochemical-based triphenylphosphonium (TPP+) conjugates. MethodsA library of phytochemical-based TPP+ conjugates was repositioned and extended, and its antimicrobial activity was evaluated against a panel of Gram-positive (methicillin-resistant Staphylococcus aureus – MRSA) and Gram-negative bacteria (Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Acinetobacter baumannii) and fungi (Candida albicans, Cryptococcus neoformans var. grubii). The compounds’ cytotoxicity and haemolytic profile were also evaluated. To unravel the mechanism of action of the best compounds, the alterations in the surface charge, bacterial membrane integrity, and cytoplasmic leakage were assessed. ResultsStructure-activity-toxicity data revealed the contributions of the different structural components (phenolic ring, carbon-based spacers, carboxamide group, alkyl linker) to the compounds’ bioactivity and safety. Dihydrocinnamic derivatives 5 m and 5n stood out as safe, potent and selective antibacterial agents against S. aureus (MIC < 0.25 µg/mL; CC50 > 32 µg/mL; HC10 > 32 µg/mL). Mechanistic studies suggest that the antibacterial activity of compounds 5 m and 5n may result from interactions with the bacterial cell wall and membrane. ConclusionsCollectively, these studies demonstrate the potential of phytochemical-based TPP+ conjugates as a new class of antibiotics.

Sensitive and selective impedimetric determination of TNT using RSM-CCD optimization

Detection of trace amounts of 2,4,6-Trinitrotoluene as a widely used explosive in the military and industrial sectors is of vital importance due to security and environmental concerns. The sensitive and selective measurement characteristics of the compound still is considered a challenge for analytical chemists. Unlike conventional optical and electrochemical methods, the electrochemical impedance spectroscopy technique (EIS), has a very high sensitivity, but it faces a significant challenge in that it requires complex and expensive steps to modify the electrode surface with selective agents. We reported the design and construction of an inexpensive, simple, sensitive, and selective impedimetric electrochemical TNT sensor based on the formation of a Meisenheimer complex between magnetic multiwalled carbon nanotubes modified with aminopropyl triethoxysilane (MMWCNTs @ APTES) and TNT. The formation of the mentioned charge transfer complex at the electrode-solution interface blocks the electrode surface and disrupts the charge transfer in [(Fe (CN) 6)] 3−/4- redox probe system. Charge transfer resistance changes (ΔRCT) were used as an analytical response that corresponded to TNT concentration. To investigate the influence of effective parameters on the electrode response, such as pH, contact time, and modifier percentage, the response surface methodology based on central composite design (RSM-CCD) was used. The calibration curve was achieved in the range of 1–500 nM with a detection limit of 0.15 nM under optimal conditions, which included pH of 8.29, contact time of 479 s, and modifier percentage of 12.38% (w/w). The selectivity of the constructed electrode towards several nitroaromatic species was investigated, and no significant interference was found. Finally, the proposed sensor was able to successfully measure TNT in various water samples with satisfactory recovery percentages.

Serine Hydrolase Activity‐Based Probes for Use in Chemical Proteomics

AbstractSerine hydrolases (SHs) comprise a large superfamily of enzymes that play critical roles in many biological processes. Despite their importance, many SHs remain uncharacterized and the vast majority of SHs lack selective inhibitors. In response, activity‐based protein profiling (ABPP) and activity‐based probes (ABPs) have been leveraged to construct a more comprehensive picture of the SH proteome. Since the utility of ABPP is largely dictated by the reactivity profile of the ABPs deployed, novel scaffolds and chemotypes are needed to expand the breadth and selectivity of SH‐targeting ABPs. In this review, we highlight recent innovations in SH probe development, covering both established and emerging electrophilic warheads. We then discuss how strategic implementation of SH‐targeting ABPs has yielded selective, potent inhibitors and imaging agents with broad use. Finally, we present methods for ABP diversification and explore cutting‐edge applications in therapeutics development and discovery biology.

Antitumor activity of triazine mimic antibiotics for DNA-binding implications (impressive activity in vitro against a variety of tumor types in the NCI-60 screen): NSC 710607 to fight HCT-116 human colon carcinoma cell lines in vivo using the hollow fiber assay and xenograft mouse models.

Successful clinical applications of DNA-directed selective cytotoxic agents disrupt the vital replication/transcription processes and ultimately lead to cancer cell death. This study aimed to examine the growth screen of two lead triazine compounds in a number of cell lines and xenografts and to develop anticancer agents with noncovalent binding affinity bringing fewer side effects. The NCI initial hollow fiber test was performed using an established procedure. The cytostatic and cytocidal capacities of the test compounds were assessed by evaluating cytotoxicity by simply performing a standard cellular viability assay. The nude mouse human tumor xenograft system was used as an in vivo model. More sensitive drug with sub-micromolar activity met the interdisciplinary criteria for testing and was referred to evaluations in subcutaneous colorectal carcinoma HCT-116 human tumor xenografted into nude mice. Principal findings of the study: total cytostasis, almost nontoxic schedules, specific working hypotheses, strong rationale for the potential use, and important general implications (relevance to human biology). NSC 710607 displayed in vivo better than Cisplatin and 5-fluorouracil abilities to significantly decrease tumor growth. Cell proliferation can be reduced or stopped in vivo in view of the xenograft results. The mimic molecule behaves as DNA-binding antitumor antibiotics with great potential as general anticancer agents and deserves further trials. NSC 710607 represents the result of a design strategy with outstanding potential. This study also identifies the prognostic significance and is likely to translate to other species or systems.

Biotinylated selenocyanates: Potent and selective cytostatic agents

Most of the currently available cytotoxic agents for tackling cancer are devoid of selectivity, thus causing severe side-effects. This situation stimulated us to develop new antiproliferative agents with enhanced affinity towards tumour cells. We focused our attention on novel chalcogen-containing compounds (thiosemicarbazones, disulfides, selenoureas, thio- and selenocyanates), and particularly on selenium derivatives, as it has been documented that this kind of compounds might act as prodrugs releasing selenium-based reactive species on tumour cells. Particularly interesting in terms of potency and selectivity was a pharmacophore comprised by a selenocyanato-alkyl fragment connected to a p-phenylenediamine residue, where the nature of the second amino moiety (free, Boc-protected, enamine-protected) provided a wide variety of antiproliferative activities, ranging from the low micromolar to the nanomolar values. The optimized structure was in turn conjugated through a peptide linkage with biotin (vitamin B7), a cellular growth promoter, whose receptor is overexpressed in numerous cancer cells; the purpose was to develop a selective vector towards malignant cells. Such biotinylated derivative behaved as a very strong antiproliferative agent, achieving GI50 values in the low nM range for most of the tested cancer cells; moreover, it was featured with an outstanding selectivity, with GI50 > 100 µM against human fibroblasts.Mechanistic studies on the mode of inhibition of the biotinylated selenocyanate revealed (Annexin-V assay) a remarkable increase in the number of apoptotic cells compared to the control experiment; moreover, depolarization of the mitochondrial membrane was detected by flow cytometry analysis, and with fluorescent microscopy, what supports the apoptotic cell death. Prior to the apoptotic events, cytostatic effects were observed against SW1573 cells using label-free cell-living imaging; therefore, tumour cell division was prevented.Multidrug resistant cell lines exhibited a reduced sensitivity towards the biotinylated selenocyanate, probably due to its P-gp-mediated efflux. Remarkably, antiproliferative levels could be restored by co-administration with tariquidar, a P-gp inhibitor; this approach can, therefore, overcome multidrug resistance mediated by the P-gp efflux system.