Chemical Inducers Research Articles

Study of white rot fungi for the production of laccase and its multifold application

Laccase benzenediol oxygen oxidoreductases are multicopper enzymes belonging to the group of blue oxidases. They catalyses the monoelectronic oxidation of substrates at the expense of molecular oxygen. intrestess in these essentially eco-friendly enzymes they work with air and produce water as the only by product has grown significantly in recent years, Carbon sources used are glucose, fructose, galactose, galacturonic acid, xylose, lactose, sucrose ,pectin and insulin for the production of laccase. Chemical inducers used are inducers used are copper, lignin, veteryl alcohol, xylidine and gualacsol.

Cardiac Differentiation of Mesenchymal Stem Cells: Impact of Biological and Chemical Inducers.

Cardiovascular disorders (CVDs) are the leading cause of global death, widely occurs due to irreparable loss of the functional cardiomyocytes. Stem cell-based therapeutic approaches, particularly the use of Mesenchymal Stem Cells (MSCs) is an emerging strategy to regenerate myocardium and thereby improving the cardiac function after myocardial infarction (MI). Most of the current approaches often employ the use of various biological and chemical factors as cues to trigger and modulate the differentiation of MSCs into the cardiac lineage. However, the recent advanced methods of using specific epigenetic modifiers and exosomes to manipulate the epigenome and molecular pathways of MSCs to modify the cardiac gene expression yield better profiled cardiomyocyte like cells in vitro. Hitherto, the role of cardiac specific inducers triggering cardiac differentiation at the cellular and molecular level is not well understood. Therefore, the current review highlights the impact and recent trends in employing biological and chemical inducers on cardiac differentiation of MSCs. Thereby, deciphering the interactions between the cellular microenvironment and the cardiac inducers will help us to understand cardiomyogenesis of MSCs. Additionally, the review also provides an insight on skeptical roles of the cell free biological factors and extracellular scaffold assisted mode for manipulation of native and transplanted stem cells towards translational cardiac research.

Optogenetic Amplification Circuits for Light-Induced Metabolic Control.

Dynamic control of microbial metabolism is an effective strategy to improve chemical production in fermentations. While dynamic control is most often implemented using chemical inducers, optogenetics offers an attractive alternative due to the high tunability and reversibility afforded by light. However, a major concern of applying optogenetics in metabolic engineering is the risk of insufficient light penetration at high cell densities, especially in large bioreactors. Here, we present a new series of optogenetic circuits we call OptoAMP, which amplify the transcriptional response to blue light by as much as 23-fold compared to the basal circuit (OptoEXP). These circuits show as much as a 41-fold induction between dark and light conditions, efficient activation at light duty cycles as low as ∼1%, and strong homogeneous light-induction in bioreactors of at least 5 L, with limited illumination at cell densities above 40 OD600. We demonstrate the ability of OptoAMP circuits to control engineered metabolic pathways in novel three-phase fermentations using different light schedules to control enzyme expression and improve production of lactic acid, isobutanol, and naringenin. These circuits expand the applicability of optogenetics to metabolic engineering.

Combined effect of β-aminobutyric acid and silver nanoparticles on eggplants, Solanum melongena, infected with Meloidogyne javanica

Summary The acquired resistance of plants to root-knot nematodes (Meloidogyne spp.) can be achieved by applying chemical inducers, such as β-aminobutyric acid (BABA). Recently, the use of metal nanoparticles with multifunctional use against plant-parasitic nematodes has been presented. In this study, silver nanoparticles (AgNPs) were synthesised using the aqueous leaf extract of Malva sylvestris. The effect of different AgNPs concentrations on hatching and mortality of the second-stage juveniles (J2) of Meloidogyne javanica was investigated in vitro. The effects of AgNPs on eggplants infected with 0, 1, 2, 4 and 8 eggs (cm3 soil)−1 were investigated under glasshouse conditions. The combined effects of AgNPs and BABA on eggplants infected with 4 and 8 eggs (cm3 soil)−1 were investigated in another study. The results showed that increasing the concentration of AgNPs the hatching of M. javanica decreased and the mortality of J2 increased. The number of eggs, galls, egg masses and reproduction factor in plants treated with AgNPs infected with 4 eggs (cm3 soil)−1 decreased by 43.4, 24.7, 23.5 and 43.2%, and in plants infected with 8 eggs (cm3 soil)−1, decreased by 64.5, 25.5, 63.1 and 64.5%, respectively, compared to control plants. The reproduction factor in infected plants with 4 and 8 eggs (cm3 soil)−1 that were treated with BABA (1 mM) and AgNPs decreased by 51.6 and 55.9%, respectively, compared to control plants. The results of this study demonstrated the effects of BABA, AgNPs and the combination of these two substances in reducing the damage of different inoculum densities of M. javanica in eggplant.

Efficient CRISPR-Cas9-based genome editing of β-globin gene on erythroid cells from homozygous β039-thalassemia patients

Gene editing by the CRISPR-Cas9 nuclease system technology can be considered among the most promising strategies to correct hereditary mutations in a variety of monogenic diseases. In this paper, we present for the first time the correction, by CRISPR-Cas9 gene editing, of the β039-thalassemia mutation, one of the most frequent in the Mediterranean area. The results obtained demonstrated the presence of normal β-globin genes after CRISPR-Cas9 correction of the β039-thalassemia mutation performed on erythroid precursor cells from homozygous β039-thalassemia patients. This was demonstrated by allele-specific PCR and sequencing. Accumulation of corrected β-globin mRNA and relevant “de novo” production of β-globin and adult hemoglobin (HbA) were found with high efficiency. The CRISPR-Cas9-forced HbA production levels were associated with a significant reduction of the excess of free α-globin chains. Genomic toxicity of the editing procedure (low indels and no off-targeting) was analyzed. The protocol might be the starting point for the development of an efficient editing of CD34+ cells derived from β039 patients and for the design of combined treatments using, together with the CRISPR-Cas9 editing of the β-globin gene, other therapeutic approaches, such as, for instance, induction of HbA and/or fetal hemoglobin (HbF) using chemical inducers.

Efficacy of some Bio-agents, Chemical Inducers and Fungicides in Controlling Tomato Root Rot Disease Caused by Rhizoctonia solani

Efficacy of some Bio-agents, Chemical Inducers and Fungicides in Controlling Tomato Root Rot Disease Caused by Rhizoctonia solani

Optimization of the Light-On system in a lentiviral platform to a light-controlled expression of genes in neurons

Abstract Background Molecular brain therapies require the development of molecular switches to control gene expression in a limited and regulated manner in time and space. Light-switchable gene systems allow precise control of gene expression with an enhanced spatio-temporal resolution compared to chemical inducers. In this work, we adapted the existing light-switchable Light-On system into a lentiviral platform, which consists of two modules: (i) one for the expression of the blue light-switchable trans-activator GAVPO and (ii) a second module containing an inducible-UAS promoter (UAS) modulated by a light-activated GAVPO. Results In the HEK293-T cell line transfected with this lentiviral plasmids system, the expression of the reporter mCherry increased between 4 to 5 fold after light induction. A time expression analysis after light induction during 24 h revealed that mRNA levels continuously increased up to 9 h, while protein levels increased throughout the experiment. Finally, transduction of cultured rat hippocampal neurons with this dual Light-On lentiviral system showed that CDNF, a potential therapeutic trophic factor, was induced only in cells exposed to blue light. Conclusions In conclusion, the optimized lentiviral platform of the Light-On system provides an efficient way to control gene expression in neurons, suggesting that this platform could potentially be used in biomedical and neuroscience research, and eventually in brain therapies for neurodegenerative diseases. How to cite: Zarate RV, Arancibia D, Fernandez A, et al. Optimization of the Light-On system in a lentiviral platform to a light-controlled expression of genes in neurons. Electron J Biotechnol 2021;51. https://doi.org/10.1016/j.ejbt.2021.03.006

Research progress and industrial application of Bacillus subtilis in systematic and synthetic biotechnology

Bacillus subtilis is a model strain for studying the physiological and biochemical mechanisms of microorganism, and is also a good chassis cell for industrial application to produce biological agents such as small molecule compounds, bulk chemicals, industrial enzymes, precursors of drugs and health product. In recent years, studies on metabolic engineering methods and strategies of B. subtilis have been increasingly reported, providing good tools and theoretical references for using it as chassis cells to produce biological agents. This review provides information on systematically optimizing the Bacillus subtilis chassis cell by regulating global regulatory factors, simplifying and optimizing the genome, multi-site and multi-dimensional regulating, dynamic regulating through biosensors, membrane protein engineering. For producing the protein reagent, the strain is optimized by optimizing the promoters, signal peptides, secretion components and building the expression system without chemical inducers. In addition, this review also prospects the important issues and directions that need to be focused on in the further optimization of B. subtilis in industrial production.

Potential metabolic resistance mechanisms to ivermectin in Anopheles gambiae: a synergist bioassay study

BackgroundDespite remarkable success obtained with current malaria vector control strategies in the last 15 years, additional innovative measures will be needed to achieve the ambitious goals for malaria control set for 2030 by the World Health Organization (WHO). New tools will need to address insecticide resistance and residual transmission as key challenges. Endectocides such as ivermectin are drugs that kill mosquitoes which feed on treated subjects. Mass administration of ivermectin can effectively target outdoor and early biting vectors, complementing the still effective conventional tools. Although this approach has garnered attention, development of ivermectin resistance is a potential pitfall. Herein, we evaluate the potential role of xenobiotic pumps and cytochrome P450 enzymes in protecting mosquitoes against ivermectin by active efflux and metabolic detoxification, respectively.MethodsWe determined the lethal concentration 50 for ivermectin in colonized Anopheles gambiae; then we used chemical inhibitors and inducers of xenobiotic pumps and cytochrome P450 enzymes in combination with ivermectin to probe the mechanism of ivermectin detoxification.ResultsDual inhibition of xenobiotic pumps and cytochromes was found to have a synergistic effect with ivermectin, greatly increasing mosquito mortality. Inhibition of xenobiotic pumps alone had no effect on ivermectin-induced mortality. Induction of xenobiotic pumps and cytochromes may confer partial protection from ivermectin.ConclusionThere is a clear pathway for development of ivermectin resistance in malaria vectors. Detoxification mechanisms mediated by cytochrome P450 enzymes are more important than xenobiotic pumps in protecting mosquitoes against ivermectin.Graphical

Constitutive secretory expression and characterization of nitrilase from Alcaligenes faecalis in Pichia pastoris for production of R-mandelic acid.

Nitrilases can directly hydrolyze nitrile compounds into carboxylic acids and ammonium. To solve the current problems of bioconversions using nitrilases, including the difficult separation of products from the resting cells used as the catalyst and high costs of chemical inducers, a nitrilase from Alcaligenes faecalis was heterologously expressed in Pichia pastoris X33. The stable nitrilase-expressing strain No.39-6-4 was obtained after three rounds of screening based on a combined detection method including dot-blot, SDS-PAGE, and western blot analyses, which confirmed the presence of recombinant nitrilase with a molecular mass of about 50 kDa. The temperature and pH optima of the nitrilase were 45°C and pH 7.5, respectively. Cu2+ , Zn2+ , and Tween 80 strongly inhibited the enzyme activity, but the optical purity of the product R-mandelic acid (R-MA) was stable, with practically 100% enantiomeric excess (ee). The nitrilase-producing P. pastoris strain developed in this study provides a basis for further research on the enzyme.

Development of a reversible regulatory system for gene expression in the cyanobacterium Synechocystis sp. PCC 6803 by quorum-sensing machinery from marine bacteria

Histidine kinases are common sensory proteins used to detect environmental changes in bacteria. They respond to specific stimuli via a signal-input domain and alter gene expression through a cognate response regulator. The modulation/control of transcriptional regulation in cyanobacteria is important to reinforce the production of useful target compounds via photosynthesis without altering the growth profiles. For instance, heavy metal ions (Ni2+ and Cu2+), chemical inducers (IPTG), and a volatile compound (toluene) have been previously applied to regulate gene expression in cyanobacteria. However, most systems/regulators are only able to regulate gene expression once because it is impossible to eliminate them from the medium. To construct a reversible regulation system, a chimeric sensor, VanN_SphS, was developed by fusing the signal input domain of a quorum-sensing (QS) sensor, VanN, from Vibrio anguillarum, responding N-3-hydroxyhexanoyl-L-homoserine lactone (OHC6-HSL), with the kinase domain of SphS, a phosphate-deficiency sensor from the cyanobacterium Synechocystis sp. PCC 6803. After expression of the chimeric sensor in Synechocystis cells, responses to the various N-acyl-homoserine-lactones (AHLs) were evaluated by measuring the alkaline phosphatase (AP) activity, which is regulated by SphS. VanN_SphS responded only to OHC6-HSL and repressed AP activity. Then, the coexpression of the AHLs-degradation enzyme, Aii20J, a lactonase from Tenacibaculum sp. 20J, resumed the activity. This is the first report on the use of AHL-mediated transcriptional regulation in Synechocystis, which could be used in the future for the controlled production of useful compounds in the cyanobacterium.

Use of anti-fungus Trichoderma viride and salicylic acid to Induce chemical controll to fight tomato fusarium wilt disease.

Tomatoes are among the most economical vegetable crops that can be affected by many diseases, for example Fusarium wilt disease, which is caused by the fungus “Fusarium oxysporum f. sp. lycopersici,” which is considered one of the most common diseases affecting tomato plants. Biocontrol and chemical inducers represent an important strategy to stimulate the plant defense system, especially when applied together. In the current study, the efficacy of the following treatments, for example, “Trichoderma viride” and salicylic acid, alone or in combination, was evaluated compared to the effectiveness of the recommended fungicide Topsin-M 70%, which was used in the recommended dose as a new strategy to increase the defense efficiency of tomatoes to combat Fusarium wilt disease under greenhouse and field conditions. The various physiological defensive changes were studied in addition to estimating some enzymes associated with the chemical defense system of tomato plants such as polyphenol oxidase, peroxidase chitinase, and total dissolved phenols). The results obtained confirmed that all the treatments used led to the protection of tomato plants completely from infection, as the risk of disease decreased significantly, the level of all physiological estimates specified changed significantly as a result of the use of anti-fungi and chemical inducers, as many defense compounds were shown, which resulted in the activation of the chemical control in tomato plants against the attack of pathogens. These treatments also led to a significant increase in the growth variables and the total yield.

Molecular identification of Peronospora viciae causing downy mildew on field pea pods and its management

In the growing season 2016–2017, downy mildew disease was initially occurred on pea pods followed by plantlets leaves that observed in different locations where pea plants were grown at Kafr-El-Dawar district, Beheria governorate, Egypt. This irregular observation leads to thought that the cause of disease is new to this particular region. Molecular identification of the isolated fungus proved that the causal agent is Peronospora viciae. The sequences of the fungal isolate sent to GenBank and registered under accession number MN620384. Under field trials, it was observed that throughout the two cultivated seasons the fungicides application had greater effect on downy mildew incidence comparing with applied chemical inducers. Along the two cultivation seasons, pea plants sprayed with salicylic acid, potassium sorbate and ascorbic acid highly suppressed the disease incidence between 12.4 and 17.1%. Moderate effect was recorded as 19.2–22.7% disease reduction with potassium bicarbonate, lemongrass oil, lemongrass oil and humic acid application. The lowest effect was recorded as 32.5% at sodium benzoate treatment; meanwhile, 54.3% disease incidence was recorded at untreated control. Under field conditions, significant reduction in pea downy mildew disease was achieved by the use of fungicide alternative as foliar application. Therefore, these approaches could be utilized successfully for controlling this foliar disease especially they distinguished with safely use and avoidance of environmental pollution.

Mixtures modeling identifies chemical inducers versus repressors of toxicity associated with wildfire smoke

Exposure to wildfire smoke continues to be a growing threat to public health, yet the chemical components in wildfire smoke that primarily drive toxicity and associated disease are largely unknown. This study utilized a suite of computational approaches to identify groups of chemicals induced by variable biomass burn conditions that were associated with biological responses in the mouse lung, including pulmonary immune response and injury markers. Smoke condensate samples were collected and characterized, resulting in chemical distribution information for 86 constituents across ten different exposures. Mixtures-relevant statistical methods included (i) a chemical clustering and data-reduction method, weighted chemical co-expression network analysis (WCCNA), (ii) a quantile g-computation approach to address the joint effect of multiple chemicals in different groupings, and (iii) a correlation analysis to compare mixtures modeling results against individual chemical relationships. Seven chemical groups were identified using WCCNA based on co-occurrence showing both positive and negative relationships with biological responses. A group containing methoxyphenols (e.g., coniferyl aldehyde, eugenol, guaiacol, and vanillin) displayed highly significant, negative relationships with several biological responses, including cytokines and lung injury markers. This group was further shown through quantile g-computation methods to associate with reduced biological responses. Specifically, mixtures modeling based on all chemicals excluding those in the methoxyphenol group demonstrated more significant, positive relationships with several biological responses; whereas mixtures modeling based on just those in the methoxyphenol group demonstrated significant negative relationships with several biological responses, suggesting potential protective effects. Mixtures-based analyses also identified other groups consisting of inorganic elements and ionic constituents showing positive relationships with several biological responses, including markers of inflammation. Many of the effects identified through mixtures modeling in this analysis were not captured through individual chemical analyses. Together, this study demonstrates the utility of mixtures-based approaches to identify potential drivers and inhibitors of toxicity relevant to wildfire exposures.

Optimization of growth conditions for enhancing the production of microbial laccase and its application in treating antibiotic contamination in wastewater.

In this work, seven indigenous macrofungal isolates were selected to screen for their laccase production capability. Among them, isolates viz., Pleurotus eryngii, Pleurotus florida, Pleurotus sajor caju and Gandoderma lucidum were found to exhibit high laccase activity in the preliminary studies and were thus selected for the optimization studies with an aim to enhance laccase production. The pH optimization studies were carried out between pH range of 4-6. The laccase activity and biomass were found to be optimum at pH 4, 4.5, 4.5 and 5 for P. eryngii, P. florida, P. sajor caju and G. lucidum, respectively. Optimization studies with chemical inducers namely, tannic acid, 2,6 dimethoxyphenol and copper sulphate at three different concentration levels were conducted and tannic acid at 2mM concentration was found to increase the laccase activity to about 45% followed by 2,6 dimethoxyphenol (2mM) with an increase of about 43% and copper sulphate (0.1mM) showing 21% increase in the yield. Biodegradation studies utilizing laccase isolated from P. eryngii, P. florida and P. sajor caju was carried out for a commonly detected fluoroquinolone antibiotic, levofloxacin, in water and pharmaceutical wastewater. The results indicated that the degradation efficiency of levofloxacin using laccase isolated from P. eryngii (88.9%) was comparable to commercial laccase (89%). When the cost economics of using crude laccase was evaluated against commercial laccase it was evident that the total cost of the treatment could be reduced by 71.7% if commercial grade laccase was replaced by crude enzyme extracted from indigenous macrofungi such Pleurotus eryngii, Pleurotus florida, and Pleurotus sajor caju indicating a promising and cost-effective alternative for wastewater treatment.

Floro-pyrazolo[3,4-d]pyrimidine derivative as a novel plant activator induces two-pathway immune system

Plants are known to develop a multi-faceted innate immune system for pathogenic defense. Systemic acquired resistance (SAR) and induced systemic resistance (ISR) are the two main pathways. Many chemical inducers known as plant activators that activate innate immunity to defend against pathogens have been discovered. Currently, the exploitation of new plant activators is mainly done to develop analogs of salicylic acid as SAR-signaling molecules; however, the ISR pathway is hardly investigated for new plant activators. Based on recent studies on 1-methyl pyrazolo [3,4-d]pyrimidine bioactivity and ATP-induced resistance to biotrophic and necrotrophic pathogens, a new lead compound, 1-methyl-4-amino-pyrazolo [3,4-d]pyrimidine, was obtained as a new scaffold of plant activators for possible inducing ISR immunity system. Additionally, fluorine atom plays an important role in the design and development of new pesticides due to the unique physical chemistry effect, a series of pyrazolo [3,4-d]pyrimidine derivatives were designed and synthesized. Several compounds showed good broad-spectrum induced resistance in vivo, but there was no direct antibacterial activity in vitro. Notably, the introduction of fluorine atom at the para-position of the benzene ring greatly enhanced the induction activity of P1d both involved in SAR and ISR pathways, which implied the inducing resistance both in defending pathogens and insects.

The histone methyltransferase inhibitor A-366 enhances hemoglobin expression in erythroleukemia cells upon co\u2010exposure with chemical inducers in culture

BackgroundErythroleukemia is caused by the uncontrolled multiplication of immature erythroid progenitor cells which fail to differentiate into erythrocytes. By directly targeting this class of malignant cells, the induction of terminal erythroid differentiation represents a vital therapeutic strategy for this disease. Erythroid differentiation involves the execution of a well-orchestrated gene expression program in which epigenetic enzymes play critical roles. In order to identify novel epigenetic mediators of differentiation, this study explores the effects of multiple, highly specific, epigenetic enzyme inhibitors, in murine and human erythroleukemia cell lines.ResultsWe used a group of compounds designed to uniquely target the following epigenetic enzymes: G9a/GLP, EZH1/2, SMYD2, PRMT3, WDR5, SETD7, SUV420H1 and DOT1L. The majority of the probes had a negative impact on both cell proliferation and differentiation. On the contrary, one of the compounds, A-366, demonstrated the opposite effect by promoting erythroid differentiation of both cell models. A-366 is a selective inhibitor of the G9a methyltransferase and the chromatin reader Spindlin1. Investigation of the molecular mechanism of action revealed that A-366 forced cells to exit from the cell cycle, a fact that favored erythroid differentiation. Further analysis led to the identification of a group of genes that mediate the A-366 effects and include CDK2, CDK4 and CDK6.ConclusionsA-366, a selective inhibitor of G9a and Spindlin1, demonstrates a compelling role in the erythroid maturation process by promoting differentiation, a fact that is highly beneficial for patients suffering from erythroleukemia. In conclusion, this data calls for further investigation towards the delivery of epigenetic drugs and especially A-366 in hematopoietic disorders.

Chemical Autophagy Regulators.

Autophagy is a catabolic process that removes aggregated proteins and damaged organelles via lysosomal degradation. Increasing evidence suggests that dysfunction of autophagy is associated with a variety of human pathologies, including aging, cancer, neurodegenerative diseases, heart diseases, diabetes, and other metabolic diseases. Current research suggests that the regulation of autophagy may be a novel target for the treatment of these diseases. For this purpose, it is essential to have a deep understanding on the molecular details of autophagy and its regulatory network in each of the disease contexts. Over the years, a variety of chemical autophagy inducers and inhibitors has been developed. The application of these autophagy regulators can assist us in the exploration of the mechanism and therapeutic potential of autophagy regulation. In this chapter, we summarize the recent advances in chemical autophagy regulators to provide methodological support for autophagy research.

Development of Split-Protein Systems: From Binary to Ternary System

Tens of thousands of protein-protein interactions (PPIs) have been found in human cells and many of these macromolecular partnerships could determine the cell growth and death. Thus there is a need to develop the methods to catalogue these macromolecules by detecting their interactions, modifications, and cellular locations. It will be helpful for scientists to compare the difference between a diseased cellular state and its normal state and to find the potential therapy treatment to intervene this status. One technology called split-protein reassembly or protein fragment complementation has been developed in the last two decades. This technology makes use of appropriate fragmentation of some protein reporters and the refolding of these reports could be detected by their function to confirm the interaction of interest. This system has been set up in cell-free systems, E. coli, yeast, mammalian cells, plants and live animals. Herein, I present the development in fluorescence- and bioluminescence-based split-protein biosensors in both binary and ternary systems. In addition, some people developed the split-protein system by combining it with chemical inducer of dimerization strategy (CID). This has been applied for identifying the enzyme inhibitors and regulating the activity of protein kinases and phosphatases. With effort from many laboratories from the world, a variety of split-protein systems have been developed for studying the PPI in vitro and in vivo, monitoring the biological process, and controlling the activity of the enzyme of interest.

Conformational Dynamics and Binding Kinetics Drive Mutant Selective Degradation of EGFR

Targeted degradation of proteins by chimeric chemical inducers of degradation (CIDEs) has emerged as a major drug discovery paradigm. Despite the increased interest in this approach, the criteria dictating target protein degradation by a CIDE remain poorly understood, and potent target engagement by a CIDE does not strongly correlate with target degradation. In this study, we present the biochemical characterization of an EGFR mutant-selective degrader that potently binds to wildtype and mutant EGFR but only degrades EGFR mutant variants. Mechanistic studies reveal that ternary complex half-life is the primary determinant driving mutant-selective degradation. We present a cryo-EM structure of a CRL2VHL:CIDE:EGFR complex and show that potent target degradation can be achieved in the absence of CIDE-induced protein-protein interactions. These results highlight the importance of considering target conformation during CIDE development as well as leveraging ligand binding kinetics to achieve robust target degradation.