Correlation of Experimental and Calculated Inhibition Constants of Protease Inhibitor Complexes.

Predicting the potency of inhibitors is key to in silico screening of promising synthetic or natural compounds. Here we describe a predictive workflow that provides calculated inhibitory values, which concord well with empirical data. Calculations of the free interaction energy ΔG with the YASARA plugin FoldX were used to derive inhibition constants Ki from PDB coordinates of protease-inhibitor complexes. At the same time, corresponding KD values were obtained from the PRODIGY server. These results correlated well with the experimental values, particularly for serine proteases. In addition, analyses were performed for inhibitory complexes of cysteine and aspartic proteases, as well as of metalloproteases, whereby the PRODIGY data appeared to be more consistent. Based on our analyses, we calculated theoretical Ki values for trypsin with sunflower trypsin inhibitor (SFTI-1) variants, which yielded the more rigid Pro14 variant, with probably higher potency than the wild-type inhibitor. Moreover, a hirudin variant with an Arg1 and Trp3 is a promising basis for novel thrombin inhibitors with high potency. Further examples from antibody interaction and a cancer-related effector-receptor system demonstrate that our approach is applicable to protein interaction studies beyond the protease field.

Brassica napus Drought-Induced 22-kD Protein (BnD22) Acts Simultaneously as a Cysteine Protease Inhibitor and Chlorophyll-Binding Protein.

Class II water-soluble chlorophyll proteins (WSCPs) from Brassicaceae are non-photosynthetic proteins that bind with chlorophyll (Chl) and its derivatives. The physiological function of WSCPs is still unclear, but it is assumed to be involved in stress responses, which is likely related to their Chl-binding and protease inhibition (PI) activities. Yet, the dual function and simultaneous functionality of WSCPs must still be better understood. Here, the biochemical functions of Brassica napus drought-induced 22-kDa protein (BnD22), a major WSCP expressed in B. napus leaves, were investigated using recombinant hexahistidine-tagged protein. We showed that BnD22 inhibited cysteine proteases, such as papain, but not serine proteases. BnD22 was able to bind with Chla or Chlb to form tetrameric complexes. Unexpectedly, BnD22-Chl tetramer displays higher inhibition toward cysteine proteases, indicating (i) simultaneous Chl-binding and PI activities and (ii) Chl-dependent activation of PI activity of BnD22. Moreover, the photostability of BnD22-Chl tetramer was reduced upon binding with the protease. Using three-dimensional structural modeling and molecular docking, we revealed that Chl binding favors interaction between BnD22 and proteases. Despite its Chl-binding ability, the BnD22 was not detected in chloroplasts but rather in the endoplasmic reticulum and vacuole. In addition, the C-terminal extension peptide of BnD22, which cleaved off post-translationally in vivo, was not implicated in subcellular localization. Instead, it drastically promoted the expression, solubility and stability of the recombinant protein.

Determination and Kinetic Characterization of a New Potential Inhibitor for AmsI Protein Tyrosine Phosphatase from the Apple Pathogen Erwinia amylovora.

Erwinia amylovora is a Gram-negative bacterium, responsible for the fire blight disease in Rosaceae plants. Its virulence is correlated with the production of an exopolysaccharide (EPS) called amylovoran, which protects the bacterium from the surrounding environment and helps its diffusion inside the host. Amylovoran biosynthesis relies on the expression of twelve genes clustered in the ams operon. One of these genes, amsI, encodes for a Low Molecular Weight Protein Tyrosine Phosphatase (LMW-PTP) called EaAmsI, which plays a key role in the regulation of the EPS production pathway. For this reason, EaAmsI was chosen in this work as a target for the development of new antibacterial agents against E. amylovora. To achieve this aim, a set of programs (DOCK6, OpenEye FRED) was selected to perform a virtual screening using a database of ca. 700 molecules. The six best-scoring compounds identified were tested in in vitro assays. A complete inhibition kinetic characterization carried out on the most promising molecule (n-Heptyl ß-D-glucopyranoside, N7G) showed an inhibition constant of 7.8 ± 0.6 µM. This study represents an initial step towards the development of new EaAmsI inhibitors able to act as antibacterial agents against E. amylovora infections.

Influence of Substitutions in the Binding Motif of Proline-Rich Antimicrobial Peptide ARV-1502 on 70S Ribosome Binding and Antimicrobial Activity.

Proline-rich antimicrobial peptides (PrAMPs) are promising candidates to treat bacterial infections. The designer peptide ARV-1502 exhibits strong antimicrobial effects against Enterobacteriaceae both in vitro and in vivo. Since the inhibitory effects of ARV-1502 reported for the 70 kDa heat-shock protein DnaK do not fully explain the antimicrobial activity of its 176 substituted analogs, we further studied their effect on the bacterial 70S ribosome of Escherichia coli, a known target of PrAMPs. ARV-1502 analogues, substituted in positions 3, 4, and 8 to 12 (underlined) of the binding motif D3KPRPYLPRP12 with aspartic acid, lysine, serine, phenylalanine or leucine, were tested in a competitive fluorescence polarization (FP) binding screening assay using 5(6)-carboxyfluorescein-labeled (Cf-) ARV-1502 and the 70S ribosome isolated from E. coli BW25113. While their effect on ribosomal protein expression was studied for green fluorescent protein (GFP) in a cell-free expression system (in vitro translation), the importance of known PrAMP transporters SbmA and MdtM was investigated using E. coli BW25113 and the corresponding knockout mutants. The dissociation constant (Kd) of 201 ± 16 nmol/L obtained for Cf-ARV-1502 suggests strong binding to the E. coli 70S ribosome. An inhibitory binding assay indicated that the binding site overlaps with those of other PrAMPs including Onc112 and pyrrhocoricin as well as the non-peptidic antibiotics erythromycin and chloramphenicol. All these drugs and drug candidates bind to the exit-tunnel of the 70S ribosome. Substitutions of the C-terminal fragment of the binding motif YLPRP reduced binding. At the same time, inhibition of GFP expression increased with net peptide charge. Interestingly, the MIC values of wild-type and ΔsbmA and ΔmdtM knockout mutants indicated that substitutions in the ribosomal binding motif altered also the bacterial uptake, which was generally improved by incorporation of hydrophobic residues. In conclusion, most substituted ARV-1502 analogs bound weaker to the 70S ribosome than ARV-1502 underlining the importance of the YLPRP binding motif. The weaker ribosomal binding correlated well with decreased antimicrobial activity in vitro. Substituted ARV-1502 analogs with a higher level of hydrophobicity or positive net charge improved the ribosome binding, inhibition of translation, and bacterial uptake.

Anti-breast cancer action of carbonic anhydrase IX inhibitor 4-[4-(4-Benzo[1,3]dioxol-5-ylmethyl-piperazin-1-yl)-benzylidene-hydrazinocarbonyl]-benzenesulfonamide (BSM-0004): in vitro and in vivo studies.

Anti-breast cancer action of novel human carbonic anhydrase IX (hCA IX) inhibitor BSM-0004 has been investigated using in vitro and in vivo models of breast cancer. BSM-0004 was found to be a potent and selective hCA IX inhibitor with a Ki value of 96 nM. In vitro anticancer effect of BSM-0004 was analysed against MCF 7 and MDA-MA-231 cells, BSM-0004 exerted an effective cytotoxic effect under normoxic and hypoxic conditions, inducing apoptosis in MCF 7 cells. Additionally, this compound significantly regulates the expression of crucial biomarkers associated with apoptosis. The investigation was extended to confirm the efficacy of this hCA IX inhibitor against in vivo model of breast cancer. The results specified that the treatment of BSM-0004 displayed an effective in vivo anticancer effect, reducing tumour growth in a xenograft cancer model. Hence, our investigation delivers an effective anti-breast cancer agent that engenders the anticancer effect by inhibiting hCA IX.

Steroidal fraction of Carissa carandas L. inhibits microbial hyaluronidase activity by mixed inhibition mechanism.

Hyaluronidase (hyase) is a hyaluronic acid (HA) depolymerizing enzyme produced by many pathogenic bacteria as a virulence factor to establish and spread infections. Present studies established that a steroidal fraction (SF) isolated from leaves of Carissa carandas act as a strong hyase inhibitor. The kinetic parameters involved in the inhibition of hyase by purified SF were studied and compared with standard hyase inhibitor quercetin. The purified SF showed the highest inhibition with an IC50 of 5.19 mM in comparison with a standard inhibitor, quercetin (IC50 8.63 mM). The inhibition constant (Ki) of purified SF determined by Dixon plot was 8.32 mM, which was significantly lower than that of quercetin standard. The kinetic behavior of enzyme hyase revealed to be more complex than classical competitive and uncompetitive inhibition where inhibitor affects both Km and Vmax. The inhibitor (I) favored the binding to the enzyme-substrate (ES) complex where Km value appeared to decrease (Kmapp < Km). The inhibitor also leads to decrease in the apparent maximum velocity of the enzyme-substrate reaction (Vmaxapp < Vmax). These results signpost toward mixed nature of inhibition of enzyme hyase by purified SF. Anti-hyaluronidase activity by a bioactive metabolite from C. carandas has not been reported so far and has high therapeutic potential against spread of pathogen and its toxins in the host.

Interpretation of results from the competitive Biacore procedure for characterizing immunochemical interactions in solution.

Rigorous consideration of the consequences of antibody bivalence in the published competitive kinetic procedure for quantifying the solution characteristics of an antigen-antibody interaction in solution has rendered redundant the practice of substituting the Fab fragment for the antibody to ensure validity of the analysis of results in terms of theory developed for a univalent analyte. Although the quantitative expressions differ for univalent and bivalent analytes, the additional contribution arising from bivalence is likely to be well within the limits of experimental uncertainty in the measured binding constant.

Electrophysiological determination of phosphodiesterase-6 inhibitor inhibition constants in intact mouse retina.

Cyclic nucleotide phosphodiesterases (PDEs) hydrolyze the second messengers cAMP and cGMP. PDEs control numerous cellular processes making them promising targets for the development of therapeutic agents. Unfortunately, many PDE inhibitor molecules are non-selective among PDE classes and efficient methods for quantitative studies on the isoform-specificity of PDE inhibitors in the natural environments of PDEs are unavailable. The PDE in photoreceptors, PDE6, mediates the conversion of photon information into electrical signals making the retina an exceptional model system for examinations of the pharmacological effects of PDE inhibitors on PDE6. Here we introduce electroretinography-based methods for determining the inhibition constants of PDE inhibitors towards the naturally occurring light-activated and spontaneously activated forms of PDE6. We compare our results to earlier biochemical determinations with trypsin-activated PDE6 with disintegrated PDE6 γ-subunit. The potencies of PDE inhibitors were determined by stimulating the photoreceptors of isolated mouse retinas with light and tracking the inhibitor-induced changes in their electrical responses. The methods were tested with three PDE inhibitors, 3-isobutyl-1-methylxanthine (IBMX), sildenafil, and zaprinast. The inhibition constants towards light-activated, spontaneously activated, and trypsin-activated PDE6 differed significantly from each other for sildenafil and zaprinast but were closely similar for IBMX. We hypothesize that this is due to the ability of the PDE6 γ-subunit to compete with sildenafil and zaprinast from the same binding sites near the catalytic domain of PDE6. The introduced methods are beneficial both for selecting potent molecules for PDE6 inhibition and for testing the drugs targeted at other PDE isoforms against adverse effects on visual function.

Soil properties influence kinetics of soil acid phosphatase in response to arsenic toxicity.

Soil phosphatase, which plays an important role in phosphorus cycling, is strongly inhibited by Arsenic (As). However, the inhibition mechanism in kinetics is not adequately investigated. In this study, we investigated the kinetic characteristics of soil acid phosphatase (ACP) in 14 soils with varied properties, and also explored how kinetic properties of soil ACP changed with different spiked As concentrations. The results showed that the Michaelis constant (Km) and maximum reaction velocity (Vmax) values of soil ACP ranged from 1.18 to 3.77mM and 0.025-0.133mMh-1 in uncontaminated soils. The kinetic parameters of soil ACP in different soils changed differently with As contamination. The Km remained unchanged and Vmax decreased with increase of As concentration in most acid and neutral soils, indicating a noncompetitive inhibition mechanism. However, in alkaline soils, the Km increased linearly and Vmax decreased with increase of As concentration, indicating a mixed inhibition mechanism that include competitive and noncompetitive. The competitive inhibition constant (Kic) and noncompetitive inhibition constant (Kiu) varied among soils and ranged from 0.38 to 3.65mM and 0.84-7.43mM respectively. The inhibitory effect of As on soil ACP was mostly affected by soil organic matter and cation exchange capacity. Those factors influenced the combination of As with enzyme, which resulted in a difference of As toxicity to soil ACP. Catalytic efficiency (Vmax/Km) of soil ACP was a sensitive kinetic parameter to assess the ecological risks of soil As contamination.

On the use of the experimentally determined enzyme inhibition constant as a measure of absolute binding affinity.

Defined as a state function representing an inhibitor's absolute affinity for its target enzyme, the experimentally determined enzyme inhibition constant (Ki) is widely used to rank order binding affinities of different inhibitors for a common enzyme or different enzymes for a common inhibitor and to benchmark computational approaches to predicting binding affinity. Herein, we report that adsorption of bis(7)-tacrine to the glass container surface increased its Ki against Electrophorus electricus acetylcholinesterase (eeAChE) to 3.2 ± 0.1 nM (n = 5) compared to 2.9 ± 0.4 pM (n = 5) that was determined using plastic containers with other assay conditions kept the same. We also report that, due to binding or "adsorption" of bis(7)-tacrine to the inactive eeAChE, the bis(7)-tacrine Ki increased from 2.9 ± 0.4 pM (n = 5) to 734 ± 70 pM (n = 5) as the specific eeAChE activity decreased from 342 U/mg to 26 U/mg while other assay conditions were kept the same. These results caution against using Kis to rank order binding potencies, define selectivity, or benchmark computational methods without knowing detailed assay conditions.

Design, synthesis and herbicidal activity study of aryl 2,6-disubstituted sulfonylureas as potent acetohydroxyacid synthase inhibitors.

A series of sulfonylurea derivatives containing a 2,6-disubstituted aryl moiety were designed, synthesized and evaluated for their herbicidal activities. Most of these compounds showed excellent inhibitory rates against both monocotyledonous and dicotyledonous weeds, especially 10a, 10h and 10i. They exhibited equivalent or superior herbicidal efficiency than commercial chlorsulfuron at the dosage of 15g/ha and the preliminary SAR was summarized. In order to illuminate the molecular mechanism of several potent compounds, their apparent inhibition constant (Kiapp) of Arabidopsis thaliana acetohydroxyacid synthase (AHAS) were determined and the results confirmed that these compounds were all potent AHAS inhibitors. 10i have a Kiapp of 11.5nM, which is about 4 times as potent as chlorsulfuron (52.4nM).

Design, Synthesis and Biological Evaluation of Potent Human Glyoxalase I Inhibitors.

Several glutathione derivatives bearing the S-(N-aryl-N-hydroxycarbamoyl) or S-(C-aryl-N-hydroxycarbamoyl) moieties (10, 10', 13-15) were synthesized, characterized, and their human glyoxalase I (hGLO1) inhibitory activity was evaluated. Compound 10 was proved to be the effective hGLO1 inhibitor with a Ki value of 1.0 nM and the inhibition effect of compound 10 on hGLO1 was nearly ten-fold higher than that of the strongest inhibitor 2 (Ki=10.0 nM) which has been reported in the field of glutathione-type hGLO1 inhibitors. Its diethyl ester prodrug 10' was able to penetrate cell membrane and had good inhibitory effect on the growth of NCI-H522 cell xenograft tumor model.

Pyrazolylbenzo[d]imidazoles as new potent and selective inhibitors of carbonic anhydrase isoforms hCA IX and XII.

Novel pyrazolylbenzo[d]imidazole derivatives (2a-2f) were designed, synthesized and evaluated against four human carbonic anhydrase isoforms belonging to α family comprising of two cytosolic isoforms hCA I and II as well as two transmembrane tumor associated isoforms hCA IX and XII. Starting from these derivatives that showed high potency but low selectivity in favor of tumor associated isoforms hCA IX and XII, we investigated the impact of removing the sulfonamide group. Thus, analogs 3a-3f without sulfonamide moiety were synthesized and biological assay revealed a good activity as well as an excellent selectivity as inhibitors for tumor associated hCA IX and hCA XII and the same was analyzed by molecular docking studies.

[Hepatotoxicity of emodin based on UGT1A1 enzyme-mediated bilirubin in liver microsomes].

To study the hepatotoxicity of emodin based on bilirubin metabolism mediated by glucuronidation of UGT1A1 enzyme. In this study, three different incubation systems were established by using RLM, HLM, and rUGT1A1, with bilirubin as the substrate. Different concentrations of bilirubin and emodin were added in the incubation systems. The double reciprocal Michaelis equation was drawn based on the total amount of bilirubin glucuronidation. The apparent inhibition constant Ki was then calculated with the slope curve to predict the hepatotoxicity. The results indicated that emodin had a significant inhibition to the UGT1A1 enzyme in all of the three systems, with Ki=5.400±0.956(P<0.05) in HLM system, Ki =10.020±0.611(P<0.05) in RLM system, Ki=4.850±0.528(P<0.05) in rUGT1A1 system. Meanwhile, emodin had no significant difference between rat and human in terms of inhibition of UGT1A1 enzyme. Emodin had a potential risk of the hepatotoxicity by inhibiting the UGT1A1 enzyme activity. And the method established in this study provides a new thought and new method to evaluate hepatotoxicity and safety of traditional Chinese medicines.

Cloning, characterization and anion inhibition studies of a γ-carbonic anhydrase from the Antarctic cyanobacterium Nostoc commune.

We report the cloning and catalytic activity of a γ-carbonic anhydrase (CA, EC 4.2.1.1) isolated from the Antarctic cyanobacterium Nostoc commune, NcoCA. The enzyme has a significant catalytic activity for the physiologic reaction, CO2 hydration to bicarbonate and protons, with a k(cat) of 9.5×10(5) s(-1) and a k(cat)/K(m) of 8.3×10(7) M(-1) × s(-1), being the most catalytically efficient γ-CA investigated so far. An anion inhibition study of NcoCA with inorganic/organic anions is also reported here. Fluoride, sulfate, perchlorate and tetrafluoroborate did not inhibit appreciably NcoCA, whereas the other halides, pseudohalides, bicarbonate, nitrate, nitrite and many complex inorganic anions showed inhibition in the millimolar range. The best NcoCA inhibitors detected so far were diethyldithiocarbamate (K(I) of 0.80 mM) as well as sulfamide, sulfamate, phenylboronic acid and phenylarsonic acid (K(I)s in the range of 70-90 µM). Since γ-CAs are present in carboxysomes, being involved in photosynthesis, this study may be relevant for a better understanding of such processes in some Antarctic organisms.

The influence of a 21 kDa Kunitz-type trypsin inhibitor from nonhost madras thorn, Pithecellobium dulce, seeds on H. armigera (Hübner) (Lepidoptera: Noctuidae).

A trypsin inhibitor purified from the seeds of the Manila tamarind, Pithecellobium dulce (PDTI), was studied for its effects on growth parameters and developmental stages of Helicoverpa armigera. PDTI exhibited inhibitory activity against bovine trypsin (∼86%; ∼1.33 ug/ml IC50). The inhibitory activity of PDTI was unaltered over a wide range of temperature, pH, and in the presence of dithiothreitol. Larval midgut proteases were unable to digest PDTI for up to 12 h of incubation. Dixon and Lineweaver-Burk double reciprocal plots analysis revealed a competitive inhibition mechanism and a Ki of ∼3.9 × 10(-8) M. Lethal dose (0.50% w/w) and dosage for weight reduction by 50% (0.25% w/w) were determined. PDTI showed a dose-dependent effect on mean larval weight and a series of nutritional disturbances. In artificial diet at 0.25% w/w PDTI, the efficiency of conversion of ingested food, of digested food, relative growth rate, and growth index declined, whereas approximate digestibility, relative consumption rate, metabolic cost, consumption index, and total developmental period were increased in larvae. This is the first report of antifeedant and antimetabolic activities of PDTI on midgut proteases of H. armigera.

Identification of novel drug scaffolds for inhibition of SARS-CoV 3-Chymotrypsin-like protease using virtual and high-throughput screenings.

We have used a combination of virtual screening (VS) and high-throughput screening (HTS) techniques to identify novel, non-peptidic small molecule inhibitors against human SARS-CoV 3CLpro. A structure-based VS approach integrating docking and pharmacophore based methods was employed to computationally screen 621,000 compounds from the ZINC library. The screening protocol was validated using known 3CLpro inhibitors and was optimized for speed, improved selectivity, and for accommodating receptor flexibility. Subsequently, a fluorescence-based enzymatic HTS assay was developed and optimized to experimentally screen approximately 41,000 compounds from four structurally diverse libraries chosen mainly based on the VS results. False positives from initial HTS hits were eliminated by a secondary orthogonal binding analysis using surface plasmon resonance (SPR). The campaign identified a reversible small molecule inhibitor exhibiting mixed-type inhibition with a K(i) value of 11.1 µM. Together, these results validate our protocols as suitable approaches to screen virtual and chemical libraries, and the newly identified compound reported in our study represents a promising structural scaffold to pursue for further SARS-CoV 3CLpro inhibitor development.

Purification and characterization of three ß-glycosidases exhibiting high glucose tolerance from Aspergillus niger ASKU28.

Production and utilization of cellulosic ethanol has been limited, partly due to the difficulty in degradation of cellulosic feedstock. ß-Glucosidases convert cellobiose to glucose in the final step of cellulose degradation, but they are inhibited by high concentrations of glucose. Thus, in this study, we have screened, isolated, and characterized three ß-glycosidases exhibiting highly glucose-tolerant property from Aspergillus niger ASKU28, namely ß-xylosidase (P1.1), ß-glucosidase (P1.2), and glucan 1,3-ß-glucosidase (P2). Results from kinetic analysis, inhibition study, and hydrolysis of oligosaccharide substrates supported the identification of these enzymes by both LC/MS/MS analysis and nucleotide sequences. Moreover, the highly efficient P1.2 performed better than the commercial ß-glucosidase preparation in cellulose saccharification, suggesting its potential applications in the cellulosic ethanol industry. These results shed light on the nature of highly glucose-tolerant ß-glucosidase activities in A. niger, whose kinetic properties and identities have not been completely determined in any prior investigations.

In silico evaluation of some commercially available terpenoids as spike glycoprotein of SARS-CoV-2 - inhibitors using molecular dynamic approach.

Coronavirus, an extremely contagious infections disease had a harmful effect on the world's population. It is a family of enveloped, single-stranded, positive-strand RNA viruses of Nidovirales order belongs to coroviridae family. At present, worldwide several lakhs of deaths and several billions of infections have been reported. Hence, the focus of the present study was to assess the SARS-CoV-2 enzyme inhibitory potential of certain commercially available terpenoids using Lamarckian genetic algorithm as a working principle and molecular dynamic studies was also performed. AutoDock 4.2 software was used to perform the computational docking calculations of terpenoids against SARS-CoV-2 enzyme. The terpenoids such as, Andrographolide, Betulonic acid, Erythrodiol, Friedelin, Mimuscopic acid, Moronic acid, and Retinol were selected based on the drug likeness properties. Remdesivir a well-known anti-viral drug was selected as the standard drug. Molecular dynamic simulation studies were carried using Desmond module of Schrodinger Suite. In the current study we observed that, Friedelin was exhibited excellent SARS-CoV-2 enzyme inhibitory potential than the standard drug and other selected terpenoids. Friedelin and the standard Remdesivir was undergone the molecular dynamic studies and Friedelin showed a good number of hydrogen bonds over the simulation time of 100 ns. Based on the in silico computational evaluation, it can be concluded that Friedelin could be worthwhile terpenoid against SARS-CoV-2 spike protein. A further study on Friedelin is required to develop a potential chemical entity against the management of COVID disease.Communicated by Ramaswamy H. Sarma.

Enhanced biodegradation of 2, 4-dichlorophenol in packed bed biofilm reactor by impregnation of polyurethane foam with Fe3O4 nanoparticles: Bio-kinetics, process optimization, performance evaluation and toxicity assessment.

In this work, an effort has been made to enhance the efficacy of biological process for the effective degradation of 2, 4-dichlorophenol (2, 4-DCP) from wastewater. The polyurethane foam was modified with Fe3O4 nanoparticles and combined with polyvinyl alcohol, sodium alginate, and bacterial consortium for biodegradation of 2, 4-DCP in a packed bed biofilm reactor. The maximum removal efficiency of 2, 4-DCP chemical oxygen demand, and total organic carbon were found to be 92.51 ± 0.83 %, 86.85 ± 1.32, and 91.78 ± 1.24 %, respectively, in 4 days and 100 mg L-1 of 2, 4-DCP concentration at an influent loading rate of 2 mg L-1h-1 and hydraulic retention time of 50 h. Packed bed biofilm reactor was effective for up to four cycles to remove 2, 4-DCP. Growth inhibition kinetics were evaluated using the Edward model, yielding maximum growth rate of 0.45 day-1, inhibition constant of 110.6 mg L-1, and saturation constant of 62.3 mg L-1.