Model-Based Virtual PK/PD Exploration and Machine Learning Approach to Define PK Drivers in Early Drug Discovery.

While poor translatability of preclinical efficacy models can be responsible for clinical phase II failures, misdefinition of the optimal PK properties required to achieve therapeutic efficacy can also be a contributing factor. In the present work, the pharmacological dependency of PK end points in driving efficacy is demonstrated for six common pharmacological processes via model-based analysis. The analysis shows that the response is driven by multiple pharmacology-specific PK end points that change with how the response is defined. Moreover, the results demonstrate that the most important chemical structural features influencing response are specific to both target and downstream pharmacology, meaning the design and screening criteria must be defined uniquely for each target and corresponding pharmacology. The model-based virtual exploration of PK/PD relationships presented in this work offers one approach to identify target pharmacology-specific PK drivers and the associated potency-ADME space early in discovery to increase the probability of success and, ultimately, clinical attrition.

Applications of Model-Based Target Pharmacology Assessment in Defining Drug Design and DMPK Strategies: GSK Experiences.

Many critical decisions faced in early discovery require a thorough understanding of the dynamic behavior of pharmacological pathways following target engagement. From fundamental decisions on the optimal target to pursue and the ultimate drug product profile (combination of modality, potency, and compound properties) expected to elicit the desired clinical outcome to tactical program decisions such as what chemical series to pursue, what chemical properties require optimization, and what compounds to synthesize and progress, all demand detailed consideration of pharmacodynamics. Model-based target pharmacology assessment (mTPA) is a computational approach centered around large-scale virtual exploration of pharmacokinetic and pharmacodynamic models built early in discovery to guide these decisions. The present work summarizes several examples (use cases) from programs at GlaxoSmithKline that demonstrate the utility of mTPA throughout the drug discovery lifecycle.

Discovery of 4-ethoxy-7H-pyrrolo[2,3-d]pyrimidin-2-amines as potent, selective and orally bioavailable LRRK2 inhibitors.

Inhibition of LRRK2 kinase activity with small molecules has emerged as a potential novel therapeutic treatment for Parkinson's disease. Herein we disclose the discovery of a 4-ethoxy-7H-pyrrolo[2,3-d]pyrimidin-2-amine series as potent LRRK2 inhibitors identified through a kinase-focused set screening. Optimization of the physicochemical properties and kinase selectivity led to the discovery of compound 7, which exhibited potent in vitro inhibition of LRRK2 kinase activity, good physicochemical properties and kinase selectivity across the kinome. Moreover, compound 7 was able to penetrate into the CNS, and in vivo pharmacology studies revealed significant inhibition of Ser935 phosphorylation in the brain of both rats (30 and 100 mg/kg) and mice (45 mg/kg) following oral administration.

Discovery of EBI-2511: A Highly Potent and Orally Active EZH2 Inhibitor for the Treatment of Non-Hodgkin's Lymphoma.

A novel series of benzofuran derived EZH2 inhibitors were discovered through a scaffold hopping approach based on the clinical compound of EPZ-6438. Further rational structure-activity relationship exploration and optimization led to the discovery of more potent EZH2 inhibitors with oral bioavailability in mice and rats. A lead compound EBI-2511 (compound 34) demonstrated excellent in vivo efficacy in Pfeiffer tumor Xenograft models in mouse and is under preclinical development for the treatment of cancers associated with EZH2 mutations.

Discovery of 5-substituent-N-arylbenzamide derivatives as potent, selective and orally bioavailable LRRK2 inhibitors.

Leucine-rich repeat kinase 2 (LRRK2) has been suggested as a potential therapeutic target for Parkinson's disease. Herein we report the discovery of 5-substituent-N-arylbenzamide derivatives as novel LRRK2 inhibitors. Extensive SAR study led to the discovery of compounds 8e, which demonstrated potent LRRK2 inhibition activity, high selectivity across the kinome, good brain exposure, and high oral bioavailability.

Nanoplasmonic biochips for rapid label-free detection of imidacloprid pesticides with a smartphone.

The widespread and intensive use of neonicotinoid insecticides induces negative cascading effects on ecosystems. It is desirable to develop a portable sensitive sensing platform for on-site screening of high-risk pesticides. We combined an indirect competitive immunoassay, highly sensitive surface plasmon resonance (SPR) biochip and a simple portable imaging setup for label-free detection of imidacloprid pesticides. The SPR biochip consists of several capped nanoslit arrays with different periods which form a spectral image on the chip. The qualitative and semiquantitative analyses of pesticides can be directly observed from the spot shift on the chip. The precise semiquantitative analyses can be further completed by using image processing in a smartphone. We demonstrate simultaneous detection of four different concentrations of imidacloprid pesticides. The visual detection limit is about 1ppb, which is well below the maximum residue concentration permitted by law (20ppb). Compared to the one-step strip assay, the proposed chip is capable of performing semiquantitative analyses and multiple detection. Compared to the enzyme-linked immunosorbent assay, our method is label-free and requires simple washing steps and short reaction time. In addition, the label-free chip has a comparable sensitivity but wider working range than those labeling techniques.

Water-assisted proton delivery and removal in bio-inspired hydrogen production catalysts.

Electrocatalysts for H2 production are envisioned to play an important role in renewable energy utilization systems. Nickel-based catalysts featuring pendant amines functioning as proton relays in the second coordination sphere of the metal center have led to catalysts achieving turnover frequencies as high as 10(7) s(-1) for H2 production. The fastest rates are observed when water is present in solution, with rates up to 10(3) times faster than those found in dry solvent. The focus of this paper is to provide mechanistic insight into the unexpected enhancement due to water. Addition of H2 to [Ni(P(Cy)2N(R')2)2](2+) was previously shown to give three isomers of a Ni(0) product with two protonated amines, where the N-H can be endo or exo to the Ni. By investigating the deprotonation of these two N-protonated Ni(0) intermediates resulting from the addition of H2 to [Ni(P(Cy)2N(R')2)2](2+), we observe by NMR spectroscopy studies an enhancement in the rate of deprotonation for protons positioned on the pendant amine next to the metal (endo) vs. protons that are positioned away from the metal (exo). Computational studies suggest that for smaller bases, the desolvation energy of the exogenous base is the primary contribution limiting the rate of endo deprotonation, while steric accessibility and facile proton movement also contribute. For more bulky bases, steric accessibility can play the dominant role. The significant reduction in these barriers observed in the presence of water has important implications for disfavoring less productive catalytic pathways and increasing catalytic rates.

Toward molecular catalysts by computer.

CONSPECTUS: Rational design of molecular catalysts requires a systematic approach to designing ligands with specific functionality and precisely tailored electronic and steric properties. It then becomes possible to devise computer protocols to design catalysts by computer. In this Account, we first review how thermodynamic properties such as redox potentials (E°), acidity constants (pKa), and hydride donor abilities (ΔGH(-)) form the basis for a framework for the systematic design of molecular catalysts for reactions that are critical for a secure energy future. We illustrate this for hydrogen evolution and oxidation, oxygen reduction, and CO conversion, and we give references to other instances where it has been successfully applied. The framework is amenable to quantum-chemical calculations and conducive to predictions by computer. We review how density functional theory allows the determination and prediction of these thermodynamic properties within an accuracy relevant to experimentalists (∼0.06 eV for redox potentials, ∼1 pKa unit for pKa values, and 1-2 kcal/mol for hydricities). Computation yielded correlations among thermodynamic properties as they reflect the electron population in the d shell of the metal center, thus substantiating empirical correlations used by experimentalists. These correlations point to the key role of redox potentials and other properties (pKa of the parent aminium for the proton-relay-based catalysts designed in our laboratory) that are easily accessible experimentally or computationally in reducing the parameter space for design. These properties suffice to fully determine free energies maps and profiles associated with catalytic cycles, i.e., the relative energies of intermediates. Their prediction puts us in a position to distinguish a priori between desirable and undesirable pathways and mechanisms. Efficient catalysts have flat free energy profiles that avoid high activation barriers due to low- and high-energy intermediates. The criterion of a flat energy profile can be mathematically resolved in a functional in the reduced parameter space that can be efficaciously calculated by means of the correlation expressions. Optimization of the functional permits the prediction by computer of design points for optimum catalysts. Specifically, the optimization yields the values of the thermodynamic properties for efficient (high rate and low overpotential) catalysts. We are on the verge of design of molecular electrocatalysts by computer. Future efforts must focus on identifying actual ligands that possess these properties. We believe that this can also be achieved through computation, using Taft-like relationships linking molecular composition and structure with electron-donating ability and steric effects. We note also that the approach adopted here of using free energy maps to decipher catalytic pathways and mechanisms does not account for kinetic barriers associated with elementary steps along the catalytic pathway, which may make thermodynamically accessible intermediates kinetically inaccessible. Such an extension of the approach will require further computations that, however, can take advantage of Polanyi-like linear free energy relationships linking activation barriers and reaction free energies.

Controlling proton movement: electrocatalytic oxidation of hydrogen by a nickel(II) complex containing proton relays in the second and outer coordination spheres.

A nickel bis(diphosphine) complex containing proton relays in the second and outer coordination spheres, Ni(P(Cy)2N((CH2)2OMe))2, (P(Cy)2N((CH2)2OMe) = 1,5-di(methoxyethyl)-3,7-dicyclohexyl-1,5-diaza-3,7-diphosphacyclooctane), is an electrocatalyst for hydrogen oxidation. The addition of hydrogen to the Ni(II) complex results in rapid formation of three isomers of the doubly protonated Ni(0) complex, [Ni(P(Cy)2N((CH2)2OMe)2H)2](2+). The three isomers show fast interconversion at 40 °C, unique to this complex in this class of catalysts. Under conditions of 1.0 atm H2 using H2O as a base, catalytic oxidation proceeds at a turnover frequency of 5 s(-1) and an overpotential of 720 mV, as determined from the potential at half of the catalytic current. Compared to the previously reported Ni(P(Cy)2N(Bn))2 complex, the new complex operates at a faster rate and at a lower overpotential.

Molecular dynamics study of the proposed proton transport pathways in [FeFe]-hydrogenase.

Possible proton transport pathways in Clostridium pasteurianum (CpI) [FeFe]-hydrogenase were investigated with molecular dynamics simulations. This study was undertaken to evaluate the functional pathway and provide insight into the hydrogen bonding features defining an active proton transport pathway. Three pathways were evaluated, two of which consist of water wires and one of predominantly amino acid residues. Our simulations suggest that protons are not transported through water wires. Instead, the five-residue motif (Glu282, Ser319, Glu279, H2O, Cys299) was found to be the likely pathway, consistent with previously made experimental observations. The pathway was found to have a persistent hydrogen bonded core (residues Cys299 to Ser319), with less persistent hydrogen bonds at the ends of the pathway for both H2 release and H2 uptake. Single site mutations of the four residues have been shown experimentally to deactivate the enzyme. The theoretical evaluation of these mutations demonstrates redistribution of the hydrogen bonds in the pathway, resulting in enzyme deactivation. Finally, coupling between the protein dynamics near the proton transport pathway and the redox partner binding regions was also found as a function of H2 uptake and H2 release states, which may be indicative of a correlation between proton and electron movement within the enzyme.

High catalytic rates for hydrogen production using nickel electrocatalysts with seven-membered cyclic diphosphine ligands containing one pendant amine.

A series of Ni-based electrocatalysts, [Ni(7P(Ph)2N(C6H4X))2](BF4)2, featuring seven-membered cyclic diphosphine ligands incorporating a single amine base, 1-para-X-phenyl-3,6-triphenyl-1-aza-3,6-diphosphacycloheptane (7P(Ph)2N(C6H4X), where X = OMe, Me, Br, Cl, or CF3), have been synthesized and characterized. X-ray diffraction studies have established that the [Ni(7P(Ph)2N(C6H4X))2](2+) complexes have a square planar geometry, with bonds to four phosphorus atoms of the two bidentate diphosphine ligands. Each of the complexes is an efficient electrocatalyst for hydrogen production at the potential of the Ni(II/I) couple, with turnover frequencies ranging from 2400 to 27,000 s(-1) with [(DMF)H](+) in acetonitrile. Addition of water (up to 1.0 M) accelerates the catalysis, giving turnover frequencies ranging from 4100 to 96,000 s(-1). Computational studies carried out on the [Ni(7P(Ph)2N(C6H4X))2](2+) family indicate the catalytic rates reach a maximum when the electron-donating character of X results in the pKa of the Ni(I) protonated pendant amine matching that of the acid used for proton delivery. Additionally, the fast catalytic rates for hydrogen production by the [Ni(7P(Ph)2N(C6H4X))2](2+) family relative to the analogous [Ni(P(Ph)2N(C6H4X)2)2](2+) family are attributed to preferred formation of endo protonated isomers with respect to the metal center in the former, which is essential to attain suitable proximity to the reduced metal center to generate H2. The results of this work highlight the importance of precise pKa matching with the acid for proton delivery to obtain optimal rates of catalysis.

The role of a dipeptide outer-coordination sphere on H2-production catalysts: influence on catalytic rates and electron transfer.

The outer-coordination sphere of enzymes acts to fine-tune the active site reactivity and control catalytic rates, suggesting that incorporation of analogous structural elements into molecular catalysts may be necessary to achieve rates comparable to those observed in enzyme systems at low overpotentials. In this work, we evaluate the effect of an amino acid and dipeptide outer-coordination sphere on [Ni(P(Ph)(2)N(Ph-R)(2))(2)](2+) hydrogen production catalysts. A series of 12 new complexes containing non-natural amino acids or dipeptides was prepared to test the effects of positioning, size, polarity and aromaticity on catalytic activity. The non-natural amino acid was either 3-(meta- or para-aminophenyl)propionic acid terminated as an acid, an ester or an amide. Dipeptides consisted of one of the non-natural amino acids coupled to one of four amino acid esters: alanine, serine, phenylalanine or tyrosine. All of the catalysts are active for hydrogen production, with rates averaging ∼1000 s(-1), 40 % faster than the unmodified catalyst. Structure and polarity of the aliphatic or aromatic side chains of the C-terminal peptide do not strongly influence rates. However, the presence of an amide bond increases rates, suggesting a role for the amide in assisting catalysis. Overpotentials were lower with substituents at the N-phenyl meta position. This is consistent with slower electron transfer in the less compact, para-substituted complexes, as shown in digital simulations of catalyst cyclic voltammograms and computational modeling of the complexes. Combining the current results with insights from previous results, we propose a mechanism for the role of the amino acid and dipeptide based outer-coordination sphere in molecular hydrogen production catalysts.

Evaluation of the Role of Water in the H2 Bond Formation by Ni(II)-Based Electrocatalysts.

We investigate the role of water in the H-H bond formation by a family of nickel molecular catalysts that exhibit high rates for H2 production in acetonitrile solvent. A key feature leading to the high reactivity is the Lewis acidity of the Ni(II) center and pendant amines in the diphosphine ligand that function as Lewis bases, facilitating H-H bond formation or cleavage. Significant increases in the rate of H2 production have been reported in the presence of added water. Our calculations show that molecular water can displace an acetonitrile solvent molecule in the first solvation shell of the metal. One or two water molecules can also participate in shuttling a proton that can combine with a metal hydride to form the H-H bond. However the participation of the water molecules does not lower the barrier to H-H bond formation. Thus these calculations suggest that the rate increase due to water in these electrocatalysts is not associated with the elementary step of H-H bond formation or cleavage but rather with the proton delivery steps. We attribute the higher barrier in the H-H bond formation in the presence of water to a decrease in direct interaction between the protic and hydridic hydrogen atoms forced by the water molecules.

Proton delivery and removal in [Ni(P(R)2N(R')2)2]2+ hydrogen production and oxidation catalysts.

To examine the role of proton delivery and removal in the electrocatalytic oxidation and production of hydrogen by [Ni(P(R)(2)N(R')(2))(2)](2+) (where P(R)(2)N(R')(2) is 1,5-R'-3,7-R-1,5-diaza-3,7-diphosphacyclooctane), we report experimental and theoretical studies of the intermolecular proton exchange reactions underlying the isomerization of [Ni(P(Cy)(2)N(Bn)(2)H)(2)](2+) (Cy = cyclohexyl, Bn = benzyl) species formed during the oxidation of H(2) by [Ni(II)(P(Cy)(2)N(Bn)(2))(2)](2+) or the protonation of [Ni(0)(P(Cy)(2)N(Bn)(2))(2)]. Three protonated isomers are formed (endo/endo, endo/exo, or exo/exo), which differ in the position of the N-H bond's with respect to nickel. The endo/endo isomer is the most productive isomer due to the two protons being sufficiently close to the nickel to proceed readily to the transition state to form/cleave H(2). Therefore, the rate of isomerization of the endo/exo or exo/exo isomers to generate the endo/endo isomer can have an important impact on catalytic rates. We have found that the rate of isomerization is limited by proton removal from, or delivery to, the complex. In particular, the endo position is more sterically hindered than the exo position; therefore, protonation exo to the metal is kinetically favored over endo protonation, which leads to less catalytically productive pathways. In hydrogen oxidation, deprotonation of the sterically hindered endo position by an external base may lead to slow catalytic turnover. For hydrogen production catalysts, the limited accessibility of the endo position can result in the preferential formation of the exo protonated isomers, which must undergo one or more isomerization steps to generate the catalytically productive endo protonated isomer. The results of these studies highlight the importance of precise proton delivery, and the mechanistic details described herein will be used to guide future catalyst design.

The role of pendant amines in the breaking and forming of molecular hydrogen catalyzed by nickel complexes.

We present the results of a comprehensive theoretical investigation of the role of pendant amine ligands in the oxidation of H(2) and formation of H(2) by [Ni(P(R)(2)N(R')(2))(2)](2+) electrocatalysts (P(R)(2)N(R')(2) is the 1,5-R'-3,7-R derivative of 1,5-diaza-3,7-diphosphacyclooctane, in which R and R' are aryl or alkyl groups). We focus our analysis on the thermal steps of the catalytic cycle, as they are known to be rate-determining for both H(2) oxidation and production. We find that the presence of pendant amine functional groups greatly facilitates the heterolytic H(2) bond cleavage, resulting in a protonated amine and a Ni hydride. Only one single positioned pendant amine is required to serve this function. The pendant amine can also effectively shuttle protons to the active site, making the redistribution of protons and the H(2) evolution a very facile process. An important requirement for the overall catalytic process is the positioning of at least one amine in close proximity to the metal center. Indeed, only protonation of the pendant amines on the metal center side (endo position) leads to catalytically active intermediates, whereas protonation on the opposite side of the metal center (exo position) leads to a variety of isomers, which are detrimental to catalysis.

Understanding the effect of magnesium ion concentration on the catalytic activity of ribonuclease H through computation: does a third metal binding site modulate endonuclease catalysis?

Ribonuclease H (RNase H) belongs to the nucleotidyl-transferase superfamily and hydrolyzes the phosphodiester linkage on the RNA strand of a DNA/RNA hybrid duplex. Due to its activity in HIV reverse transcription, it represents a promising target for anti-HIV drug design. While crystallographic data have located two ions in the catalytic site, there is ongoing debate concerning just how many metal ions bound at the active site are optimal for catalysis. Indeed, experiments have shown a dependency of the catalytic activity on the Mg(2+) concentration. Moreover, in RNase H, the glutamate residue E188 has been shown to be essential for full enzymatic activation, regardless of the Mg(2+) concentration. The catalytic center is known to contain two Mg(2+) ions, and E188 is not one of the primary metal ligands. Herein, classical molecular dynamics (MD) simulations are employed to study the metal-ligand coordination in RNase H at different concentration of Mg(2+). Importantly, the presence of a third Mg(2+) ion, bound to the second-shell ligand E188, is a persistent feature of the MD simulations. Free energy calculations have identified two distinct conformations, depending on the concentration of Mg(2+). At standard concentration, a third Mg(2+) is found in the catalytic pocket, but it does not perturb the optimal RNase H active conformation. However, at higher concentration, the third Mg(2+) ion heavily perturbs the nucleophilic water and thereby influences the catalytic efficiency of RNase H. In addition, the E188A mutant shows no ability to engage additional Mg(2+) ions near the catalytic pocket. This finding likely explains the decrease in catalytic activity of E188A and also supports the key role of E188 in localizing the third Mg(2+) ion at the active site. Glutamate residues are commonly found surrounding the metal center in the endonuclease family, which suggests that this structural motif may be an important feature to enhance catalytic activity. The present MD calculations support the hypothesis that RNase H can accommodate three divalent metal ions in its catalytic pocket and provide an in-depth understanding of their dynamic role for catalysis.

Quantum Mechanical and Quantum Mechanical/Molecular Mechanical Studies of the Iron-Dioxygen Intermediates and Proton Transfer in Superoxide Reductase.

Classical and quantum-chemical computations are employed to probe the reaction intermediates and proton-transfer processes in superoxide reductase (SOR) from Desulfoarculus baarsii. Ab initio studies of the SOR active site, as well as classical and QM/MM MD simulations on the overall enzymatic reaction, are performed. We explore the use of a Hubbard U correction to standard density functional theory (DFT) in order to obtain a better description of the strongly correlated d electrons in the transition-metal center. The results obtained from the standard and Hubbard-U-corrected DFT approaches are compared with those obtained using different hybrid-DFT functionals. We show that the Hubbard U correction gives a significant improvement in the description of the structural, energetic, and electronic properties of SOR. We establish that adopting the Hubbard U correction in the QM/MM approach leads to increased accuracy with essentially no additional computational cost. Our results suggest that Lys(48) is one of the likely sources of the first proton donation to the superoxide, either directly or through an interstitial water molecule. Our QM/MM calculations highlight the important role of the interactions and hydrogen-bond network created by the imidazole rings of the His ligands and the internal water molecules. Whereas the hydrogen-bonding pattern due to internal waters can facilitate the protonation event, the interactions with the His ligands and the hydrogen bonds with water can stabilize the dioxygen ligand in a side-on conformation, which, in turn, prevents the immediate proton transfer from Lys(48), as indicated by recent experimental studies.

Bulk and interfacial aqueous fluoride: an investigation via first principles molecular dynamics.

Using first principles molecular dynamics simulation, we have studied a fluoride anion embedded in a periodically replicated water slab composed of 215 water molecules to mimic both bulk and interfacial solvation. In contrast to some recent experiments, our findings suggest that there are only small structural changes for fluoride and its first solvation shell in the bulk. Moreover, the presence of fluoride does not significantly alter the rotational dynamics of nearby water. In addition, we have computed the molecular dipole moments using Wannier centers. At the interface, the presence of fluoride increases the molecular dipole moments of nearby water molecules, whereas in the bulk, the dipole moments for water appear to be essentially invariant to the presence of fluoride in the vicinity. Previous studies of the air-water interface have showed interfacial water to have higher average HOMO energies and, thus, likely to be more prone to electrophilic attack. With the addition of fluoride, the most likely reactive site for electrophilic reactions shifts to the anion. This finding could explain the known large increase in reaction rates for heterogeneous process of interest in atmospheric science. The reactive properties of other anions near the air-water interface are of general interest in heterogeneous chemistry and can be elucidated using a similar type of analysis, as performed here for the fluoride anion.

Unraveling the Catalytic Pathway of Metalloenzyme Farnesyltransferase through QM/MM Computation.

The protein farnesyltransferase (FTase) is a Zn(2+)-metalloenzyme that catalyzes the farnesylation reaction, i.e., the transfer of the 15-carbon atom farnesyl group from farnesyl diphosphate (FPP) to a specific cysteine of protein substrates. Oncogenic Ras proteins, which are among the FTase substrates, are observed in about 20-30% of human cancer cells. Thus, FTase represents a target for anticancer drug design. Herein, we present a classical force-field-based and quantum mechanics/molecular mechanics (QM/MM) computational study of the FTase reaction mechanism. Our findings offer a detailed picture of the FTase catalytic pathway, describing structural features and the energetics of its saddle points. A moderate dissociation of the diphosphate group from the FPP is observed during the nucleophilic attack of the zinc-bound thiolate. At the transition state, a resonance structure is observed, which indicates the formation of a metastable carbocation. However, no stable intermediate is found along the reaction pathway. Thus, the reaction occurs via an associative mechanism with dissociative character, in agreement with the mechanism proposed by Fierke et al. ( Biochemistry 2000, 39, 2593-2602 and Biochemistry 2003, 42, 9741-9748 ). Moreover, a fluorine-substituted FPP analogue (CF3-FPP) is used to investigate the inhibitory effect of fluorine, which in turn provides additional agreement with experimental data.

Formulation factors that can reduce the formation of the phytotoxic impurity, N,N'-dibutylurea, from benomyl.

Fungicide benomyl is easily decomposed to carbendazim (MBC) and butyl isocyanate (BIC) in formulation, BIC is further hydrolyzed to butylamine. The BIC also reacts with butylamine to form N,N'-dibutylurea (DBU), a phytotoxic compound. The purpose of this study was to investigate the effects of selected additives and the manufacturing method of benomyl water dispersible granules (WG) on reducing DBU content in benomyl formulations. The manufacturing methods studied were granulation by extrusion, fluid bed spray, and spray dry. For the extrusion method, each benomyl powder formulation was homogenized by kneading with 20% v/w of 95% ethanol instead of water. After granulation, the percentages of the active ingredient benomyl and its degradation product carbendazim in each formulation were determined. For the fluid bed spray method, two formulations of wettable powders were formed. The first sample was granulated using 5% Na(2)SO(4) as the binder solution; the second sample used 2% urea. Changes in the active ingredient content after granulation were determined for each sample. For the spray dry method, four basic formulations of 70% benomyl, 5% sodium dodecyl sulfate (SDS) and 10% or 20% sodium sulfate were prepared, to study the effects of HMTA, urea and dispersant on reducing DBU formation in formulation. The DBU content of each formulation was measured for the fresh samples and after 1 year of storage. The results showed that urea had a stabilizing effect on benomyl, and reduced DBU formation. BIC increased benomyl yield during manufacturing, which reduced DBU content in fresh samples but allowed a greater potential for future DBU formation since it did not stabilize the extra benomyl. HMTA was found to reduce DBU in both aqueous BIC and prepared formulations. The study discusses how each of the selected constituents affected DBU formation and how commercial formulations can be improved to reduce DBU formation. From this study, it is clear that a safer benomyl formulation can be developed.