Aedes aegypti is the species of greatest concern for mosquito-borne disease in the Florida Keys. Previous locally transmitted dengue outbreaks in Key West (2009-2010) and Key Largo (2020) illustrate the need for an immediate and effective response plan to maintain Ae. aegypti populations below threshold levels. An important part of the Florida Keys Mosquito Control District\'s vector response plan is adulticide application because it can provide an immediate reduction in Ae. aegypti adults in the community. It has become apparent that in the Florida Keys, and throughout Florida, Ae. aegypti resistance to the adulticide permethrin is prevalent. This study uses the CDC bottle bioassay method to look at resistance in Ae. aegypti collected from Key Largo, Vaca Key, and Key West, FL. Resistance was found in all three populations when exposed to permethrin and Sumithrin® but not malathion. Inhibitor testing revealed that esterase and glutathione transferase activity is involved in resistance to permethrin in Key Largo and Key West Ae. aegypti populations while oxidase activity is involved in resistance to permethrin in Ae. aegypti from Vaca Key. Lack of knockdown at the diagnostic time and previous studies detecting the presence of kdr-associated allele mutations suggest knockdown resistance in all three populations. Results from this study show that there are multiple factors involved with resistance in the Ae. aegypti populations in the Florida Keys and that resistance mechanisms vary between islands. Continued surveillance will remain important so the most effective active ingredients can be used in response to future disease transmission.

Enzymes are important drug targets and inhibition of enzymatic activity is an important therapeutic strategy. Enzyme assays measuring catalytic activity are utilized in both the discovery and development of new drugs. Colorimetric assays based on the release of 4-nitrophenol from substrates are commonly used. 4-Nitrophenol is only partly ionized to 4-nitrophenolate under typical assay conditions (pH 7-9) leading to under-estimation of product formation rates due to the much lower extinction coefficient of 4-nitrophenol compared to 4-nitrophenolate. Determination of 4-nitrophenol pKa values based on absorbance at 405 nm as a function of experimental pH values is reported, allowing for calculation of a corrected extinction coefficient at the assay pH. Characterization of inhibitor properties using steady-state enzyme kinetics is demonstrated using calf intestine alkaline phosphatase and 4-nitrophenyl phosphate as substrate at pH ∼8.2. The following kinetic parameters were determined: Km= 40±3 µM; Vmax= 72.8±1.2 µmolmin-1mg protein-1; kcat= 9.70±0.16 s-1; kcat/Km= 2.44±0.16 × 105 M-1s-1 (mean± SEM, N = 4). Sodium orthovanadate and EDTA were used as model inhibitors and the following pIC50 values were measured using dose-response curves: 6.61±0.08 and 3.07±0.03 (mean±SEM, N = 4). Rapid dilution experiments determined that inhibition was reversible for sodium orthovanadate and irreversible for EDTA. A Ki value for orthovanadate of 51±8 nM (mean±SEM, N = 3) was determined. Finally, data analysis and statistical design of experiments are discussed.

4-hydroxyphenylpyruvate dioxygenase (HPD) is a key enzyme in the catabolism of tyrosine and therefore of great importance as a drug target to treat tyrosine-related inherited metabolic disorders (TIMD). Inhibition of this enzyme is therapeutically applied to prevent accumulation of toxic metabolites in TIMD patients. Nowadays an ex-herbicide, nitisinone, is used for this purpose and many more inhibitors are being explored and need to be tested. Here, we describe a colorimetric bacterial whole-cell screening system that allows quantifying the inhibitory effects of new human HPD inhibitors in a high-throughput and robust fashion. For this high-throughput screening (HTS) system we rely on the capability of recombinant E. coli that express human HPD, to generate a brown ochronotic pigment after the addition of tyrosine, whereafter this brown pigment can be quantified in a very specific and sensitive way by spectrophotometric analysis. Altogether, this robust and simple HTS screening system can be described as non-harmful, non-laborious and cost-effective with the aim to identify and evaluate novel therapeutic human HPD inhibitors for the treatment of TIMD.•This robust high-throughput screening system enables rapid identification and evaluation of potential inhibitors of human 4-hydroxyphenylpyruvate dioxygenase.•Simple and fast colorimetric quantification of the formation of ochronotic pigment.

Human protein kinase CK2 is an emerging target for the development of novel anti-cancer therapeutics. CK2 is a tetramer composed of two catalytically active α- and/or α\'-subunits, bound to a dimer of the regulatory β-subunit. Inhibitors targeting one of the two isoforms of the catalytically active CK2-subunit (α- and α\') are important to study the distinct functions of these isoforms toward different CK2 associated pathologies. The present study for the first time describes the successful Autodisplay of the CK2α\'-subunit, the paralogous isoform of CK2α. Expression on the cell surface of E. coli of CK2α\' alone and in combination with the regulatory CK2β-subunit was confirmed by outer membrane isolation and protease accessibility test. Kinase activity of surface displayed CK2 could be detected with a CE-based assay and was found to be 3.06×10(-6) μmol/min for CK2α\' alone and 1.02×10(-5) μmol/min when expressed in combination with CK2β. The comparison of the influence of NaCl on activity of the α\'-subunit alone and in combination with the non-catalytically active β-subunit indicated interaction of both subunits on the cell surface. TMCB (4,5,6,7-tetrabromo-2-(dimethylamino)-1H-benzo[d]imidazol-1-yl)acetic acid), a known CK2 inhibitor described with distinct Ki values of 83 nM and 21 nM for the two different catalytic CK2 subunits α and α\' was used for testing. First, inhibition of TMCB toward the purified CK2 holoenzyme CK2α2β2 was determined and resulted in a Ki value of 10.1 nM. Second, Ki values were determined with the surface displayed isoform CK2 holoenzymes and turned out to be of 31.1 nM for CK2α2β2 and 19.6 nM for CK2α\'2β2. The inhibition data as obtained represented the distinct affinities of TMCB toward the two isoform holoenzymes. This indicated, that the surface display of CKα and CK2α\', in the context of the corresponding holoenzymes, can be used to identify selective compounds. A set of twelve ATP competitive CK2 inhibitors with an indeno[1,2-b]indole scaffold was tested in order to demonstrate suitability for this application.