Enzyme inhibition kinetics

Malaria is a deadly disease killing worldwide hundreds of thousands people each year and the responsible parasite has acquired resistance to the available drug combinations. The four vacuolar plasmepsins (PMs) in Plasmodium falciparum involved in hemoglobin catabolism represent promising targets to combat drug resistance. High antimalarial activities can be achieved by developing a single drug that would simultaneously target all the vacuolar PMs. We have demonstrated for the first time the use of soluble recombinant plasmepsin II (PMII) for structure-guided drug discovery with KNI inhibitors. Compounds used in this study (KNI-10742, 10743, 10395, 10333, and 10343) exhibit nanomolar inhibition against PMII, and are also effective in blocking the activities of PMI and PMIV with the low nanomolar K i values. The high resolution * Corresponding author, pbhaumik@iitb.ac.in. Structural data are available in the PDB under the accession numbers 5YIE, 5YIB, 5YID, 5YIC, and 5YIA. Conflict of Interest The authors declare no competing financial interests. Author contributions V.M. and I.R. have contributed equally. V.M, I.R., and P.B. conceived the idea and coordinated the study. V.M. and I.R. prepared the proteins and undertook the purification and crystallographic experiments under the supervision of P.B. The structures were solved and structural analysis were done by V.M. and I.R. with input from P.B., A.G., and A.W. The kinetics experiments were performed by V.M. with assistance from I.R. and suggestions from H.X. and R.Y.Y. under the supervision of P.B. The antiparasitic assay was performed by A.A. with an advice from S.P. The cytotoxicity assay was done by L.K.S. under the supervision of S.S. The inhibitors were synthesized by K.H and Y.K. The paper was jointly written by V.M., I.R., and P.B. with inputs from the remaining authors.

A salmon protein hydrolysate (SPH) was produced from consecutive enzymatic hydrolysis of salmon muscle proteins with pepsin followed by trypsin + chymotrypsin. The SPH was separated into four (SF1-SF4) reverse-phase HPLC peptide fractions. The SF3 peptide fraction was the most active against both angiotensin I-converting enzyme (ACE) and renin activities during in vitro tests. SPH and SF3 inhibited ACE activity uncompetitively but renin inhibition was non-competitive. SPH and SF3 were orally administered (200 and 30 mg/kg body weight, respectively) to spontaneously hypertensive rats, followed by systolic blood pressure (SBP) measurements. The SF3 significantly (p < 0.05) reduced SBP by a maximum value of −42.1 ± 3.4 mmHg when compared to −21.1 ± 4.5 mmHg for SPH 2 h after oral administration. Mass spectrometry analysis showed some differences in the peptide ion composition, which may have contributed to the observed differences in SBP-reducing effects of SPH and SF3. Salmon Protein hydrolysate Enzyme inhibition kinetics Systolic blood pressure Spontaneously hypertensive rats Mass spectrometry

Acetohydroxyacid synthase (AHAS) is a thiamin diphosphate (ThDP)-and flavin adenine dinucleotide (FAD)dependent plant and microbial enzyme that catalyzes the first common step in the biosynthesis of essential amino acids such as leucine, isoleucine and valine. To identify strong potent inhibitors against Shigella sonnei (S. sonnei) AHAS, we cloned and characterized the catalytic subunit of S. sonnei AHAS and found two potent chemicals (KHG20612, KHG25240) that inhibit 87-93% S. sonnei AHAS activity at an inhibitor concentration of 100 uM. The purified S. sonnei AHAS had a size of 65 kDa on SDS-PAGE. The enzyme kinetics revealed that the enzyme has a K m of 8.01 mM and a specific activity of 0.117 U/mg. The cofactor activation constant (K s ) for ThDP and (K c ) for Mg ++ were 0.01 mM and 0.18 mM, respectively. The dissociation constant (K d ) for ThDP was found to be 0.14 mM by tryptophan fluorescence quenching. The inhibition kinetics of inhibitor KHG20612 revealed an un-competitive inhibition mode with a K ii of 2.65 mM and an IC 50 of 9.3 μM, whereas KHG25240 was a non-competitive inhibitor with a K ii of 5.2 mM, K is of 1.62 mM and an IC 50 of 12.1 μM. Based on the S. sonnei AHAS homology model structure, the docking of inhibitor KHG20612 is predicted to occur through hydrogen bonding with Met 257 at a 1.7 Å distance with a low negative binding energy of −9.8 kcal/ mol. This current study provides an impetus for the development of a novel strong antibacterial agent targeting AHAS based on these potent inhibitor scaffolds.

Aurora kinases play pivotal role in mitosis. Aurora kinase A controls centrosome duplication, spindle pole integrity as well as bipolar spindle formation. Aurora kinase B is part of the chromosome passenger complex (CPC) and through its co-factors, drives chromosome congression, bidirectional tension on kinetochores and spindle checkpoint signalling, as well as cytokinesis completion.
Both CPC and Aurora kinases are exclusively expressed during mitosis and are up regulated in many tumours. Their over-expression correlates with the level of genomic instability within tumours and these proteins are therefore proposed as potential targets for cancer therapy.
This review describes the interplay between Aurora kinases in mitosis and the different strategies that have been developed towards their targeting.