New series of pyrrolidine mercaptosulfide, 2-mercaptocyclopentane arylsulfonamide, and 3-mercapto-4-arylsulfonamidopyrrolidine matrix metalloproteinase inhibitors (MMPIs) were designed, synthesized, and evaluated. Exhibiting unique properties over other MMPIs (e.g., hydroxamates), these newly reported compounds are capable of modulating activities of several MMPs in the low nanomolar range, including MMP-2 (∼2 to 50 nM), MMP-13 (∼2 to 50 nM), and MMP-14 (∼4 to 60 nM). Additionally these compounds are selective to intermediate- and deep-pocket MMPs but not shallow-pocketed MMPs (e.g., MMP-1, ∼850 to >50 000 nM; MMP-7, ∼4000 to >25 000 nM). Our previous work with the mercaptosulfide functionality attached to both cyclopentane and pyrrolidine frameworks demonstrated that the cis-(3S,4R)-stereochemistry was optimal for all of the MMPs tested. However, in our newest compounds an interesting shift of preference to the trans form of the mercaptosulfonamides was observed with increased oxidative stability and biological compatibility. We also report several kinetic and biological characteristics showing that these compounds may be used to probe the mechanistic activities of MMPs in disease.

Many basic proteins (pI > 7) and putative disease biomarkers are not identified using conventional proteomic methods. This study applied a new method to improve the identification of such proteins. Prefractionated basic proteins were compared with total tissue lysates from human ductal carcinoma in situ tissue loaded on basic immobilized pH gradient strips prior to two-dimensional gel electrophoresis (2-DE). Extraction of alkaline proteins was achieved in less than 20 min using a chromatofocusing resin and two buffers in a microcentrifuge tube. Prefractionation showed improved resolution and visualization of low-abundance proteins on 2-DE gels, allowing proteins to be excised, accumulated, trypsin-digested, and identified by liquid chromatography–tandem mass spectrometry. Proteins identified in the prefractionated samples had a higher number of peptides and three times the number of unique basic proteins when compared with total lysates. Low-molecular-weight (LMW, <26 kDa) unique alkaline proteins comprise 75% of those identified in prefractionated samples compared with 25% identified in total lysates, representing a 9-fold increase of LMW proteins due to prefractionation. Prefractionation ultimately increases loading capacity of samples onto the 2-DE gel and leads to better resolution, visualization, and identification of proteins with pI values greater than 7.

Matrix metalloproteinases (MMPs) are a family of hydrolytic enzymes that play significant roles in development, morphogenesis, inflammation, and cancer invasion. Endometase (matrilysin 2 or MMP-26) is a putative early biomarker for human carcinomas. The effects of the ionic and nonionic detergents on catalytic activity of endometase were investigated. The hydrolytic activity of endometase was detergent concentration dependent, exhibiting a bell-shaped curve with its maximum activity near the critical micelle concentration (CMC) of nonionic detergents tested. The effect of Brij-35 on human gelatinase B (MMP-9), matrilysin (MMP-7), and membrane-type 1 MMP (MT1-MMP) was further explored. Their maximum catalysis was observed near the CMC of Brij-35 (∼ 90 μM). Their IC50 values were above the CMC. The inhibition mechanism of MMP-7, MMP-9, and MT1-MMP by Brij-35 was a mixed type as determined by Dixon’s plot; however, the inhibition mechanism of endometase was noncompetitive with a Ki value of 240 μM. The catalytic activities of MMPs are influenced by detergents. Monomer of detergents may activate and stabilize MMPs to enhance catalysis, but micelle of detergents may sequester enzyme and block the substrate binding site to impede catalysis. Under physiological conditions, a lipid or membrane microenvironment may regulate enzymatic activity.