No abstract

No abstract

Cathepsin K, a lysosomal cysteine protease, is abundantly expressed in osteoclasts and involved in bone remodeling and resorption. It has been reported that inhibitors of cathepsin K show great potential in the treatment of osteoporosis. The objective of this research is to prepare functional recombinant cathepsin K enzyme which will be used in the development of new cathepsin K inhibitors. Human procathepsin K cDNA has been amplified by PCR and successfully subcloned into pET15b expression vector. Overexpression of the recombinant His‐tagged fusion protein in BL21(DE3)pLysS host cells was found mainly in inclusion bodies. The inclusion bodies were solubilized by 6 M guanidine hydrochloride and the recombinant procathepsin K protein purified using Ni‐NTA affinity column. The purified protein was refolded by dilution followed by dialysis. The procathepsin K protein has been autoactivated and its proteolytic activity assayed by monitoring the increase in absorbance at 405 nm due to hydrolysis of chromogenic substrate, Z‐Phe‐Arg‐pNA. The availability of the functional cathepsin K enzyme will permit a drug discovery program for development of newer effective therapy for the treatment of osteoporosis in the near future.Supported by National Cancer Institute at NIH (Grant No. 3R15CA086933‐04 and 3R15CA086933‐04A2S1)

Francisella tularensis (F. tularensis) has been identified by the United States Centers for Disease Control (CDC) as one of the top six bacteria that could potentially be used as biological weapons. This research focuses on an enzyme of fatty acid biosynthesis in F. tularensis, namely FabI. FabI is an enoyl‐ACP reductase which catalyzes the last reduction step in each elongation cycle of type‐II fatty acid synthesis. The enzyme has been identified as a potential target for antibacterial drug development. In this research, the gene encoding of FabI was custom synthesized and cloned in pET15b expression vector. The enzyme was over‐expressed in E. coli, purified using Ni‐NTA affinity column, and its activity assayed spectrophotometrically. The pure FabI was tested with a set of indole‐2‐carboxylic acid compounds for their ability to inhibit FabI. We have discovered several new compounds with promising activity against F. tularensis FabI. The best result was obtained with indole‐2‐carboxylic acid analog WIUAKP‐031. The compound at 37.5 μM inhibited 98% of the FabI enzyme with IC50 value of 6 μM. The availability of the functional F. tularensis FabI will greatly aid in structural studies of this essential bacterial enzyme and facilitate the identification of small molecule inhibitors of type II fatty acid synthase.Supported in parts by seed grants from University Research Council, Western Illinois University.

Cathepsin B is a lysosomal cysteine proteinase that is implicated in the processes of tumor invasion, metastasis, and Alzheimer's disease. Development of specific inhibitors of cathepsin B could lead to the discovery of therapeutic agents for treatment of many types of carcinomas, as well as Alzheimer's disease. The objective of this research is to produce functional recombinant cathepsin B enzyme which will be used in screening of new cathepsin B inhibitors. Human procathepsin B cDNA has been successfully amplified by PCR and sub‐cloned into pET15b expression vector. Overexpression of the procathepsin B protein has been accomplished by IPTG induction. The ability to produce large quantities of procathepsin B enzyme will permit a drug discovery program for development of newer effective inhibitors of cathepsin B.Supported by National Cancer Institute at NIH (Grant No. 3R15CA086933‐04 and 3R15CA086933‐04A2S1)