Therapeutics

Cancer researchers are focusing their efforts on identifying central pathways that trigger or enhance the invasiveness of tumor cells. One such target is EMMPRIN (Extracellular Matrix Metalloproteinase Inducer). It has been shown that EMMPRIN expression is linked to various cell signaling pathways that lead to an increase in tumor cell vascularization. Researchers are focusing efforts on elucidating the role of EMMPRIN in a number of disorders including cancer, arthritis, tissue repair and numerous inflammation-dependent diseases. DESCRIPTION/DETAILS Basigin is a member of the immunoglobulin superfamily thatis also known as EMMPRIN. Human basigin is expressed as two differentiallyspliced isoforms encoded by a single gene found on chromosome, renamedrespectively as basigin-1 and basigin-2. Attempts to demonstrate specifichemophilic on separate cells, or between soluble forms of recombinant basiginusing surface Plasmon resonance have not been successful, suggesting thatbasigin-2 cannot function as a receptor for itself. In order to identify possible receptors for soluble basiginligand, an affinity purification approach was employed. This approach resulted inthe labeling of several potential interacting proteins, and MALDI MS/MSsequencing of one of these proteins identified a novel isoform of human basigin(basigin-3).

Immunoprecipitation studies using cell fractions revealed thatrBSG interacts with basigin-2 at the cell membrane, and subsequently interactswith the basigin-3 isoform within the cell. Small-interfering RNA (siRNA)knockdown of basigin-2 protein reduced, but did not eliminate, rBSG-mediatedERK activation in HESCs, suggesting that additional cell surface receptors forsoluble basigin may exist. Taken together, these results support the hypothesisthat basigin-2 can function as a receptor for soluble basigin and demonstratethat the hemophilic interactions between basigin proteins are not dependentupon glycosylation of the basigin ligand. It has also been shown that soluble EMMPRIN binds tomembrane-bound EMMPRIN and the bound complex is internalized presenting apotential means to deliver therapeutic compounds to the cell in a highlytargeted manner. A therapeutic scheme isproposed that aims to design EMMPRIN-conjugates designed to deliver cancertherapeutics in a targeted fashion.

Applications

Conjugation oftherapeutics to EMMPRIN increases specificity and targets cancers cells cancer (skin, bladder,breast); other inflammatory diseases (arthritis, endometriosis); Upregulationof inflammatory response.

Benefits

It is possible to design therapeutic agents directed toward human basigin. Possible therapeutic scheme to block tumor progressionor inflammation.

Phosphonic and phosphinic acids are bioreactive compounds that have been used as antibiotics, antifungal agents, antimalarial drugs, and herbicides. Rhizocticins are naturally occurring phosphonates produced by Bacillus subtilus that have antifungal properties and interfere with the biosynthesis of the amino acid threonine. By varying the N-terminal amino acid group in the rhizocticin compound, it is possible to alter its biological activity. In particular, oligopeptides that are created by varying this amino acid could potentially allow enhanced capability for controlling selectivity for targeting particular organisms. As a result, compounds are less toxic to the host species and hold more promise for creating therapeutics. Despite the potential for making these types of compounds, organic synthesis has never been achieved due largely in part to the fact that the biosynthetic pathway has not been well understood. University of Illinois scientists have now identified the gene cluster responsible for biosynthesis of rhizocticin in B. subtilis, making biosynthesis of rhizocticin compounds possible.

Details

Original attempts to isolate and identify this unquie cluster failed, however, the genome of a specific strain of B. subtilis was sequenced and open reading frame locations were determined and annotated. Upon comparison with a non-rhizocticin-producing strain of B. subtilis, the rhizocticin biosynthetic gene cluster was able to be identified and isolated. There are more than 10 genes in the B. subtilis rhizocticin cluster. The biosynthetic pathway was proposed using sequence homology (similarity) for each of the genes. One particular gene in the cluster was identified by the inventors as being unique to this biosynthetic pathway. The cluster responsible for rhizocticin was verified by successfully inserting it into a non-producing strain of B. subtilis.

Applications

• Pharmaceutical industry: Synthesis of biologically active compounds and analogs of rhizocticin.
• Production of novel oligopeptides with therapeutic properties
• Agricultural industry: Herbicide

Benefits

The identification of specific gene cluster for biosynthesis of rhizocticin allows:

• Specific synthesis of compounds;
• Control of biological activity of compounds; and
• Control of compound toxicity in mammals

Dr. Metcalf and Dr. van der Donk from the University of Illinois have discovered 12 novel phosphonate natural products as well as the genes responsible for the biosynthesis of hundreds of additional compounds of other natural product classes. Phosphonic and phosphinic acids represent an underexploited group of bioactive compounds with great promise for the treatment of human disease.

One of the most challenging problems in biochemical science are studies that involve membranes, including small molecules and proteins which are associated with these cellular structures. Using designed amphipathic helical peptides, termed membrane scaffold proteins (MSPs), the Sligar laboratory has developed a simple and robust system that self-assembles phospholipids into soluble discoidal phospholipid bilayers of defined size (8-16 nm in diameter) termed Nanodiscs. Importantly, the same self-assembly process can be used to directly incorporate a variety of integral membrane proteins and small molecules into Nanodiscs.

The result is a native-like environment that provides stability and full functionality for these integral membrane protein targets. Using MSPs of varying lengths, the size and composition of the Nanodiscs can also control the oligomerization state of incorporated targets.

Additionally, the Sligar laboratory has demonstrated the usefulness of Nanodiscs in a variety of in vivo applications including imaging, delivery of therapeutics and in providing a naturalistic presentation of antigens for control of the immune response.

Applications

All classes of membrane proteins can be effectively solubilized using the Nanodisc technology, including; receptors, for example G-protein coupled receptors (GPCRs) which account for over 50 percent of drug targets; membrane bound enzymes such as the cytochromes P450 and in providing a precisely controlled membrane surface for control of biological processes such as blood coagulation and hemostasis.

Additionally, since the Nanodisc incorporated target can be easily affixed to surfaces without loss of activity, the technology has direct application to uses in high-throughput screening modalities for drug discovery. All assay formats utilized for soluble targets are now applicable to those involving membranes and membrane proteins. Nanodiscs can be generated with natural or synthetic phospholipids, and can be formed pyrrogen free and suitable for in vivo therapeutic delivery allowing numerous applications in the fields of biologicals and small molecule drugs. The availability of lipid or covalent anchoring has also enabled use in the delivery of nucleic acid based therapeutics.