Molecular agents that specifically bind and neutralize misfolded and toxic amyloids may find application in attenuating progression of neuronal diseases. However, high structural similarities between the wild-type and mutant amyloid limit the utility of this approach. We are addressing this challenge by converting promiscuous proteins into highly specific aggregation inhibitors of SOD1, Aβ42 and Tau. We utilize computational algorithms for mapping protein surfaces predisposed to inhibitor intermolecular interactions to construct a focused protein inhibitor library, complemented with an experimental platform based on yeast surface display for affinity and specificity screening.
The pathophysiological functions of the signaling molecules matrix metalloproteinase-14 (MMP-14) and integrin αvβ3 in various types of cancer are believed to derive from their collaborative activity in promoting invasion, metastasis, and angiogenesis, as shown and . To address the lack of multitarget inhibitors, we establish a combinatorial approach that is based on flow cytometry screening of a yeast-displayed N-TIMP2 (N-terminal domain variant of tissue inhibitor of metalloproteinase-2) mutant library. On the basis of this screening, we identify protein monomers and generate protein heterodimers that contain monovalent and bivalent binding epitopes to MMP-14 and integrin αvβ3.
Enhanced activation of the signaling pathways that mediate the differentiation of mononuclear monocytes into osteoclasts is an underlying cause of several bone diseases and bone metastasis. In particular, dysregulation and overexpression of macrophage colony-stimulating factor (M-CSF) and its c-FMS tyrosine kinase receptor, proteins that are essential for osteoclast differentiation, are known to promote bone metastasis and osteoporosis, making both the ligand and its receptor attractive targets for therapeutic intervention. We develop M-CSF ligand-based, high-affinity antagonists for c-FMS that retain its binding ability but prevent the ligand dimerization that leads to receptor dimerization and activation. These mutants act as functional inhibitors of M-CSF-dependent c-FMS activation and osteoclast differentiation and .
We develop and characterize nanobodies that present tight and specific binding and internalization into PSMA or RTK positive cells and that accumulate specifically in PSMA or RTK positive tumors. We then conjugate these nanobodies to cytotoxic drugs, and we show that the conjugate internalizes specifically into PSMA or RTK positive cells, where the drug is released and induces cytotoxic activity both and in preclinical models of cancer.
Characterizing the binding selectivity landscape of interacting proteins is crucial both for elucidating the underlying mechanisms of their interaction and for developing selective inhibitors. However, current mapping methods are laborious and cannot provide a sufficiently comprehensive description of the landscape. We develop novel and efficient strategies for comprehensively mapping the binding landscape of proteins using a combination of experimental multi-target selective library screening and next-generation sequencing analysis. Our protocols combine protein randomization, yeast surface display technology, deep sequencing, and a few experimental ΔΔGbind data points on purified proteins to generate ΔΔGbind values for the remaining numerous mutants of the same protein complex.
SERINE PROTEASE INHIBITORS
Engineered protein therapeutics offer advantages, including strong target affinity, selectivity and low toxicity, but like natural proteins can be susceptible to proteolytic degradation, thereby limiting their effectiveness. A compelling therapeutic target is mesotrypsin, a protease up-regulated with tumour progression, associated with poor prognosis, and implicated in tumour growth and progression of many cancers. However, with its unique capability for cleavage and inactivation of proteinaceous inhibitors, mesotrypsin presents a formidable challenge to the development of biological inhibitors. We are using a powerful yeast display platform for directed evolution, employing novel multi-modal library screening strategies, to engineer the human amyloid precursor protein Kunitz protease inhibitor domain (APPI) simultaneously for increased proteolytic stability, stronger binding affinity and improved selectivity for mesotrypsin inhibition.
STABILIZATION OF GPCRS
The most efficient approach in drug discovery remains structure-based (SB), in which design of new drugs is guided by the available structures of protein-ligand complexes. SB approach in G protein-coupled receptors (GPCRs), however, is hampered by the tremendous difficulties associated with the expression, isolation, and crystallization of these proteins. GPCRs are extracted from membrane by detergents. For crystallization purposes, short chain (< C9) detergents such as n-Octyl-β-D-glucoside (OG) are particularly useful, but they are poor mimics of the lipid bilayer and most GPCRs are unstable in the detergent-solubilized form. To overcome this problem, mutagenesis is employed to improve structural stability of GPCRs in the presence of short-chain detergents.. We develop a HTP yeast-based methodology for a directed evolution of structurally stable GPCRs amenable to crystallization and structural analysis in the presence of short-chain detergents.
Matrix metalloproteinases (MMPs) are a family of zinc-dependent enzymes that regulate the degradation of the ECM components. Imbalance in their activity might lead to a variety of diseases, including cancer and cardiovascular diseases. As the family of natural tissue inhibitors of metalloproteinases (TIMPs) regulates MMPs activity, they have become an attractive target for therapeutic drug design. Yet, their short circulation half-life makes a serious limitation for this purpose. Therefore, we chose to focus on N-TIMP2 and to apply protein PEGylation in order to increase its size and to extend its circulation half-life. Since the common approaches for protein PEGylation are not site-specific and randomly target amino acids on the target protein, which might affect the protein functionality and stability, we are incorporating a non-canonical amino acid (NCAA), Propargyl lysine (PrK), into N-TIMP2 at different positions, allowing a site-specific PEGylation. Our preliminary results show significant improvements in the pharmacokinetic profile using our new approach.