Rational design of endogenous-like inhibitors and modulators targeting matrix enzymes
Rational design of potent and selective inhibitors for human matrix metalloproteases, ADAMs, LOXLs as well as other extracellular matrix enzymes is based on detailed mechanistic information obtained from our molecular and biophysical studies. Controlling the enzymatic activity of specific MMPs by antagonist molecules is highly desirable, first, for studying their individual function and mode of action, and second as putative therapeutic agents. Along these lines, we solve the molecular architecture of the natural inhibitors of matrix proteases, TIMP and enzyme pro-domains by protein crystallography/NMR techniques and study their mechanism of inhibition in our biophysics, biochemistry and cell biology set ups. In addition, we are using directed evolution experimental approach to re-design and stabilize recombinant protein-based modulators targeting key enzymes which remodel the cellular microenvironment. This includes the engineering of function-blocking antibodies which bind and inhibit specific ECM enzymes (Sela-Passwell et al, Nature Medicine 2012). These inhibitors are currently tested in cancer and developed as therapeutics for clinical trials in inflammatory diseases.
Complex of MT1-MMP (yellow) with TIMP1 (grey), listed as 3MA2 in the protein data base.
Our x-ray absoption analysis of the crystal structure of MT1-MMP with tissue inhibitor of
metalloproteinase 1(TIMP1) combined with molecular dynamics simulation elucidates the effect of a single point mutation on the protein conformational dynamics and function of the inhibitor
TIMP1. We suggest that controlling the intrinsic protein dynamics of MMP endogenous inhibitors may
be utilized for rationalizing the design of selective novel protein inhibitors for this family of metalloenzymes.
Please check our page in proteopedia on the MT1-MMP-TIMP1-complex. There you will find our high resolution crystal structure of an engineered TIMP1 which was mutated in order to achieve increased binding affinity to MT1-MMP.
Drugging the undruggable: New opportunities for inhibition of MMPs
Monitoring enzymatic activity in vivo of individual homologous enzymes such as MMPs by target-specific antagonist molecules is highly desired for defining and controlling physiological and pathophysiological pathways. However, the rational design of antagonists targeting enzyme catalytic moieties specific to one of the homologous often appears to be an extremely difficult task. This is mainly due to the high structural homology at the enzyme active sites shared by members of the protein family. Accordingly, controlling enzymatic activity via alternative allosteric sites has become an attractive proposition for drug design targeting individual homologous enzymes. Despite these advances in rational drug design, it remains a challenge to assign and identify potential regulatory alternative sites which are often hidden and scattered over different locations on the protein's surface.
We have designed branched amphiphilic molecules exhibiting specific inhibitory activity towards individual members of the MMP family. These amphiphilic isomers allow to probe hidden regulatory residues on a given protein surface. Using the advantage provided by amphiphilic ligands, here we explore a new approach for determining hidden regulatory sites. Using diverse experimental techniques, such as structural spectroscopic analyses, NMR and protein crystallography combined with computational prediction we were able to identify potential inhibitory sites distinct for each MMP.