This short review highlights some recent advances in matrix metalloproteinase inhibitor (MMPi) design and development. Three distinct approaches to improved MMP inhibition are discussed: (1) the identification and investigation of novel zinc-binding groups (ZBGs), (2) the study of non-zinc-binding MMPi, and (3) mechanism-based MMPi that form covalent adducts with the protein. Each of these strategies is discussed and their respective advantages and remaining challenges are highlighted. The studies discussed here bode well for the development of ever more selective, potent, and well-tolerated MMPi for treating several important disease pathologies.

Fragment-based lead design (FBLD) has been used to identify new metal-binding groups for metalloenzyme inhibitors. When screened at 1 mM, a chelator fragment library (CFL-1.1) of 96 compounds produced hit rates ranging from 29-43% for five matrix metalloproteases (MMPs), 24% for anthrax lethal factor (LF), 49% for 5-lipoxygenase (5-LO), and 60% for tyrosinase (TY). The ligand efficiencies (LE) of the fragment hits are excellent, in the range of 0.4-0.8 kcal/mol. The MMP enzymes all generally elicit the same chelators as hits from CFL-1.1; however, the chelator fragments that inhibit structurally unrelated metalloenzymes (LF, 5-LO, TY) vary considerably. To develop more advanced hits, one hit from CFL-1.1, 8-hydroxyquinoline, was elaborated at four different positions around the ring system to generate new fragments. 8-Hydroxyquinoline fragments substituted at either the 5-or 7-positions gave potent hits against MMP-2, with IC 50 values in the low micromolar range. The 8-hydroxyquinoline represents a promising, new chelator scaffold for the development of MMP inhibitors that was discovered by use of a metalloprotein-focused chelator fragment library.

The need for selective matrix metalloproteinase (MMP) inhibition is of interest because of the range of pathologies mediated by different MMP isoforms. The development of more selective MMP inhibitors (MMPi) may help to overcome some of the undesired side effects that have hindered the clinical success of these compounds. In an effort to devise new approaches to selective inhibitors, herein we describe several novel MMPi and show that their selectivity is dependent on the nature of the zinc-binding group (ZBG). This is in contrast to most current MMPi, which obtain isoform selectivity solely from the peptidomimetic backbone portion of the compound. In the present study, six different hydroxypyrone and hydroxypyridinone ZBGs were appended to a common biphenyl backbone and the inhibition efficiency of each inhibitor was determined in vitro (IC(50) values) against MMP-1, -2, -3, -7, -8, -9, -12, and -13. The results show that the selectivity profile of each inhibitor is different as a result of the various ZBGs. Computational modeling studies were used to explain some trends in the observed selectivity profiles. To assess the importance of the ZBG in a biological model, two of the semiselective, potent MMPi (and one control) were evaluated using an isolated perfused rat heart system. Hearts were subjected to ischemia reperfusion injury, and recovery of contractile function was examined. In this model, only one of the two MMPi showed significant and sustained heart recovery, demonstrating that the choice of ZBG can have a significant effect in a relevant pathophysiological endpoint.

A chelator fragment library based on a variety of metal binding groups was screened against a metalloproteinase. Lead hits were identified and an expanded library of select compounds was synthesized, resulting in numerous high-affinity hits against several metalloprotein targets. The findings clearly demonstrate that chelators can be used to generate libraries suitable for fragmentbased lead design (FBLD) directed at important metalloproteins. Keywordschelators; fragment-based lead design; libraries; metalloproteins; zinc Fragment-based lead design (FBLD), sometimes referred to as fragment-based drug discovery (FBDD), is an increasingly important strategy for the discovery of biologically active compounds.[1] FBLD generally uses libraries consisting of modest collections (100-1000 compounds) of small molecular fragments (MW <300 amu) that are screened against targets of interest.[2] Although such fragments do not bind as tightly (K d values in the micro-to millimolar range) as more complex molecules, including those used in high-throughput screening (HTS) approaches, they can provide 'hits' that serve as efficient starting structures for the development of potent inhibitors. Fragments that bind to a target are identified, after which one of two approaches is generally pursued: a) a single fragment can be elaborated in order to obtain a tight binder; or b) multiple fragments binding at adjacent and distinct sites can be connected by an appropriate linker to obtain a potent inhibitor. Compared to HTS, FBLD is purported to have several advantages, including a more efficient exploration of chemically diverse space and higher ligand efficiencies.Although the application of FBLD to metalloprotein targets of medicinal interest has been described, [3] Matrix metalloproteinases (MMPs) represent one of the most well-established targets in the realm of metalloproteins. These zinc(II)-dependent enzymes have been extensively studied, and the development of MMP inhibitors (MMPi) has played an important role in the discovery of inhibitors for other zinc(II)-dependent metalloproteins, such as anthrax lethal factor (LF), histone deacetylases (HDACs), and others.[10] Indeed, the earliest application of FBLD to a metalloprotein target was directed at MMP-3.[3] Therefore, we focused our preliminary library screening efforts on MMPs, as a representative system for identifying fragments that would bind zinc(II) metalloproteins. Based on widely-reported criteria for fragment libraries, [2] an initial chelator fragment library (CFL-1) was assembled. A modest library containing 96 structural cores was prepared from chelators with two to four donor atoms for binding metal ions and sufficient solubility for screening ( Figure 1). The chelating groups included picolinic acids, hydroxyquinolones, pyrimidines, hydroxypyrones, hydroxypyridinones and salicylic acids, in addition to other compounds that are well-established components of metalloprotein inhibitors, such as hydroxamic acids and sulfonamides ( Figure 1). The CFL-1 librar...

The histone methyltransferase EZH2 is required for B and T cell development; however the molecular mechanisms underlying this requirement remain elusive. In a murine model of lymphoid specific EZH2-deficiency we found that EZH2 was required for proper development of adaptive, but not innate, lymphoid cells. In adaptive lymphoid cells EZH2 prevented the premature expression of Cdkn2a and the consequent stabilization of p53, an effector of the pre-antigen receptor checkpoints. Deletion of Cdkn2a in Ezh2-deficient lymphocytes prevented p53 stabilization, extended lymphocyte survival, and restored differentiation resulting in the generation of mature B and T lymphocytes. Our results uncover a crucial role for EZH2 in adaptive lymphocytes to control the developmental timing of effectors of the pre-antigen receptor checkpoints.