Joseph W. Becker

Platensimycin is a selective FabF inhibitor with potent antibiotic properties

Bacterial infection remains a serious threat to human lives because of emerging resistance to existing antibiotics. Although the scientific community has avidly pursued the discovery of new antibiotics that interact with new targets, these efforts have met with limited success since the early 1960s. Here we report the discovery of platensimycin, a previously unknown class of antibiotics produced by Streptomyces platensis. Platensimycin demonstrates strong, broad-spectrum Gram-positive antibacterial activity by selectively inhibiting cellular lipid biosynthesis. We show that this anti-bacterial effect is exerted through the selective targeting of β-ketoacyl-(acyl-carrier-protein (ACP)) synthase I/II (FabF/B) in the synthetic pathway of fatty acids. Direct binding assays show that platensimycin interacts specifically with the acyl-enzyme intermediate of the target protein, and X-ray crystallographic studies reveal that a specific conformational change that occurs on acylation must take place before the inhibitor can bind. Treatment with platensimycin eradicates Staphylococcus aureus infection in mice. Because of its unique mode of action, platensimycin shows no cross-resistance to other key antibiotic-resistant strains tested, including methicillin-resistant S. aureus, vancomycin-intermediate S. aureus and vancomycin-resistant enterococci. Platensimycin is the most potent inhibitor reported for the FabF/B condensing enzymes, and is the only inhibitor of these targets that shows broad-spectrum activity, in vivo efficacy and no observed toxicity.

A combinatorial approach for determining protease specificities: application to interleukin-1beta converting enzyme (ICE).

BACKGROUND Interleukin-1beta converting enzyme (ICE/caspase-1) is the protease responsible for interleukin-1beta (IL-1beta) production in monocytes. It was the first member of a new cysteine protease family to be identified. Members of this family have functions in both inflammation and apoptosis. RESULTS A novel method for identifying protease specificity, employing a positional-scanning substrate library, was used to determine the amino-acid preferences of ICE. Using this method, the complete specificity of a protease can be mapped in the time required to perform one assay. The results indicate that the optimal tetrapeptide recognition sequence for ICE is WEHD, not YVAD, as previously believed, and this led to the synthesis of an unusually potent aldehyde inhibitor, Ac-WEHD-CHO (Ki = 56 pM). The structural basis for this potent inhibition was determined by X-ray crystallography. CONCLUSIONS The results presented in this study establish a positional-scanning library as a powerful tool for rapidly and accurately assessing protease specificity. The preferred sequence for ICE (WEHD) differs significantly from that found in human pro-interleukin-1beta (YVHD), which suggests that this protease may have additional endogenous substrates, consistent with evidence linking it to apoptosis and IL-1alpha production.

Structural basis for p38alpha MAP kinase quinazolinone and pyridol-pyrimidine inhibitor specificity.

The quinazolinone and pyridol-pyrimidine classes of p38 MAP kinase inhibitors have a previously unseen degree of specificity for p38 over other MAP kinases. Comparison of the crystal structures of p38 bound to four different compounds shows that binding of the more specific molecules is characterized by a peptide flip between Met109 and Gly110. Gly110 is a residue specific to the α, β and γ isoforms of p38. The δ isoform and the other MAP kinases have bulkier residues in this position. These residues would likely make the peptide flip energetically unfavorable, thus explaining the selectivity of binding. To test this hypothesis, we constructed G110A and G110D mutants of p38 and measured the potency of several compounds against them. The results confirm that the selectivity of quinazolinones and pyridol-pyrimidines results from the presence of a glycine in position 110. This unique mode of binding may be exploited in the design of new p38 inhibitors.

The structure of JNK3 in complex with small molecule inhibitors: structural basis for potency and selectivity

The c-Jun terminal kinases (JNKs) are members of the mitogen-activated protein (MAP) kinase family and regulate signal transduction in response to environmental stress. Activation of JNK3, a neuronal-specific isoform, has been associated with neurological damage, and as such, JNK3 may represent an attractive target for the treatment of neurological disorders. The MAP kinases share between 50% and 80% sequence identity. In order to obtain efficacious and safe compounds, it is necessary to address the issues of potency and selectivity. We report here four crystal structures of JNK3 in complex with three different classes of inhibitors. These structures provide a clear picture of the interactions that each class of compound made with the kinase. Knowledge of the atomic interactions involved in these diverse binding modes provides a platform for structure-guided modification of these compounds, or the de novo design of novel inhibitors that could satisfy the need for potency and selectivity.

The three-dimensional structure of human granzyme B compared to caspase-3, key mediators of cell death with cleavage specificity for aspartic acid in P1

BACKGROUND Granzyme B, one of the most abundant granzymes in cytotoxic T-lymphocyte (CTL) granules, and members of the caspase (cysteine aspartyl proteinases) family have a unique cleavage specificity for aspartic acid in P1 and play critical roles in the biochemical events that culminate in cell death. RESULTS We have determined the three-dimensional structure of the complex of the human granzyme B with a potent tetrapeptide aldehyde inhibitor. The Asp-specific S1 subsite of human granzyme B is significantly larger and less charged than the corresponding Asp-specific site in the apoptosis-promoting caspases, and also larger than the corresponding subsite in rat granzyme B. CONCLUSIONS The above differences account for the variation in substrate specificity among granzyme B, other serine proteases and the caspases, and enable the design of specific inhibitors that can probe the physiological functions of these proteins and the disease states with which they are associated.

Mouse and Human Granzyme B Have Distinct Tetrapeptide Specificities and Abilities to Recruit the Bid Pathway

Granzyme B is an important mediator of cytotoxic lymphocyte granule-induced death of target cells, accomplishing this through cleavage of Bid and cleavage and activation of caspases as well as direct cleavage of downstream substrates. Significant controversy exists regarding the primary pathways used by granzyme B to induce cell death, perhaps arising from the use of different protease/substrate combinations in different studies. The primary sequence of human, rat, and mouse granzymes B is well conserved, and the substrate specificity and crystal structure of the human and rat proteases are extremely similar. Although little is known about the substrate specificity of mouse granzyme B, recent studies suggest that it may differ significantly from the human protease. In these studies we show that the specificities of human and mouse granzymes B differ significantly. Human and mouse granzyme B cleave species-specific procaspase-3 more efficiently than the unmatched substrates. The distinct specificities of human and mouse granzyme B highlight a previously unappreciated requirement for Asp192 in the acquisition of catalytic activity upon cleavage of procaspase-3 at Asp175. Although human granzyme B efficiently cleaves human or mouse Bid, these substrates are highly resistant to cleavage by the mouse protease, strongly indicating that the Bid pathway is not a major primary mediator of the effects of mouse granzyme B. These studies provide important insights into the substrate specificity and function of the granzyme B pathway in different species and highlight that caution is essential when designing and interpreting experiments with different forms of granzyme B.

Identification of a potent synthetic FXR agonist with an unexpected mode of binding and activation.

The farnesoid X receptor (FXR), a member of the nuclear hormone receptor family, plays important roles in the regulation of bile acid and cholesterol homeostasis, glucose metabolism, and insulin sensitivity. There is intense interest in understanding the mechanisms of FXR regulation and in developing pharmaceutically suitable synthetic FXR ligands that might be used to treat metabolic syndrome. We report here the identification of a potent FXR agonist (MFA-1) and the elucidation of the structure of this ligand in ternary complex with the human receptor and a coactivator peptide fragment using x-ray crystallography at 1.9-Å resolution. The steroid ring system of MFA-1 binds with its D ring-facing helix 12 (AF-2) in a manner reminiscent of hormone binding to classical steroid hormone receptors and the reverse of the pose adopted by naturally occurring bile acids when bound to FXR. This binding mode appears to be driven by the presence of a carboxylate on MFA-1 that is situated to make a salt-bridge interaction with an arginine residue in the FXR-binding pocket that is normally used to neutralize bound bile acids. Receptor activation by MFA-1 differs from that by bile acids in that it relies on direct interactions between the ligand and residues in helices 11 and 12 and only indirectly involves a protonated histidine that is part of the activation trigger. The structure of the FXR:MFA-1 complex differs significantly from that of the complex with a structurally distinct agonist, fexaramine, highlighting the inherent plasticity of the receptor.

The differential interactions of peroxisome proliferator-activated receptor gamma ligands with Tyr473 is a physical basis for their unique biological activities.

Despite their proven antidiabetic efficacy, widespread use of peroxisome proliferator-activated receptor (PPAR)γ agonists has been limited by adverse cardiovascular effects. To overcome this shortcoming, selective PPARγ modulators (SPPARγMs) have been identified that have antidiabetic efficacy comparable with full agonists with improved tolerability in preclinical species. The results of structural studies support the proposition that SPPARγMs interact with PPARγ differently from full agonists, thereby providing a physical basis for their novel activities. Herein, we describe a novel PPARγ ligand, SPPARγM2. This compound was a partial agonist in a cell-based transcriptional activity assay, with diminished adipogenic activity and an attenuated gene signature in cultured human adipocytes. X-ray cocrystallography studies demonstrated that, unlike rosiglitazone, SPPARγM2 did not interact with the Tyr473 residue located within helix 12 of the ligand binding domain (LBD). Instead, SPPARγM2 was found to bind to and activate human PPARγ in which the Tyr473 residue had been mutated to alanine (hPPARγY473A), with potencies similar to those observed with the wild-type receptor (hPPARγWT). In additional studies, we found that the intrinsic binding and functional potencies of structurally distinct SPPARγMs were not diminished by the Y473A mutation, whereas those of various thiazolidinedione (TZD) and non-TZD PPARγ full agonists were reduced in a correlative manner. These results directly demonstrate the important role of Tyr473 in mediating the interaction of full agonists but not SPPARγMs with the PPARγ LBD, thereby providing a precise molecular determinant for their differing pharmacologies.

The interleukin‐1β converting enzyme family of cysteine proteases

Interleukin‐1β converting enzyme (ICE) is the first enzyme of a new family of cysteine endoproteinases to be isolated and characterized. An overview of the structure and activity of ICE is outlined together with highlights of salient features common to members of each of the family members. J. Cell. Biochem. 64:2–10.

CHAPTER 24. INHIBITION OF MATRIX METALLOPROTEINASES

Publisher Summary This chapter briefly describes the metalloproteinase (MMP) family of enzymes and the putative role it plays in both physiology and pathology, the structures of these enzymes and the current status of MMP inhibitor development for the treatment of this wide range of diseases. The MMP family currently includes fourteen members encoded by unique genes, 10 of which are secreted from cells in a soluble form and 4 new members that are bound to the cell membrane. The MMPs are zinc dependent, calcium requiring enzymes, which are expressed as inactive zymogens. These enzymes are also inhibited by one or more members of the tissue inhibitor of metalloproteinase (TIMP) family, of which three have been cloned and sequenced. The physiological activators of many of these enzymes remain unknown; however, some of the members of the MMP family have the capacity to activate other family members. Therefore, inhibition of the enzyme(s), ultimately responsible for this activation cascade, could result in blocking the activity of multiple enzymes without directly inhibiting their activity. Recently four membrane type MMPs (MT-MMPs) have been cloned and sequenced. Two of these enzymes have the capacity to activate MMP-2 suggesting that they may be a target to consider for the intervention of tumor metastasis. These enzymes are unique among the MMPs because they appear to be associated with the cell membrane and not released into the extracellular space. Using MMP inhibitors, shedding of a number of cell associated proteins, including TNFα, IL-6 and tumor necrosis factors (TNF) receptors, FAS ligand, ACE, TSH receptor ectodomain, and CD-23, may be mediated by membrane associated MMPs.