Paul A. Sprengeler

Discovery of Selective Irreversible Inhibitors for Bruton’s Tyrosine Kinase

The importance of B cells in rheumatoid arthritis (RA) pathogenesis has been recently demonstrated in several clinical studies using the anti-CD20 antibody rituximab, which selectively depletes B cells. A recent phase III clinical trial led to the FDA approval of rituximab for a subset of RA patients. Bruton’s tyrosine kinase (Btk), a member of Tec family kinases, is a key component in the B-cell receptor signal pathway (BCR). Upon activation by upstream kinases (for example, Lyn and Syk), Btk phosphorylates and thereby activates phospholipase-Cg (PLCg), leading to several important downstream events including calcium ion transportation, NF-kB activation, and (auto)antibody generation. Previous biological studies (genetic loss of function and siRNA knockdown) strongly suggest that Btk is also a mediator of proinflammatory signals. Taken together, these studies indicate Btk may be a potential target for the treatment of RA. However, despite the previous discovery of LFM-A13 as a selective Btk inhibitor, there is no published study that has demonstrated that inhibition of Btk activity leads to in vivo efficacy in an animal model of rheumatoid arthritis. As ATP binding sites in kinases are highly conserved, it is a formidable task to develop selective ATP competitive kinase inhibitors. Among several approaches, the use of electrophilic inhibitors has been shown as a viable method to achieve selectivity. Considering the relative scarcity of knowledge on “chemical knockdown” of Btk activity, it is crucial to discover a potent and selective tool compound for this kinase. Herein, we describe the discovery of a selective, irreversible Btk inhibitor and its efficacy in a mouse RA model. An initial campaign to scan for scaffolds capable of inhibiting Btk’s kinase activity identified compound 1 as having

Structural basis for selectivity of a small molecule, S1-binding, submicromolar inhibitor of urokinase-type plasminogen activator.

BACKGROUND Urokinase-type plasminogen activator (uPA) is a protease associated with tumor metastasis and invasion. Inhibitors of uPA may have potential as drugs for prostate, breast and other cancers. Therapeutically useful inhibitors must be selective for uPA and not appreciably inhibit the related, and structurally and functionally similar enzyme, tissue-type plasminogen activator (tPA), involved in the vital blood-clotting cascade. RESULTS We produced mutagenically deglycosylated low molecular weight uPA and determined the crystal structure of its complex with 4-iodobenzo[b]thiophene 2-carboxamidine (K(i) = 0.21 +/- 0.02 microM). To probe the structural determinants of the affinity and selectivity of this inhibitor for uPA we also determined the structures of its trypsin and thrombin complexes, of apo-trypsin, apo-thrombin and apo-factor Xa, and of uPA, trypsin and thrombin bound by compounds that are less effective uPA inhibitors, benzo[b]thiophene-2-carboxamidine, thieno[2,3-b]-pyridine-2-carboxamidine and benzamidine. The K(i) values of each inhibitor toward uPA, tPA, trypsin, tryptase, thrombin and factor Xa were determined and compared. One selectivity determinant of the benzo[b]thiophene-2-carboxamidines for uPA involves a hydrogen bond at the S1 site to Ogamma(Ser190) that is absent in the Ala190 proteases, tPA, thrombin and factor Xa. Other subtle differences in the architecture of the S1 site also influence inhibitor affinity and enzyme-bound structure. CONCLUSIONS Subtle structural differences in the S1 site of uPA compared with that of related proteases, which result in part from the presence of a serine residue at position 190, account for the selectivity of small thiophene-2-carboxamidines for uPA, and afford a framework for structure-based design of small, potent, selective uPA inhibitors.

The Structure of the Extracellular Region of Human Hepsin Reveals a Serine Protease Domain and a Novel Scavenger Receptor Cysteine-Rich (SRCR) Domain

Hepsin is an integral membrane protein that may participate in cell growth and in maintaining proper cell morphology and is overexpressed in a number of primary tumors. We have determined the 1.75 A resolution structure of the extracellular component of human hepsin. This structure includes a 255-residue trypsin-like serine protease domain and a 109-residue region that forms a novel, poorly conserved, scavenger receptor cysteine-rich (SRCR) domain. The two domains are associated with each other through a single disulfide bond and an extensive network of noncovalent interactions. The structure suggests how the extracellular region of hepsin may be positioned with respect to the plasma membrane.

Elaborate manifold of short hydrogen bond arrays mediating binding of active site-directed serine protease inhibitors.

An extensive structural manifold of short hydrogen bond-mediated, active site-directed, serine protease inhibition motifs is revealed in a set of over 300 crystal structures involving a large suite of small molecule inhibitors (2-(2-phenol)-indoles and 2-(2-phenol)-benzimidazoles) determined over a wide range of pH (3.5-11.4). The active site hydrogen-bonding mode was found to vary markedly with pH, with the steric and electronic properties of the inhibitor, and with the type of protease (trypsin, thrombin or urokinase type plasminogen activator (uPA)). The pH dependence of the active site hydrogen-bonding motif is often intricate, constituting a distinct fingerprint of each complex. Isosteric replacements or minor substitutions within the inhibitor that modulate the pK(a) of the phenol hydroxyl involved in short hydrogen bonding, or that affect steric interactions distal to the active site, can significantly shift the pH-dependent structural profile characteristic of the parent scaffold, or produce active site-binding motifs unique to the bound analog. Ionization equilibria at the active site associated with inhibitor binding are probed in a series of the protease-inhibitor complexes through analysis of the pH dependence of the structure and environment of the active site-binding groups involved in short hydrogen bond arrays. Structures determined at high pH (>11), suggest that the pK(a) of His57 is dramatically elevated, to a value as high as approximately 11 in certain complexes. K(i) values involving uPA and trypsin determined as a function of pH for a set of inhibitors show pronounced parabolic pH dependence, the pH for optimal inhibition governed by the pK(a) of the inhibitor phenol involved in short hydrogen bonds. Comparison of structures of trypsin, thrombin and uPA, each bound by the same inhibitor, highlights important structural variations in the S1 and active sites accessible for engineering notable selectivity into remarkably small molecules with low nanomolar K(i) values.

2-(2-Hydroxy-3-alkoxyphenyl)-1H-benzimidazole-5-carboxamidine derivatives as potent and selective urokinase-type plasminogen activator inhibitors.

The development of potent and selective urokinase-type plasminogen activator (uPA) inhibitors based on the lead molecule 2-(2-hydroxy-3-ethoxyphenyl)-1H-benzimidazole-5-carboxamidine (3a) is described.