The paracaspase MALT1 (mucosa associated lymphoid tissue lymphoma translocated gene 1) plays an important role in various immune pathways and is proposed as a therapeutic target for autoimmune disorders as well as different types of cancer, such as diffuse large B-cell lymphoma (DLBCL). Different mechanisms are explored to inhibit the protease activity of MALT1 and two unrelated chemical scaffolds are discovered. Biophysical and structural studies reveal that both scaffolds stabilize the protease in an inactive conformation. While one ligand binds to the allosteric site at the interface between the caspase and the Ig3 domain, the other ligand binds to the active site using a so far undescribed mechanism. Iterative structure-based drug discovery on one scaffold results in the identification of a potent, selective, and orally bioavailable MALT1 inhibitor.

The formation of the CBM (CARD11-BCL10-MALT1) complex is pivotal for antigen-receptor-mediated activation of the transcription factor NF-κB. Signaling is dependent on MALT1 (mucosa-associated lymphoid tissue lymphoma translocation protein 1), which not only acts as a scaffolding protein but also possesses proteolytic activity mediated by its caspase-like domain. It remained unclear how the CBM activates MALT1. Here, we provide biochemical and structural evidence that MALT1 activation is dependent on its dimerization and show that mutations at the dimer interface abrogate activity in cells. The unliganded protease presents itself in a dimeric yet inactive state and undergoes substantial conformational changes upon substrate binding. These structural changes also affect the conformation of the C-terminal Ig-like domain, a domain that is required for MALT1 activity. Binding to the active site is coupled to a relative movement of caspase and Ig-like domains. MALT1 binding partners thus may have the potential of tuning MALT1 protease activity without binding directly to the caspase domain.

Neurodegenerative diseases pose one of the most pressing unmet medical needs today. It has long been recognized that caspase-6 may play a role in several neurodegenerative diseases for which there are currently no disease-modifying therapies. Thus it is a potential target for neurodegenerative drug development. In the present study we report on the biochemistry and structure of caspase-6. As an effector caspase, caspase-6 is a constitutive dimer independent of the maturation state of the enzyme. The ligand-free structure shows caspase-6 in a partially mature but latent conformation. The cleaved inter-domain linker remains partially inserted in the central groove of the dimer, as observed in other caspases. However, in contrast with the structures of other caspases, not only is the catalytic machinery misaligned, but several structural elements required for substrate recognition are missing. Most importantly, residues forming a short anti-parallel beta-sheet abutting the substrate in other caspase structures are part of an elongation of the central alpha-helix. Despite the dramatic structural changes that are required to adopt a canonical catalytically competent conformation, the pre-steady-state kinetics exhibit no lag phase in substrate turnover. This suggests that the observed conformation does not play a regulatory role in caspase-6 activity. However, targeting the latent conformation in search for specific and bio-available caspase-6 inhibitors might offer an alternative to active-site-directed approaches.

Neonicotinoids do not act as a homogenous class of insecticides. Radioligand receptor binding assays revealed two classes of neonicotinoids described here as 'competitive' and 'non-competitive', respectively, relative to [3H]imidacloprid. Differences in affinity, mode of displacement, number of binding sites and temperature sensitivity suggest that thiamethoxam binds in a way unique among the commercial neonicotinoids. Metabolic transformation is not relevant for its insecticidal effects.

Neonicotinoids bind selectively to insect nicotinic acetylcholine receptors with nanomolar affinity to act as potent insecticides. While the members of the neonicotinoid class have many structural features in common, it is not known whether they also share the same mode of binding to the target receptor. Previous competition studies with [3H]imidacloprid, the first commercialised neonicotinoid, indicated that thiamethoxam, representing a novel structural sub-class, may bind in a different way from that of other neonicotinoids. In the present work we analysed the mode of [3H]imidacloprid displacement by established neonicotinoids and newly synthesized analogues in the aphids Myzus persicae Sulzer and Aphis craccivora Koch. We found two classes of neonicotinoids with distinct modes of interference with [3H]imidacloprid, described as direct competitive inhibition and non-competitive inhibition, respectively. Competitive neonicotinoids were acetamiprid, nitenpyram, thiacloprid, clothianidin and nithiazine, whereas thiamethoxam and the N-methyl analogues of imidacloprid and clothianidin showed non-competitive inhibition. The chloropyridine or chlorothiazole heterocycles, the polar pharmacophore parts, such as nitroimino, cyanoimino and nitromethylene, and the cyclic or acyclic structure of the pharmacophore were not relevant for the mode of inhibition. Consensus structural features of the neonicotinoids were defined for the two mechanisms of interaction with [3H]imidacloprid binding. Furthermore, two sub-classes of non-competitive inhibitors can be discriminated on the basis of their Hill coefficients for imidacloprid displacement. We conclude from the present data that the direct competitors share the binding site with imidacloprid, whereas non-competitive compounds, like thiamethoxam, bind to a different site or in a different mode.

The neonicotinoids are the most successful chemical class of insecticides reaching sales of more than 630 Mio $ in 2001, mainly due to the excellent market performance of imidacloprid and thiamethoxam. The insect nicotinic acetylcholine receptors (nAChRs) are the targets for these compounds, which are highly effective against a variety of sucking and chewing insects. Compared with the other neonicotinoid sales products, thiamethoxam binds in a different way, possibly to a different site of nAChRs in aphids. To gain further insight into the different modes of binding, a research program applying the photoaffinity labeling technique was started. A series of novel candidate photoaffinity probes containing a 5-azido-6-chloropyridin-3-ylmethyl group were prepared from 5-azido-6-chloropyridin-3-ylmethyl chloride, which was obtained in three steps from 6-chloropyridin-3-ylmethyl chloride. These probes showed good to excellent contact/feeding and systemic activity against Myzus persicae, however, they were at least 4- to 16-fold less effective against Aphis craccivora, Nilaparvata lugens, Spodoptera littoralis, and Diabrotica balteata than the neonicotinoid sales products. In general, the introduction of an azide group at C(5) of the 6-chloropyridin-3-ylmethyl substituent resulted in reduced potency as well as in a narrower pest spectrum. In competition binding assays with [3H]imidacloprid, analogues of imidacloprid, clothianidin, thiacloprid and thiamethoxam containing a 5-azido-6-chloropyridin-3-ylmethyl group showed high displacing potency with nAChRs from Aphis and Myzus (Ki values: 2 to 27 nM) suggesting that these compounds are valuable candidate photoaffinity probes. Taking into account the biological screening activity as well as the receptor binding potency, 1-(5-azido-6-chloropyridin-3-ylmethyl)-2-nitroimino-imidazolidine, N-(5-azido-6-chloropyridin-3-ylmethyl)-N?-methyl-N?-nitroguanidine and 3-(5-azido-6-chloropyridin-3-ylmethyl)-2-cyanoimino-thiazolidine were identified as the preferred candidate neonicotinoid photoaffinity probes to study the imidacloprid binding site.