Structural basis of SARM1 activation, substrate recognition, and inhibition by small molecules.

The NADase SARM1 (sterile alpha and TIR motif containing 1) is a key executioner of axon degeneration and a therapeutic target for several neurodegenerative conditions. We show that a potent SARM1 inhibitor undergoes base exchange with the nicotinamide moiety of nicotinamide adenine dinucleotide (NAD+) to produce the bona fide inhibitor 1AD. We report structures of SARM1 in complex with 1AD, NAD+ mimetics and the allosteric activator nicotinamide mononucleotide (NMN). NMN binding triggers reorientation of the armadillo repeat (ARM) domains, which disrupts ARM:TIR interactions and leads to formation of a two-stranded TIR domain assembly. The active site spans two molecules in these assemblies, explaining the requirement of TIR domain self-association for NADase activity and axon degeneration. Our results reveal the mechanisms of SARM1 activation and substrate binding, providing rational avenues for the design of new therapeutics targeting SARM1.

Small Molecule SHP2 Inhibitor LXQ-217 Affects Lung Cancer Cell Proliferation in Vitro and in Vivo.

BACKGROUND: SHP2 is highly expressed in a variety of cancer and has emerged as a potential target for cancer therapeutic agents. The identification of uncharged pTyr mimics is an important direction for the development of SHP2 orthosteric inhibitors. METHODS: Surface plasmon resonance analysis and cellular thermal shift assay were employed to verify the direct binding of LXQ-217 to SHP2. The inhibitory effect of LXQ-217 was characterized by linear Weaver-Burke enzyme kinetic analysis and BIOVIA Discovery Studio. The inhibition of tumor cell proliferation by LXQ-217 was characterized by cell viability assay, colony formation assays and hoechst 33258 staining. The inhibition of lung cancer proliferation in vivo was studied in nude mice after oral administration of LXQ-217. RESULTS: An electroneutral bromophenol derivative, LXQ-217, was identified as a competitive SHP2 inhibitor. LXQ-217 induced apoptosis and inhibited growth of human pulmonary epithelial cells by affecting the RAS-ERK and PI3 K-AKT signaling pathways. Long-term oral administration of LXQ-217 significantly inhibited the proliferation ability of lung cancer cells in nude mice. Moreover, mice administered LXQ-217 orally at high doses exhibited no mortality or significant changes in vital signs. CONCLUSIONS: Our findings on the uncharged orthosteric inhibitor provide a foundation for further development of a safe and effective anti-lung cancer drug.

Systematic analysis and molecular profiling of EGFR allosteric inhibitor cross-reactivity across the proto-oncogenic ErbB family kinases by integrating dynamics simulation, energetics calculation and biochemical assay.

Human ErbB family of proteins contains four receptor tyrosine kinases (EGFR, Her2, Her3 and Her4) and has been established as a group of attractive druggable targets against diverse cancers. Over the past decades, a variety of ATP-competitive inhibitors have been discovered to target the orthosteric site of EGFR, which, however, would eventually develop acquired drug resistance due to the missense mutations T790M/C797S occurring in orthosteric site. In recent years, a number of forth-generation inhibitors have been successfully designed to overcome the T790M/C797S-induced drug resistance by targeting EGFR allosteric site instead of orthosteric site. Considering that the four proto-oncogenic ErbB kinases share a high conservation in sequence, structure and function, we herein attempted to investigate the binding potency and cross-reactivity of cognate EGFR allosteric inhibitors over noncognate Her2, Her3 and Her4 kinases--they are closely related to gynecological tumors such as ovarian cancer but no allosteric inhibitors have been reported specifically for them to date. A systematic affinity profile of 12 allosteric inhibitors and 4 orthosteric inhibitors to the 4 ErbB kinases was created by integrating dynamics simulations, energetics calculations and biochemical assays, which was then used to derive a systematic inhibitor selectivity profile for EGFR over other three kinases. It is found that allosteric and orthosteric inhibitors exhibit moderate and modest cross-reactivity across the ErbB family, respectively, but the former generally has a higher binding potency than the latter due to the additional energy cost used for inducing kinase conformational change. Moreover, most allosteric inhibitors can be sensitized by Her2 T798M gatekeeper mutation, a counterpart of EGFR T790M gatekeeper mutation that has been previously reported to cause generic drug resistance for orthosteric inhibitors.

Selective inhibition of matrix metalloproteinase 10 (MMP10) with a single-domain antibody.

Since their discovery, the matrix metalloproteinase (MMP) family proteases have been considered as therapeutic targets in numerous diseases and disorders. Unfortunately, clinical trials with MMP inhibitors have failed to yield any clinical benefits of these inhibitors. These failures were largely due to a lack of MMP-selective agents; accordingly, it has become important to identify a platform with which high selectivity can be achieved. To this end, we propose using MMP-targeting antibodies that can achieve high specificity in interactions with their targets. Using a scaffold of single-domain antibodies, here we raised a panel of MMP10-selective antibodies through immunization of llamas, a member of the camelid family, whose members generate conventional heavy/light-chain antibodies and also smaller antibodies lacking light-chain and CH1 domains. We report the generation of a highly selective and tightly binding MMP10 inhibitor (Ki < 2 nm). Using bio-layer interferometry-based binding assays, we found that this antibody interacts with the MMP10 active site. Activity assays demonstrated that the antibody selectively inhibits MMP10 over its closest relative, MMP3. The ability of a single-domain antibody to discriminate between the most conserved MMP pair via an active site-directed mechanism of inhibition reported here supports the potential of this antibody as a broadly applicable scaffold for the development of selective, tightly binding MMP inhibitors.

Binding of small molecules at interface of protein-protein complex - A newer approach to rational drug design.

Protein-protein interaction is a vital process which drives many important physiological processes in the cell and has also been implicated in several diseases. Though the protein-protein interaction network is quite complex but understanding its interacting partners using both in silico as well as molecular biology techniques can provide better insights for targeting such interactions. Targeting protein-protein interaction with small molecules is a challenging task because of druggability issues. Nevertheless, several studies on the kinetics as well as thermodynamic properties of protein-protein interactions have immensely contributed toward better understanding of the affinity of these complexes. But, more recent studies on hot spots and interface residues have opened up new avenues in the drug discovery process. This approach has been used in the design of hot spot based modulators targeting protein-protein interaction with the objective of normalizing such interactions.