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To get a better understanding of the possibility of developing selective carbonic anhydrase (CA) inhibitors, interactions between 17 benzenesulphonamide ligands and 6 human CAs (full-length CA I, II, VII, and XIII and catalytic domains of CA IX and XII) were characterized using surface plasmon resonance and fluorescent-based thermal shift assays. Kinetics revealed that the strongest binders had subnanomolar affinities with low dissociation rates (i.e., kd values around 1 × 10(-3) s(-1)) or were essentially irreversible. Chemodynamic analysis of the interactions highlighted an intrinsic mechanism of the CA-sulphonamide interaction kinetics and showed that slow dissociation rates were mediated by large hydrophobic contacts. The studied inhibitors demonstrated a high cross-reactivity within the protein family. However, according to chemical phylogenetic analysis developed for kinetic data, several ligands were found to be selective against certain CA isozymes, indicating that it should be possible to develop selective CA inhibitors suitable for clinical use.

Structure kinetic relationship analyses and identification of dominating interactions for optimization of lead compounds should ideally be based on intrinsic rate constants instead of the more easily accessible observed kinetic constants, which also account for binding linked reactions. The intrinsic rate constants for sulfonamide inhibitors and pharmacologically relevant isoforms of carbonic anhydrase were determined by a novel surface plasmon resonance (SPR) biosensor-based approach, using chemodynamic analysis of binding-linked pH-dependent effects. The observed association rates (k(a)(obs)) were pH-dependent and correlated with the fraction of deprotonated inhibitor and protonated zinc-bound water molecule. The intrinsic association rate constants (k(a)(intr)) were pH independent and higher than k(a)(obs). By contrast, the observed and intrinsic dissociation rate constants were identical and pH-independent, demonstrating that the observed association and dissociation mechanisms are inherently different. A model accounting for the differences between intrinsic and observed rate constants was developed, useful also for other interactions with binding-linked protonation reactions.

Calmodulin (CaM) functions depend on interactions with CaM-binding proteins, regulated by Ca2+. Induced structural changes influence the affinity, kinetics, and specificities of the interactions. The dynamics of CaM interactions with neurogranin (Ng) and the CaM-binding region of Ca2+/calmodulin-dependent kinase II (CaMKII290-309) have been studied using biophysical methods. These proteins have opposite Ca2+ dependencies for CaM binding. Surface plasmon resonance biosensor analysis confirmed that Ca2+ and CaM interact very rapidly, and with moderate affinity (KDSPR=3M). Calmodulin-CaMKII290-309 interactions were only detected in the presence of Ca2+, exhibiting fast kinetics and nanomolar affinity (KDSPR7.1nM). The CaM-Ng interaction had higher affinity under Ca2+-depleted (KDSPR480nM,3.4x105M-1s-1 and k(-1) = 1.6 x 10(-1)s(-1)) than Ca2+-saturated conditions (KDSPR19M). The IQ motif of Ng (Ng(27-50)) had similar affinity for CaM as Ng under Ca2+-saturated conditions (KDSPR=14M), but no interaction was seen under Ca2+-depleted conditions. Microscale thermophoresis using fluorescently labeled CaM confirmed the surface plasmon resonance results qualitatively, but estimated lower affinities for the Ng (KDMST890nM) and CaMKII290-309(KDMST190nM) interactions. Although CaMKII290-309 showed expected interaction characteristics, they may be different for full-length CaMKII. The data for full-length Ng, but not Ng(27-50), agree with the current model on Ng regulation of Ca2+/CaM signaling.

Lysine demethylase 1 (LSD1) regulates the degree of methylation of Lys4 of histone 3 in the nucleosome core particle. As LSD1 is overexpressed in certain cancers,  inhibitors have potential for use as drugs. Guided by the structures of two peptidic ligands bound to LSD1 we prepared truncated, mono-substituted and macrocyclic peptides to find leads for development of specific and revserible inhibitors. Surface plasmon resonance biosensor analysis revealed that some stapled, macrocyclic peptides had up to 10-fold higher affinity for LSD1 than the corresponding linear native peptide. Furthermore, peptides cyclized by lactamization were low mM inhibitors of LSD1, with the most effective one being >25-fold more potent than the linear native reference.

SET-and MYND-domain containing protein 3 (SMYD3) is a lysine methyltransferase that plays a role in epigenetic regulation. The protein was shown to have cancerogenic activities and is considered to be a perspective drug target. Here, we propose a Surface Plasmon Resonance-based (SPR) biophysical platform to aid SMYD3 drug discovery. The SPR screening assay was validated with a small subset of drug-like compounds, and resulted in an assay hit. The hit compound-SMYD3 complex structure was solved, and a new allosteric ligand binding site of the protein was revealed. The interaction was found localized within the previously reported SMYD3-heat shock protein 90 (HSP90) recognition site, thereby rendering the hit compound as a perspective candidate for a development of a protein-protein interface inhibitor.