Multiplexed experimental strategies for fragment library screening against challenging drug targets using SPR biosensors

Surface plasmon resonance (SPR) biosensor methods are ideally suited for fragment-based lead discovery. However, generally applicable experimental procedures and detailed protocols are lacking, especially for structurally or physico-chemically challenging targets or when tool compounds are not available. Success depends on accounting for the features of both the target and the chemical library, purposely designing screening experiments for identification and validation of hits with desired specificity and mode-of-action, and availability of orthogonal methods capable of confirming fragment hits. The range of targets and libraries amenable to an SPR biosensor-based approach for identifying hits is considerably expanded by adopting multiplexed strategies, using multiple complementary surfaces or experimental conditions. Here we illustrate principles and multiplexed approaches for using flow-based SPR biosensor systems for screening fragment libraries of different sizes (90 and 1056 compounds) against a selection of challenging targets. It shows strategies for the identification of fragments interacting with 1) large and structurally dynamic targets, represented by acetyl choline binding protein (AChBP), a Cys-loop receptor ligand gated ion channel homologue, 2) targets in multi protein complexes, represented by lysine demethylase 1 and a corepressor (LSD1/CoREST), 3) structurally variable or unstable targets, represented by farnesyl pyrophosphate synthase (FPPS), 4) targets containing intrinsically disordered regions, represented by protein tyrosine phosphatase 1B (PTP1B), and 5) aggregation-prone proteins, represented by an engineered form of human tau (tau K18M). Practical considerations and procedures accounting for the characteristics of the proteins and libraries, and that increase robustness, sensitivity, throughput and versatility are highlighted. The study shows that the challenges for addressing these types of targets is not identification of potentially useful fragments per se, but establishing methods for their validation and evolution into leads

Interaction kinetic analysis in drug design, enzymology and protein research

The work presented here is focused on the phenomenon of molecular recognition – the mutual ability of biological molecules to recognize each other through their chemical signatures. Here, the kinetic aspects of recognition were evaluated, as interaction kinetics reveal valuable dimensions in the description of molecular events in biological systems. The primary objects studied in this thesis were human proteins and their interaction partners. Proteins serve a fundamental role in living organisms, supporting the biochemical machinery by means of catalysis, signalling and transport; additionally, proteins are the main targets for drugs. In the first study, carbonic anhydrase (CA) isozymes were employed as a model system to address the problem of drug selectivity. Kinetic signatures preferable for the design of selective sulphonamide-based inhibitors were identified. In a follow up study, the recognition between CA and sulphonamides was separated into two parts, uncovering intrinsic recognition features that genuinely reflect the interaction mechanism. For the first time, the concept of intrinsic interaction kinetics was applied to a drug-target system. Another model protein studied in this thesis was calmodulin (CaM), as its interactions with other proteins should have specific kinetic signatures to support the dynamics of calcium-dependent signalling. The study evolved around calcium-dependent CaM interactions with the neuronal protein neurogranin (Ng), and revealed its complex nature. Ng was found to interact with CaM both in presence and absence of calcium, but with different kinetics and affinity. This finding supports development of a mechanistic model of calcium sensitivity regulation. The last two projects were more applied, exploring the druggability of an emerging class of pharmaceutical targets – epigenetic enzymes. Expertise and methodology for biophysically guided drug discovery towards histone demethylase LSD1 and histone methyltransferase SMYD3 were developed. For LSD1, the project assisted the rational design of active site-targeting macrocyclic peptides, and resulted in the development of competitive inhibitors with a well described mechanism of action. A novel biophysical platform for screening was developed for SMYD3. It proved to be successful, as it identified previously unknown allosteric ligand binding site. Both projects were supported by structural studies, expanding the druggable space of epigenetic targets

Interaction kinetic analysis in drug design, enzymology and protein research

The work presented here is focused on the phenomenon of molecular recognition – the mutual ability of biological molecules to recognize each other through their chemical signatures. Here, the kinetic aspects of recognition were evaluated, as interaction kinetics reveal valuable dimensions in the description of molecular events in biological systems. The primary objects studied in this thesis were human proteins and their interaction partners. Proteins serve a fundamental role in living organisms, supporting the biochemical machinery by means of catalysis, signalling and transport; additionally, proteins are the main targets for drugs. In the first study, carbonic anhydrase (CA) isozymes were employed as a model system to address the problem of drug selectivity. Kinetic signatures preferable for the design of selective sulphonamide-based inhibitors were identified. In a follow up study, the recognition between CA and sulphonamides was separated into two parts, uncovering intrinsic recognition features that genuinely reflect the interaction mechanism. For the first time, the concept of intrinsic interaction kinetics was applied to a drug-target system. Another model protein studied in this thesis was calmodulin (CaM), as its interactions with other proteins should have specific kinetic signatures to support the dynamics of calcium-dependent signalling. The study evolved around calcium-dependent CaM interactions with the neuronal protein neurogranin (Ng), and revealed its complex nature. Ng was found to interact with CaM both in presence and absence of calcium, but with different kinetics and affinity. This finding supports development of a mechanistic model of calcium sensitivity regulation. The last two projects were more applied, exploring the druggability of an emerging class of pharmaceutical targets – epigenetic enzymes. Expertise and methodology for biophysically guided drug discovery towards histone demethylase LSD1 and histone methyltransferase SMYD3 were developed. For LSD1, the project assisted the rational design of active site-targeting macrocyclic peptides, and resulted in the development of competitive inhibitors with a well described mechanism of action. A novel biophysical platform for screening was developed for SMYD3. It proved to be successful, as it identified previously unknown allosteric ligand binding site. Both projects were supported by structural studies, expanding the druggable space of epigenetic targets

Biophysical analysis of the dynamics of calmodulin interactions with neurogranin and Ca2+/calmodulin-dependent kinase II

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

Introduction of Intrinsic Kinetics of Protein–Ligand Interactions and Their Implications for Drug Design

Structure–kinetic relationship analyses and identification of dominating interactions for optimization of lead compounds should ideally be based on <i>intrinsic</i> rate constants instead of the more easily accessible <i>observed</i> 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 (<i>k</i>_a^obs) were pH-dependent and correlated with the fraction of deprotonated inhibitor and protonated zinc-bound water molecule. The intrinsic association rate constants (<i>k</i>_a^intr) were pH independent and higher than <i>k</i>_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

Kinetically Selective Inhibitors of Human Carbonic Anhydrase Isozymes I, II, VII, IX, XII, and XIII

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., <i>k</i>_d 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

Crystal Structure of Non-Structural Protein 10 from Severe Acute Respiratory Syndrome Coronavirus-2

Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2), causing Coronavirus Disease 19 (COVID-19), emerged at the end of 2019 and quickly spread to cause a global pandemic with severe socio-economic consequences. The early sequencing of its RNA genome revealed its high similarity to SARS, likely to have originated from bats. The SARS-CoV-2 non-structural protein 10 (nsp10) displays high sequence similarity with its SARS homologue, which binds to and stimulates the 3'-to-5' exoribonuclease and the 2'-O-methlytransferase activities of nsps 14 and 16, respectively. Here, we report the biophysical characterization and 1.6 Å resolution structure of the unbound form of nsp10 from SARS-CoV-2 and compare it to the structures of its SARS homologue and the complex-bound form with nsp16 from SARS-CoV-2. The crystal structure and solution behaviour of nsp10 will not only form the basis for understanding the role of SARS-CoV-2 nsp10 as a central player of the viral RNA capping apparatus, but will also serve as a basis for the development of inhibitors of nsp10, interfering with crucial functions of the replication-transcription complex and virus replication

Identification of fragments binding to SARS-CoV-2 nsp10 reveals ligand-binding sites in conserved interfaces between nsp10 and nsp14/nsp16

Since the emergence of SARS-CoV-2 in 2019, Covid-19 has developed into a serious threat to our health, social and economic systems. Although vaccines have been developed in a tour-de-force and are now increasingly available, repurposing of existing drugs has been less successful. There is a clear need to develop new drugs against SARS-CoV-2 that can also be used against future coronavirus infections. Non-structural protein 10 (nsp10) is a conserved stimulator of two enzymes crucial for viral replication, nsp14 and nsp16, exhibiting exoribonuclease and methyltransferase activities. Interfering with RNA proofreading or RNA cap formation represents intervention strategies to inhibit replication. We applied fragment-based screening using nano differential scanning fluorometry and X-ray crystallography to identify ligands targeting SARS-CoV-2 nsp10. We identified four fragments located in two distinct sites: one can be modelled to where it would be located in the nsp14–nsp10 complex interface and the other in the nsp16–nsp10 complex interface. Microscale thermophoresis (MST) experiments were used to quantify fragment affinities for nsp10. Additionally, we showed by MST that the interaction by nsp14 and 10 is weak and thereby that complex formation could be disrupted by small molecules. The fragments will serve as starting points for the development of more potent analogues using fragment growing techniques and structure-based drug design

Macrocyclic Peptides Uncover a Novel Binding Mode for Reversible Inhibitors of LSD1

Lysine-specific demethylase 1 (LSD1) is an epigenetic enzyme which regulates the methylation of Lys4 of histone 3 (H3) and is overexpressed in certain cancers. We used structures of H3 substrate analogues bound to LSD1 to design macrocyclic peptide inhibitors of LSD1. A linear, Lys4 to Met-substituted, 11-mer (4) was identified as the shortest peptide distinctly interacting with LSD1. It was evolved into macrocycle 31, which was &gt;40 fold more potent K-i = 2.3 mu M) than 4. Linear and macrocyclic peptides exhibited unexpected differences in structure-activity relationships for interactions with LSD1, indicating that they bind LSD1 differently. This was confirmed by the crystal structure of 31 in complex with LSD1-CoREST1, which revealed a novel binding mode at the outer rim of the LSD1 active site and without a direct interaction with FAD. NMR spectroscopy of 31 suggests that macrocyclization restricts its solution ensemble to conformations that include the one in the crystalline complex. Our results provide a solid basis for the design of optimized reversible LSD1 inhibitors

Discovery of an allosteric ligand binding site in SMYD3 lysine methyltransferase

SMYD3 is a multifunctional epigenetic enzyme with lysine methyl transferase activity and various interaction partners. It is implicated in the pathophysiology of cancers but with an unclear mechanism. To discover tool compounds for clarifying its biochemistry and potential as a therapeutic target, a set of drug-like compounds was screened using a biosensor-based competition assay. Diperodon was identified as an allosteric ligand. The ( R )-and ( S )-enantiomers of the racemic drug were isolated and their affinities determined ( K D &gt; = 42 and 84 ÎŒM). Co-crystallization revealed that both enantiomers bind to a previously unidentified allosteric site in the C-terminal protein binding domain, consistent with its weak inhibitory effect. No competition between diperodon and HSP90 (a known SMYD3 interaction partner) was observed although HSP90-SMYD3 binding was confirmed ( K D = 13 ÎŒM). The allosteric site appears to be druggable and suitable for exploration of non-catalytic SMYD3 functions and therapeutics with new mechanisms of action