Computer- and NMR-aided design of small-molecule inhibitors of the Hub1 protein

By binding to the spliceosomal protein Snu66, the human ubiquitin-like protein Hub1 is a modulator of the spliceosome performance and facilitates alternative splicing. Small molecules that bind to Hub1 would be of interest to study the protein-protein interaction of Hub1/Snu66, which is linked to several human pathologies, such as hypercholesterolemia, premature aging, neurodegenerative diseases, and cancer. To identify small molecule ligands for Hub1, we used the interface analysis, peptide modeling of the Hub1/Snu66 interaction and the fragment-based NMR screening. Fragment-based NMR screening has not proven sufficient to unambiguously search for fragments that bind to the Hub1 protein. This was because the Snu66 binding pocket of Hub1 is occupied by pH-sensitive residues, making it difficult to distinguish between pH-induced NMR shifts and actual binding events. The NMR analyses were therefore verified experimentally by microscale thermophoresis and by NMR pH titration experiments. Our study found two small peptides that showed binding to Hub1. These peptides are the first small-molecule ligands reported to interact with the Hub1 protein

Oligomeric interfaces as a tool in drug discovery:Specific interference with activity of malate dehydrogenase of Plasmodium falciparum in vitro

Malaria remains a major threat to human health, as strains resistant to current therapeutics are discovered. Efforts in finding new drug targets are hampered by the lack of sufficiently specific tools to provide target validation prior to initiating expensive drug discovery projects. Thus, new approaches that can rapidly enable drug target validation are of significant interest. In this manuscript we present the crystal structure of malate dehydrogenase from Plasmodium falciparum (PfMDH) at 2.4 Å resolution and structure-based mutagenic experiments interfering with the inter-oligomeric interactions of the enzyme. We report decreased thermal stability, significantly decreased specific activity and kinetic parameters of PfMDH mutants upon mutagenic disruption of either oligomeric interface. In contrast, stabilization of one of the interfaces resulted in increased thermal stability, increased substrate/cofactor affinity and hyperactivity of the enzyme towards malate production at sub-millimolar substrate concentrations. Furthermore, the presented data show that our designed PfMDH mutant could be used as specific inhibitor of the wild type PfMDH activity, as mutated PfMDH copies were shown to be able to self-incorporate into the native assembly upon introduction in vitro, yielding deactivated mutant:wild-type species. These data provide an insight into the role of oligomeric assembly in regulation of PfMDH activity and reveal that recombinant mutants could be used as probe tool for specific modification of the wild type PfMDH activity, thus offering the potential to validate its druggability in vivo without recourse to complex genetics or initial tool compounds. Such tool compounds often lack specificity between host or pathogen proteins (or are toxic in in vivo trials) and result in difficulties in assessing cause and effect-particularly in cases when the enzymes of interest possess close homologs within the human host. Furthermore, our oligomeric interference approach could be used in the future in order to assess druggability of other challenging human pathogen drug targets

Oligomeric protein interference validates druggability of aspartate interconversion in Plasmodium falciparum

The appearance of multi-drug resistant strains of malaria poses a major challenge to human health and validated drug targets are urgently required. To define a protein's function in vivo and thereby validate it as a drug target, highly specific tools are required that modify protein function with minimal cross-reactivity. While modern genetic approaches often offer the desired level of target specificity, applying these techniques is frequently challenging-particularly in the most dangerous malaria parasite, Plasmodium falciparum. Our hypothesis is that such challenges can be addressed by incorporating mutant proteins within oligomeric protein complexes of the target organism in vivo. In this manuscript, we provide data to support our hypothesis by demonstrating that recombinant expression of mutant proteins within P. falciparum leverages the native protein oligomeric state to influence protein function in vivo, thereby providing a rapid validation of potential drug targets. Our data show that interference with aspartate metabolism in vivo leads to a significant hindrance in parasite survival and strongly suggest that enzymes integral to aspartate metabolism are promising targets for the discovery of novel antimalarials

Benchmark of Generic Shapes for Macrocycles

Macrocycles target proteins that are otherwise considered undruggable because of a lack of hydrophobic cavities and the presence of extended featureless surfaces. Increasing efforts by computational chemists have developed effective software to overcome the restrictions of torsional and conformational freedom that arise as a consequence of macrocyclization. Moloc is an efficient algorithm, with an emphasis on high interactivity, and has been constantly updated since 1986 by drug designers and crystallographers of the Roche biostructural community. In this work, we have benchmarked the shape-guided algorithm using a dataset of 208 macrocycles, carefully selected on the basis of structural complexity. We have quantified the accuracy, diversity, speed, exhaustiveness, and sampling efficiency in an automated fashion and we compared them with four commercial (Prime, MacroModel, molecular operating environment, and molecular dynamics) and four open-access (experimental-torsion distance geometry with additional "basic knowledge" alone and with Merck molecular force field minimization or universal force field minimization, Cambridge Crystallographic Data Centre conformer generator, and conformator) packages. With three-quarters of the database processed below the threshold of high ring accuracy, Moloc was identified as having the highest sampling efficiency and exhaustiveness without producing thousands of conformations, random ring splitting into two half-loops, and possibility to interactively produce globular or flat conformations with diversity similar to Prime, MacroModel, and molecular dynamics. The algorithm and the Python scripts for full automatization of these parameters are freely available for academic use

A fragment-based approach identifies an allosteric pocket that impacts malate dehydrogenase activity

Malate dehydrogenases (MDHs) sustain tumor growth and carbon metabolism by pathogens including Plasmodium falciparum. However, clinical success of MDH inhibitors is absent, as current small molecule approaches targeting the active site are unselective. The presence of an allosteric binding site at oligomeric interface allows the development of more specific inhibitors. To this end we performed a differential NMR-based screening of 1500 fragments to identify fragments that bind at the oligomeric interface. Subsequent biophysical and biochemical experiments of an identified fragment indicate an allosteric mechanism of 4-(3,4-difluorophenyl) thiazol-2-amine (4DT) inhibition by impacting the formation of the active site loop, located >30 Å from the 4DT binding site. Further characterization of the more tractable homolog 4-phenylthiazol-2-amine (4PA) and 16 other derivatives are also reported. These data pave the way for downstream development of more selective molecules by utilizing the oligomeric interfaces showing higher species sequence divergence than the MDH active site

Fig 6. Western Blot analysis demonstrates the ability of His₆-tagged <i>Pf</i>MDH-V190W mutants to incorporate into pre-formed native Strep-tagged oligomeric assembly post-expression.

<p>Both proteins were recombinantly expressed and the lysate were mixed and sequentially purified via Ni-NTA and Strep-tactin chromatography. (Left) Mixed lysate and wild-type <i>Pf</i>MDH were first purified via Strep-Tactin (IBA Lifesciences) chromatography and subsequently analyzed by Western Blot using α-strep antibodies (See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0195011#sec009" target="_blank">Materials and methods</a>), confirming the presence of Strep-tagged <i>Pf</i>MDH-WT in both samples. (Right) Strep-purified samples were further purified via Ni-NTA chromatography. Western Blot with α-His antibodies showed no signal for the wild type sample (as expected) and confirmed the presence of His₆-tagged V190W mutant in co-purified sample.</p

Oligomeric interfaces as a tool in drug discovery: Specific interference with activity of malate dehydrogenase of <i>Plasmodium falciparum in vitro</i> - Fig 2

<p>Figs 2 a-c show the interfaces formed between individual subunits of <i>Pf</i>MDH: AB (a), AC (b) and AD (c); residues involved in the oligomeric contact are shown in blue. Evolutionary conservation of the interface residues is shown in red (absolutely conserved), orange (strictly conserved) and green (slightly conserved). For more details on sequence conservation please refer to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0195011#pone.0195011.t002" target="_blank">Table 2</a>. (d) Positions of the active sites of adjacent subunits A (yellow) and B (Magenta) are shown. Active sites from A and B subunits are mirror reflections of each other, well separated and distal to AC interface. (e) Structural superposition of <i>Pf</i>MDH AB subassembly (green) and dimeric malate dehydrogenases from <i>E</i>. <i>coli</i> MDH (29% sequence identity, 2.5 Å rmsd, <u><b>2PWZ</b></u>, primary citation unavailable) shown in gold. In order to highlight the active site positions, the NADH and pyruvate molecules were modeled into the active sites using superposition with homologous MDH structure (<u><b>4PLC</b></u>, rmds 1 Å) [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0195011#pone.0195011.ref026" target="_blank">26</a>]. Structure superposition was performed using GESAMT [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0195011#pone.0195011.ref048" target="_blank">48</a>] package from CCP4 suite [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0195011#pone.0195011.ref044" target="_blank">44</a>] and visualized using PyMol [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0195011#pone.0195011.ref049" target="_blank">49</a>].</p

Fig 4 shows examples of TSA melting curves of <i>Pf</i>MDH WT (dark bold lines), <i>Pf</i>MDH-V190W (dotted red lines), <i>Pf</i>MDH-E18W (dotted blue lines) and <i>Pf</i>MDH-E18Q (faint lines) in various buffer conditions: (a) PBS, (b) 400 mM NaCl, (c) 100 mM Na-Citrate pH 5.5 and (d) Assay Buffer (100 mM Na-Phosphate pH 7.4, 400 mM NaCl).

<p>Melting temperatures of each sample are displayed next to the respective curves. Analysis of these curves shows that <i>Pf</i>MDH is rather unstable in PBS (a) and requires optimized buffer conditions for further experiments. This effect is more pronounced for its mutant forms, where native oligomeric assembly has been disrupted (dotted lines). <i>Pf</i>MDH-E18Q mutant shows higher thermal stability, thus supporting the hypothesis that introduction of the additional hydrogen bond pair at the AB interface has had the desired stabilization effect. (b) 400 mM NaCl has significantly stabilized the wild type <i>Pf</i>MDH (ΔT_m = 10 K), dimeric V190W mutant (ΔT_m = 7 K) and tetrameric E18Q mutant (ΔT_m = 8.5 K), while having minor effect of the E18W dimeric mutant (ΔT_m = 2.5 K). (c) 100 mM Na-Citrate pH 5.5 significantly stabilized the wild type enzyme (ΔT_m = 17.5 K) and the E18Q mutant (ΔT_m = 15.5 K), while having lesser effect on V190W mutant (ΔT_m = 6 K) and negligible effect on E18W (ΔT_m = 0.5 K). (d) Selection of the Assay Buffer allowed further experiments to be performed for all four <i>Pf</i>MDH constructs used in this study in the same stabilizing buffer conditions (WT ΔT_m = 13 K, V190W ΔT_m = 7 K, E18W ΔT_m = 5 K, E18Q ΔT_m = 12.5 K). TSA assays were performed in 96-well PCR plates (VWR) using SFX96 Real-Time PCR reactor (BioRad). Melting curve (in terms of increased fluorescence, RFU) of each sample was plotted against the temperature gradient (293–363 K) using BioRad SFX96 software and the temperatures of the inflection points (T_m’s) were used as indicators of the thermal stability of each sample. ΔT_m’s reflect stabilization effect of each condition compared to the control experiments performed in PBS. For more details refer to Materials and Methods section.</p

Primers used in this study.

<p>Primers used in this study.</p