The transcriptional activity of TEAD4 (transcriptional enhancer associated domain proteins), one of the final effectors of the Hippo pathway, can be dysregulated or mutated in cancer. Consequently, targeting the interaction between TEAD and its co-activator YAP (Yes Associated Protein) to disrupt the YAP:TEAD (Y:T) heterodimer has emerged as a promising anti-cancer strategy. Therefore, in this study, we aimed to identify novel scaffolds targeting the TEAD Interface 3 surface as effective anticancer agents against colorectal and ovarian cancer. Employing virtual screening, molecular dynamics, computational ADME-T prediction, and synthetic chemistry, we initially identified novel pyrazolo-piperidinone compounds exhibiting binding affinities (Kd) between 0.6 and 10 μM towards TEAD4 Interface 3 in a fluorescence anisotropy assay, with 6a as the initial lead for a library of 20 compounds. A few of them demonstrated effective and well-characterized cellular antiproliferative activity against HCT116 and A2780 cancer cells. They demonstrated efficacy against colorectal cancer cells resistant to the KRAS-dependent clinical candidate IAG933 and confirmed the ability to inhibit TEAD4 target genes expression. Mass spectrometry-based proteomics of HCT116 cells treated with verteporfin and 6a analogs revealed a proteome modulation consistent with an anti-TEAD mechanism of action, revealing the downregulation of CTGF and TGF-β1. Bioinformatic metabolic pathway analysis further differentiated the mechanism of action of the 6a analogs from verteporfin, a known indirect modulator of the Y:T complex. These findings establish the pyrazolo-piperidinone class as novel Y:T disruptors and provide insights into their metabolic impact on downstream effectors, offering a valuable framework for future hit-to-lead optimization and mechanism of action studies.

Pteridine reductase 1 (PTR1) is a folate pathway enzyme essential for pathogenic trypanosomatids and a promising drug target for diseases such as sleeping sickness and leishmaniasis. Previous studies have shown that the 2-aminobenzothiazole moiety targets the PTR1 biopterin pocket, while 3,4-dichlorophenyl-containing compounds, such as I bind a different region of the Trypanosoma brucei PTR1 (TbPTR1) pocket. This study combines both moieties via various linkers, creating two compound series screened in silico against TbPTR1 and Leishmania major PTR1 (LmPTR1). In the first series, five compounds were synthesized, and 1a and 1b emerged as potent TbPTR1 inhibitors, with 1b also being active against LmPTR1 and moderately effective against Leishmania infantum. Furthermore, structure-activity relationship analysis, supported by quantum calculations and crystallography, revealed meta-halogenation to be more favorable than para, although single halogenation reduced antiparasite effects. Our fragment hybridization approach led to less toxic, more effective compounds than I.

Pteridine reductase 1 (PTR1) is a catalytic protein belonging to the folate metabolic pathway in Trypanosmatidic parasites. PTR1 is a known target for the medicinal chemistry development of antiparasitic agents against Trypanosomiasis and Leishmaniasis. In previous studies, new nitro derivatives were elaborated as PTR1 inhibitors. The compounds showing a diamino-pyrimidine core structure were previously developed but they showed limited efficacy. Therefore, a new class of phenyl-, heteroaryl- and benzyloxy-nitro derivatives based on the 2-nitroethyl-2,4,6-triaminopyrimidine scaffold were designed and tested. The compounds were assayed for their ability to inhibit T. brucei and L. major PTR1 enzymes and for their antiparasitic activity towards T. brucei and L. infantum parasites. To understand the structure-activity relationships of the compounds against TbPTR1, the X-ray crystallographic structure of the 2,4,6-triaminopyrimidine (TAP) was obtained and molecular modelling studies were performed. As a next step, only the most effective compounds against T. brucei were then tested against the amastigote cellular stage of T. cruzi, searching for a broad-spectrum antiprotozoal agent. An early ADME-Tox profile evaluation was performed. The early toxicity profile of this class of compounds was investigated by measuring their inhibition of hERG and five cytochrome P450 isoforms (CYP1A2, CYP2C9, CYP2C19, CYP2D6 and CYP3A4), cytotoxicity towards A549 cells and mitochondrial toxicity. Pharmacokinetic studies (SNAP-PK) were performed on selected compounds using hydroxypropyl-β-cyclodextrins (50 % w/v) to preliminarily study their plasma concentration when administered per os at a dose of 20 mg/kg. Compound 1p, showed the best pharmacodynamic and pharmacokinetic properties, can be considered a good candidate for further bioavailability and efficacy studies.

Folate enzymes, namely, dihydrofolate reductase (DHFR) and pteridine reductase (PTR1) are acknowledged targets for the development of antiparasitic agents against Trypanosomiasis and Leishmaniasis. Based on the amino dihydrotriazine motif of the drug Cycloguanil (Cyc), a known inhibitor of both folate enzymes, we have identified two novel series of inhibitors, the 2-amino triazino benzimidazoles (1) and 2-guanidino benzimidazoles (2), as their open ring analogues. Enzymatic screening was carried out against PTR1, DHFR, and thymidylate synthase (TS). The crystal structures of TbDHFR and TbPTR1 in complex with selected compounds experienced in both cases a substrate-like binding mode and allowed the rationalization of the main chemical features supporting the inhibitor ability to target folate enzymes. Biological evaluation of both series was performed against T. brucei and L. infantum and the toxicity against THP-1 human macrophages. Notably, the 5,6-dimethyl-2-guanidinobenzimidazole 2g resulted to be the most potent (K-i = 9 nM) and highly selective TbDHFR inhibitor, 6000-fold over TbPTR1 and 394-fold over hDHFR. The 5,6-dimethyl tricyclic analogue 1g, despite showing a lower potency and selectivity profile than 2g, shared a comparable antiparasitic activity against T. brucei in the low micromolar domain. The dichloro-substituted 2-guanidino benzimidazoles 2c and 2d revealed their potent and broad-spectrum antitrypanosomatid activity affecting the growth of T. brucei and L. infantum parasites. Therefore, both chemotypes could represent promising templates that could be valorized for further drug development.

Glutaminyl-peptide cyclotransferases (QCs) convert the N-terminal glutamine or glutamate residues of protein and peptide substrates into pyroglutamate (pE) by releasing ammonia or a water molecule. The N-terminal pE modification protects peptides/proteins against proteolytic degradation by amino- or exopeptidases, increasing their stability. Mammalian QC is abundant in the brain and a large amount of evidence indicates that pE peptides are involved in the onset of neural human pathologies such as Alzheimer’s and Huntington’s disease and synucleinopathies. Hence, human QC (hQC) has become an intensively studied target for drug development against these diseases. Soon after its characterization, hQC was identified as a Zn-dependent enzyme, but a partial restoration of the enzyme activity in the presence of the Co(II) ion was also reported, suggesting a possible role of this metal ion in catalysis. The present work aims to investigate the structure of demetallated hQC and of the reconstituted enzyme with Zn(II) and Co(II) and their behavior in the presence of known inhibitors. Furthermore, our structural determinations provide a possible explanation for the presence of the mononuclear metal binding site of hQC, despite the presence of the same conserved metal binding motifs present in distantly related dinuclear aminopeptidase enzymes.

α-Synuclein (αSyn) constitutes the main protein component of Lewy bodies, which are the pathologic hallmark in Parkinson's disease. αSyn is unstructured in solution but the interaction of αSyn with lipid membrane modulates its conformation by inducing an α-helical structure of the N-terminal region. In addition, the interaction with metal ions can trigger αSyn conformation upon binding and/or through the metal-promoted generation of reactive oxygen species which lead to a cascade of structural alterations. For these reasons, the ternary interaction between αSyn, copper, and membranes needs to be elucidated in detail. Here, we investigated the structural properties of copper-αSyn binding through NMR, EPR, and XAS analyses, with particular emphasis on copper(I) coordination since the reduced state is particularly relevant for oxygen activation chemistry. The analysis was performed in different membrane model systems, such as micellar sodium dodecyl sulfate (SDS) and unilamellar vesicles, comparing the binding of full-length αSyn and N-terminal peptide fragments. The presence of membrane-like environments induced the formation of a copper:αSyn = 1:2 complex where Cu+ was bound to the Met1 and Met5 residues of two helical peptide chains. In this coordination, Cu+ is stabilized and is unreactive in the presence of O2 in catechol substrate oxidation.

Article Figures and data Abstract Editor's evaluation Introduction Results Discussion Materials and methods Appendix 1 Appendix 2 Appendix 3 Appendix 4 Data availability References Decision letter Author response Article and author information Metrics Abstract Drugs that target human thymidylate synthase (hTS), a dimeric enzyme, are widely used in anticancer therapy. However, treatment with classical substrate-site-directed TS inhibitors induces over-expression of this protein and development of drug resistance. We thus pursued an alternative strategy that led us to the discovery of TS-dimer destabilizers. These compounds bind at the monomer-monomer interface and shift the dimerization equilibrium of both the recombinant and the intracellular protein toward the inactive monomers. A structural, spectroscopic, and kinetic investigation has provided evidence and quantitative information on the effects of the interaction of these small molecules with hTS. Focusing on the best among them, E7, we have shown that it inhibits hTS in cancer cells and accelerates its proteasomal degradation, thus causing a decrease in the enzyme intracellular level. E7 also showed a superior anticancer profile to fluorouracil in a mouse model of human pancreatic and ovarian cancer. Thus, over sixty years after the discovery of the first TS prodrug inhibitor, fluorouracil, E7 breaks the link between TS inhibition and enhanced expression in response, providing a strategy to fight drug-resistant cancers. Editor's evaluation Drugs that therapeutically target human thymidylate synthase are widely used to treat cancer. In this manuscript, the authors have discovered and validated an alternative strategy to target this enzyme using dimer destabilizers. By disrupting homodimerization of these proteins using a series of biophysical assays, this research team identified an analog E7 that inhibits hTS in cancer cells and demonstrates a superior profile to approved drugs targeting this pathway. The work is innovative and novel and could lead to the development of new classes of drugs targeting this key enzyme for cancer therapy. https://doi.org/10.7554/eLife.73862.sa0 Decision letter eLife's review process Introduction Small molecules able to bind at specific pockets of the monomer/monomer interface of a dimeric protein may perturb the dimeric assembly to the limit of disrupting it, and thus alter metabolic pathways associated with the cellular functions of the monomeric and dimeric protein forms (Taddia et al., 2015). Such a drastic structural change of the protein may open the way to unexpected events, such as a higher liability to intracellular degradation of the protein monomers relative to the dimers. Human thymidylate synthase (hTS) is an obligate, stable homodimer (Kd = 80 nM Genovese et al., 2010) with two active sites, each including residues from both monomers (Costi et al., 2005). As a dimeric enzyme, it provides the sole de novo pathway to deoxythymidylate (dTMP) synthesis in human cells by catalyzing the reductive methylation of deoxyuridylate (dUMP) to dTMP using methylenetetrahydrofolate (mTHF) as the one-carbon methyl donor (Costi et al., 2005; Carreras and Santi, 1995). By interacting with its own and other mRNAs, this protein regulates its own levels and those of other proteins involved in apoptotic processes, including bcl2, c-myc, and p53 (Brunn et al., 2014; Jennings and Willis, 2015). Its inhibition is usually achieved with compounds that bind at the protein active-site, competing either with the dUMP substrate, such as 5-fluorodeoxyurdine 5’-monophosphate (FdUMP), or with the folate cofactor, such as raltitrexed (RTX) and pemetrexed (PMX) (Carreras and Santi, 1995; Jennings and Willis, 2015; Figure 1A and B). However, these drugs induce cells to develop drug resistance associated with increased hTS levels which eventually leads to therapy failure (Peters, 2018). A drastic change of strategy, based on the design of new compounds with different mechanisms of action, is thus necessary (Voeller et al., 2002; Cardinale et al., 2011). Here we report the discovery of molecules that bind at the hTS monomer/monomer interface and, despite their small size, markedly shift the monomer-dimer equilibrium of the enzyme towards the inactive monomeric form. Acting as destabilizers of the hTS dimer, they not only inhibit the activity of this obligate homodimeric enzyme but also favor its intracellular degradation and, hence, decrease its level. Figure 1 with 1 supplement see all Download asset Open asset Drug target sites on hTS: the active-site (A,B) and the Y202 pocket (C–F). Drugs that are directed to the active site of hTS can mimic either the cofactor MTHF or the substrate dUMP and bind in their binding pockets. (A) hTS (in cartoon with the A and B subunits colored white and grey, respectively) inhibited by the cofactor analogue pemetrexed (PMX, in sticks, purple carbons), which occupies the cofactor binding pocket by establishing a π-π interaction with the dUMP substrate (in sticks, green carbons) (PDB ID: 1JU6). The substrate and cofactor binding sites are represented as green and orange surfaces, respectively. The catalytic cysteine, C195, is highlighted in sticks. (B) The substrate analogue 5-fluoro-2’-deoxyuridine monophosphate (FdUMP) (in sticks, purple carbons) binds inside the catalytic cavity, mainly filling the substrate binding pocket (PDB ID: 3H9K, hTS variant R163K). (C) Location of F59, Y202 and CME202 residues at the hTS dimer interface by superimposition of native hTS and hTS-C195S-Y202C double mutant structures (CME: S,S(2-hydroxyethyl)cysteine; PDB-ID: 3N5G, 4O1X). (D) Surface of the Y202 pocket (cyan) filled by F59’ from the opposite monomer (cyan sticks). The surface of Y202 is highlighted in green (PDB-ID: 3N5G). (E) Surface of the C202 pocket (cyan) filled by F59’ from the opposite monomer (cyan sticks) in the C195S-Y202C double mutant (PDB-ID: 4O1X). The surface of C202 (oxidized as CME) is highlighted in green. (F) Superimposition of the hTS-C195S-Y202C and hTS-Y202C crystal structures with the catalytic loop in hTS-Y202C (inactive conformation, in orange) and in hTS-C195S-Y202C (active conformation, in violet); notice the different orientations of the catalytic residue couples S195/SCH195 and S195’/SCH195’ (PDB-ID: 4O1X, 4O1U) (SCH: S-methyl-thio-cysteine). The one-letter code is used for the standard amino-acids, while for the chemically modified amino-acid, a conventional three-letter code is used as described above. Using a tethering approach in which sulfhydryl-containing fragments were initially identified by reaction with a cysteine residue inserted by mutation around the target site, we identified fragments anchored by disulfide bond formation at the interfacial mutant cysteine residue. We then employed molecular modeling and medicinal chemistry to modify the fragments and develop inhibitors able to bind WT hTS guided solely by their affinities for the interface region. For these small molecules, we propose a model able to quantitatively account both for the dissociation of the hTS dimer, observed spectroscopically, and for the corresponding activity inhibition. We have also shown that their ability to induce the TS-dimer dissociation in cells lies below their efficacy in inhibiting growth of colon, ovarian and pancreatic cancer cells. One of our hTS-dimer destabilizers, compound E7, induced apoptosis in cancer cells and a decrease of hTS levels due to enhanced proteasomal degradation of the enzyme monomers with respect to the dimers. Remarkably, in a mouse model of orthotopic pancreatic cancer, this dimer disrupter caused a higher reduction of cancer growth and had lower toxicity than 5-fluorouracil (5-FU), the prodrug of 5-FdUMP. Like what we observed in vitro, the molecular analysis of exported treated cancer tissues demonstrated decreased hTS protein and mRNA levels. These findings establish E7 as a new lead with an hTS dimer-disruptive ability and link the dimer-to-monomer equilibrium shift of this protein to its faster intracellular degradation. Results Choice of the ligand binding-site on the target protein surface To target the monomer-monomer interface and dissociate the hTS dimer, we applied the ‘cysteine tethering’ approach to fragment-based drug design (Erlanson et al., 2000). We investigated the crystal structures of the active and inactive hTS (PDB-IDs 1HVY and 1YPV, respectively) and carried out molecular dynamics (MD) simulations of the monomeric and dimeric forms of the enzyme to explore the monomer-monomer interface of homodimeric hTS and identify targetable binding pockets and residues suitable to be mutated to cysteine for the tethering experiments (Supplementary file 1). We selected the ‘Y202 pocket’, which accommodates the side chain of F59’ from the other monomer, as the most promising region to target (Figure 1C–F, Figure 2A–B, Figure 1—figure supplement 1; Salo-Ahen et al., 2015). Figure 2 with 1 supplement see all Download asset Open asset Disulfide library design and screening on the hTS C195S-Y202C variant. (A) Crystal structure of the hTS C195S-Y202C dimer (in the cartoon, the A and B subunits are colored pale blue and grey, respectively; the catalytic loops, in active conformation, are highlighted in cyan and pink, respectively) showing the positions of the cysteine residues (in sticks, carbon atoms are colored cyan and pink in subunits A and B, respectively). Compounds A5, A10, A20 and the reference compound N-tosyl-D-prolinecysteamine (TPC) (Appendix 2—figure 1) were selected through the tethering experiments. They are indicated close to the peptide sequence found in the mass spectrometry identification process (Supplementary file 3B and C). Their chemical structures are reported in Supplementary file 3A. (B) Sequence of the hTS C195S-Y202C variant and cysteine-containing peptides generated by trypsin digestion. Cysteine residues are displayed in red (the residue number is reported below each cysteine) and the mutated S195 (the catalytic residue) in blue. Three cysteine-containing peptides were generated by proteolytic cleavage of the hTS C195S-Y202C variant: peptide I177-R185 and peptide R176-R185 containing C180 and peptide D186-R215, containing C199, C202, and C210. Ligand (Lig)-bound cysteines and those alkylated as carbamidomethyl (Cam)-cysteines are displayed. (C) Screening cascade from the 6 million compounds from the ZINC database (Irwin and Shoichet, 2005) to the final four fragments expected to bind in the Y202 pocket. (D, E) Representative MS spectra in the hTS-binding evaluation step. Maldi (D) and ESI (E) MS spectra for the reference compound TPC (Erlanson et al., 2000). The three labeled peaks are due to hTS C195S-Y202C and to the protein bound to BME and one and two molecules of TPC. This pocket lies in the perimeter space of the intermonomer interface and is accessible in the dimeric form of the protein. It is large enough (22 Å3) to accommodate a phenyl group and has several smaller crevices around it. During the MD simulations of monomeric hTS, the side chain of Y202 moved away leaving a larger pocket and behaving like a gate. We therefore made the Y202C mutant and screened for compounds able to form a covalent disulfide bond with C202 from a library of organic disulfides. We also mutated the active site cysteine to serine to prevent the selection of compounds that bound at the active site (C195S). We made mutants with single point mutations at C195S and Y202C to test their functional and structural properties (Supplementary file 1, Figure 1—figure supplement 1) as well as the double mutant, hTS C195S-Y202C. This was used for the ligand selection according to the above-described tethering approach. As expected from removal of the catalytic C195 residue, this double mutant was inactive, but X-ray crystallography showed that it takes the active conformation of hTS (Figure 1F, Figure 2—figure supplement 1, Supplementary file 2A and B). We employed the C195S-Y202C double mutant to capture compounds that, driven by some affinity for the region near residue 202, formed a covalent disulfide bond with C202 (Supplementary file 3A). These compounds were selected by their stability towards reduction by β-mercaptoethanol (BME), and the C202-S-S-small-compound disulfides formed were identified by two mass spectrometric (MS) experiments, MALDI-TOF and ESI-QTOF (Supplementary file 3B and C). Mass-spectrometric ligand selection To identify molecules able to covalently bind near Y202, we designed a library of commercially available compounds from the ZINC database (Irwin and Shoichet, 2005; Figure 2C). We first selected 1066 disulfide compounds that were either symmetric (RSSR) or asymmetric (R1SSR2) and had drug-like or fragment-like properties. 1066 molecules were screened for their molecular properties (PCA, MW, logP, size) and 55 were evaluated with the MS techniques. Thirteen out of the 55 compounds were found to bind the protein according to the results of both MS techniques (Figure 2D and E, Appendix 1, Supplementary file 3A, B and C). Among them, seven compounds, A5, A6, A10, A15, A20, A38, and TPC, were selected based on the feasibility of their chemical modification and were further analyzed by a bottom-up MALDI-TOF and ESI-QTOF MS proteomic analysis to identify the cysteine residues they had bound (Supplementary file 3C). The 7 compounds bound either peptides (I177-R185) IIMCAWNPR (sequence A) or (R176-R185) RIIMCAWNPR (sequence B), both containing the C180 residue of the digested protein (Supplementary file 3C). Four molecules, A5, A10, A20, and TPC also bound peptide (D186-R215) DLPLMALPPSHALCQFCVVNSELSCQLYQR (sequence C) (Supplementary file 3C), containing the engineered Y202C together with the native C199 and C210. In agreement with the described computational and crystallographic data (see above), Y202C is the most exposed of these three cysteines and is thus easily available for disulfide-bond formation with the RS fragments, being therefore suitable for tethering experiments. Other reasons to select the Y202 pocket are that Y202 is not predicted to be part of the mRNA binding region and has the capability to interact with various functional groups. Hence, despite the difficulties in the identification of the precise cysteine to which the fragments could bind, all the above arguments support the choice of the Y202 pocket at the hTS interface as a suitable binding site for further structure-based studies. Therefore, we considered the fragments A5, A10, A20, and TPC for further medicinal chemistry modifications (Figure 3A). Figure 3 with 2 supplements see all Download asset Open asset From hits to dimer-destabilizing compounds. (A) Structures of the compounds identified in MS studies (in red the fragments used for the virtual screening approach to the design of the compound library without the disulfide bonds). (B) Virtual screening cascade and chemical structures of the identified compounds, B12 and B26. (C) Detail of the binding mode of compound C3 (orange sticks) with the hTS interface residues R64, Y202 and R175 (positive and negative potential regions are colored in blue and red, respectively) obtained with docking methods. (D) Stereo view of the superimposition of the calculated binding mode of C3 onto the first monomer (white cartoon) with the opposite monomer overlapping (lime cartoon). Atom color code: nitrogen – blue; oxygen – red; sulphur – yellow. (E) Results of anion exchange chromathography showing co-elution experiments of C3 with hTS dimer and monomer through anion exchange chromatography. Absorbances at 280 (blue) and 350 nm (red) of chromatographic fractions obtained by loading a 50 μM hTS and 4 μM C3 solution on an anion-exchange AEX HiPrep Q HP 16/10 mm column. (Figure 3—figure supplement 1, Figure 3—figure supplement 1—source data 1). From low-affinity compounds to TS homodimer destabilizers Starting from the four fragments identified by tethering/MS (TPC, A5, A10, A20) (Figure 3A), we performed an analogue search that yielded a dataset of 331,600 commercially available compounds (Specs dataset, https://www.specs.net) potentially able to bind non-covalently at the Y202 pocket of the monomer/monomer interface. The initial dataset was progressively reduced to 5,774 candidates using MW, structural and chemical criteria (Figure 3B). These were then docked into the Y202 pocket with the software FLAP (Cross et al., 2010) using a receptor-based pharmacophore model built from the X-ray crystal structure of hTS (PDB: 1HVY). A final set of 26 molecules (B1-B26, Supplementary file 4A) was selected to be tested for their ability to inhibit recombinant hTS and destabilize its dimeric form using, respectively, a kinetic and a spectroscopic, FRET (Förster resonance energy transfer)-based assays (Ponterini et al., 2016). For the FRET-based assay (Genovese et al., 2010), we conjugate one protein monomer with red-emitting tetramethylrhodamine (T) and the other with green-emitting fluorescein (F). Only when the monomers are close to each other, that is, combined to form a dimer, F-to-T excitation energy transfer occurs, a FRET signal is detected, and its efficiency determined. We can thus investigate the dimer/monomer equilibrium of hTS, characterize it by the corresponding dissociation equilibrium constant and evaluate the effect of a tested compound thereupon. Among the 26 compounds selected by VS, B12 and B26 (Figure 3B, Supplementary file 4A), caused low but reproducible decreases in the efficiency of FRET (ΔΦFRET = -0.13 and ΔΦFRET = -0.02, respectively), thus indicating some ability to perturb the enzyme dimeric assembly. In the docked poses, interactions were established by B12 with Y202 through the o-chloro-phenyl ring and with R175 through the p-nitro-pyridyl ring, and by B26 with Y202 through the piperidin-1-yl ring and with R175 through the quinoline ring (Figure 3C and D). Next, we designed and synthesized four sets of compounds (C-F) (Supplementary file 4) to develop B26 into biologically active compounds that exhibited the ability to both disrupt the dimer and inhibit recombinant hTS (Appendix 2—figures 2–7). The most relevant compounds were C2, C3, C4, E5, E6, and E7 (Supplementary file 4B-E). The former three, differing only for the position of the carboxylic group on the phenyl ring, featured IC50 values 5.25, 83 and 246 μM vs. 300 nM hTS (Supplementary file 4) and were selected for a quantitative mechanistic analysis of FRET and inhibition data with recombinant hTS (see the next paragraph). It was however compounds E5, E6, and E7 that proved to be the most interesting to continue our study. They featured IC50 values against 300 nM recombinant hTS of 40, 10 and 7 μM respectively, and decreased FRET efficiency by –0.24 at 50 μM (E5, E6) and –0.1 at 10 μM (E7, Supplementary file 4). Compound E7, that was the most active on cells, was determined to be a racemic mixture of two enantiomers that equilibrated with a half-life of 56 min (Appendix 3, Appendix 3—figure 1, Appendix 3—figure 2). Additional evidence of the ability of compounds C3 and E5 to bind hTS and destabilize its dimers was provided by anion-exchange (AE) chromatographic experiments. Two bands, separated by ca. 2 min, both corresponding to fractions characterized by the absorption spectrum of hTS, were found in the AE chromatogram based on absorbance at 280 nm (Figure 3—figure supplement 1A). The relative area of the second band was only about 3.5% that of the first one and decreased further (Figure 3—figure supplement 1B) when the protein was loaded together with dUMP and Raltitrexed, both at 250 μM, a concentration more than one order of magnitude larger than their dissociation constants from hTS. Because binding of hTS to its nucleotidic substrate stabilizes the active, dimeric form of the enzyme, and consistently with the expectations based on protein size and the correlated elution times, we attribute the two bands to the hTS dimers the first one, and monomers the second. In keeping with this assignment, when hTS was loaded on the AE column after a 1 hr incubation with compound C3 at three concentrations, namely 4, 40, and 80 μM, the monomer band was delayed by additional 3–4 min relative to the dimer band in the chromatograms (Figure 3—figure supplement 1A and Figure 3—figure supplement 1C–E) and its relative area increased markedly, from 0.035 to 0.08, to 0.24 and to 1.09, respectively. Co-elution, hence stable non covalent binding of the C3 inhibitor with hTS, was established by the observation that the fractions containing the hTS dimers and monomers also exhibited absorption at 350 nm (Figure 3E), a wavelength at which no absorption comes from the protein. Accumulation of compound C3 in these fractions was confirmed by identifying and quantifying it in the AE chromatographic fractions by high-resolution mass spectrometry (Supplementary file 5, Figure 3—figure supplement 2). Absorbances at 350 nm in Figure 3E indicated similar concentrations of the compound in the fractions corresponding to the protein dimers and monomers, in spite of the fact that the 280 nm dimer chromatographic band was much more intense than the monomer band. This finding suggests a somewhat larger affinity of the ligand for the latter. Consistently, parallel MS experiments in which 4 μM E5 was loaded on the AE column with hTS identified this ligand mainly in the fractions containing the protein monomers (see additional information in the Supplementary file 5). So, overall, the results synthesized here show that these small compounds co-elute with hTS, thus proving beyond doubt that they remain bound to it, possibly to the monomer more tightly than to the dimer, throughout the AE chromatographic runs. Also, they qualitatively show the ability of these ligands to de-stabilize the hTS dimers in favor of the monomers. Mechanistic analysis of dissociative inhibition The nitrothiophenyl derivative, C3 and its isomers, C2 and C4 (Supplementary file 4B), are structurally similar to compound E7 (Supplementary file 4D) and are much more soluble in water. So, they were used for quantitatively investigating the molecular mechanistic basis of their ability to both inhibit recombinant hTS and disrupt its dimer. The three isomeric compounds showed different inhibitory potencies against 300 nM hTS, with IC50 values of 5.25, 83 and 246 μM, respectively. Consistently, C3 caused the largest decrease in FRET efficiency (ΔΦFRET) at concentrations lower than 50 μM (Figure 4A, Figure 4B). Figure 4 Download asset Open asset Spectroscopic and mechanistic analysis of hTS dissociative inhibition for compounds C2, C3 and C4. (A) Emission spectra of fluorescein(F)- and tetramethylrhodamine (T)-labelled hTS (λexc = 450 nm): the decreasing emission contribution of T relative to F with increasing concentrations of C3 (0, 4.5, 9.5, 15, 25, 100 μM) parallels the decrease in the hTS dimer mole fraction. (B) Dependence of the observed FRET efficiency on the concentrations of three inhibitors (C3, black; C4, blue; C2, red); the hTS dimer concentration was 100 nM. The lines represent the best fittings of the experimental results to Equation S3 in the Appendix 4. The most significant fitting parameters are reported. (C) Cartoon, and standard chemical representations of the dissociative inhibition mechanism of hTS. Blue shapes represent enzyme monomers (M) in the active conformation bound to dUMP; red dots indicate a dissociative inhibitor (I), yellow hexagons, the folate substrate (S), green octagons, the product. (D) Dependence of FRET efficiency on the concentration of C3 with total protein concentrations (ET) of 490 nM (blue circles), 250 nM (green circles), 100 nM (red circles) and 50 nM (black circles). The horizontal segments represent the values of the efficiency in the limit of high inhibitor concentration. At ET = 50 nM and IT = 5 μM the vertical arrow indicates the evolution of the measured value of the FRET efficiency 5, 7, and 12 minu after inhibitor addition (grey and black circles). (E) Analysis of the hTS monomer-dimer equilibrium according to eq. ΦFRET = 1–0.5(ΦFRET/ET)1/2K1/2 (Genovese et al., 2010) and corresponding equilibrium constants without C3 (white circles, K=KD) and at saturating C3 concentrations (black circles, K=K’’D, ΦFRET = FRET efficiency). (F) Dependence of the initial reaction rate (v) on mTHF concentration (free substrate, [S], and total, ST) at four different C3 concentrations (from top to bottom, [I]=0, 12, 24, and 36 μM). The fitting curves represent equation v = xr2STD derived in the Appendix 4 and computed with ET (total enzyme dimer concentration)=300 nM, k=k’=0.9 s–1, k’’=0.1 s–1, K1=K2=10–5 M, K3=2 × 10–5 M, KI = 10–6 M, KI’ = KI’’=10–5 M,x = [M2]1/2 and using for xr the only acceptable root of the quadratic equation in panel 4 F (Figure 4—source data 2). Figure 4—source data 1 Spectroscopic and mechanistic analysis of hTS dissociative inhibition for compounds C2, C3 and C4. https://cdn.elifesciences.org/articles/73862/elife-73862-fig4-data1-v2.xlsx Download elife-73862-fig4-data1-v2.xlsx Figure 4—source data 2 Inhibition of hTS by compound C3. https://cdn.elifesciences.org/articles/73862/elife-73862-fig4-data2-v2.xlsx Download elife-73862-fig4-data2-v2.xlsx The dependence of ΦFRET, that in our conditions is a direct measure of the mole fraction of hTS dimers, on each inhibitor concentration (Figure 4B) was fitted according to the dimer-monomer equilibrium model sketched in Figure 4C. This was analyzed as reported in the Appendix 4. We searched for evidence of the disruptive character of compound C3 by investigating the dependence of its dose-effect curves on total protein concentration, ET (Figure 4D). As expected for a dissociative inhibition mechanism, the limit values of ΦFRET at high C3 concentrations were consistent with a 5-fold increase in the equilibrium dissociation constant for the protein bound to C3 with respect to the free protein (Figure 4E). From the common limit values of ΦFRET at high concentrations (Figure 4B), we conclude that, when saturating hTS, compounds C2-C4 have similar effects on the dimer stability. The fivefold increase in the protein-dimer dissociation equilibrium constant and the corresponding decrease in ΔG° for dimer disruption, from 40.5 to about 36.5 kJ mol–1, ΔΔG° = –4 kJ mol–1, are evidence of the dimer-destabilizing ability of compounds C2-C4. On the other hand, their different abilities to decrease the FRET efficiency at lower concentrations can be attributed to different affinities for the protein dimers, with KI’’s of 2.8x10–5, 1x10–5 and 2x10–5 M, for C2, C3, and C4, respectively. Thus, the observed overall effect of such an inhibitor results from a combination of affinity for the protein and ability to destabilize its dimeric assembly by impairing crucial attractive inter-monomer interactions. Binding of compounds C2-C4 at the Y202 pocket of the hTS monomer/monomer interface is supported by computational docking (Figure 3C and D). In the hTS dimer, the aromatic ring of the Y202 residue forms a π-π interaction with the phenyl ring of the F59’ residue from the opposite subunit. Such a ring is replaced by the phenyl ring of the benzoic acid portions of C2-C4 in their modeled complexes with hTS. Thus, these compounds mimic some of the residue side chains from the opposite monomer in the dimer, providing similar interactions in this region. This finding is supported by the higher affinities of the inhibitors for the hTS monomer (KI = 10–6 M) than for the dimer (KI’=1.7 x 10–5 M), as obtained by fitting of the FRET data in Figure 4B. The reliability of our docking virtual experiments was strengthened by their ability to explain the observed higher affinity for hTS of compound C3 relative to its isomers, C2 and C4. While the nitro-groups of all these ligands can make head-to-head hydrogen-bonding interactions with R64 (Figure 3C–D), only the para carboxylic group of compound C3 arranges properly to establish an additional H-bond with R175. Instead, the meta and ortho carboxylic groups of C2 and C4 do not have the correct orientations for interacting with this arginine residue. As for the mechanism of inhibition of the enzymatic activity of hTS by compounds C2-C4, we show that the scheme in Figure 4C can account for the dependence of the initial reaction rate (v) on the substrate concentration at different inhibitor concentrations with very reasonable fitting parameters (Figure 4F). Simple observation of the dependence of v on [C3] with total enzyme concentration (ET) 300 nM (Figure 4F) indicates a regular decrease with increasing [C3] to a limiting rate that is about 1/5 the rate in the absence of inhibitor. On the other hand, the ET-dependent limiting value of ΦFRET was about half the full one (Figure 4B). This difference in the limit behaviors of v and ΦFRET suggests that, when bound to the inhibitor, the dimeric enzyme is less catalytically active than when free. Or, in terms of the dissociative/noncompetitive model in Figure 4C, K2 and K3 are larger than K1, or k’ and/or k’’ are smaller than k. The curves that go through the kinetic data in Figure 4F are plots of eq. v = xr2STD that was obtained by fast-equilibrium solution of the scheme in Figure 4C (for the derivation of the equation and the meanings of the symbols therein, see the Appendix 4). Remarkably, the fitting parameters, provided in the caption, are consistent with the results of the FRET-based analysis of the equilibria in Figure 4C and the limit behaviors of the initial rate and of ΦFRET. More in detail: (i) k’’ is much smaller than k and k’, that is, M2I2 is almost catalytically inactive, a feature

The optimization of compounds with multiple targets in the drug discovery cycle is a difficult multidimensional problem. Here, we present a systematic, multidisciplinary approach to the development of selective anti-parasitic compounds. Efficient microwave-assisted synthesis of pteridines along with iterations of crystallographic structure determination were used to validate computational docking predictions and support derivation of a structure-activity relationship for multitarget inhibition. This approach yielded compounds showing picomolar inhibition of T. brucei pteridine reductase 1 (PTR1), nanomolar inhibition of L. major PTR1, along with selective submicromolar inhibition of parasitic dihydrofolate reductase (DHFR). Moreover, by combining design for polypharmacology with a property-based on-parasite optimization, we found three compounds that exhibited micromolar EC 50 values against T. brucei brucei , whilst retaining their target inhibition. Our results provide a basis for the further development of pteridine-based compounds and we expect our multitarget approach to be generally applicable to the design and optimization of anti-infective agents.

Metallo-β-lactamases (MBLs) contribute to the resistance of Gram-negative bacteria to carbapenems, last-resort antibiotics at hospital, and MBL inhibitors are urgently needed to preserve these important antibacterial drugs. Here, we describe a series of 1,2,4-triazole-3-thione-based inhibitors displaying an α-amino acid substituent, which amine was mono- or disubstituted by (hetero)aryl groups. Compounds disubstituted by certain nitrogen-containing heterocycles showed submicromolar activities against VIM-type enzymes and strong NDM-1 inhibition (Ki = 10-30 nM). Equilibrium dialysis, native mass spectrometry, isothermal calorimetry (ITC), and X-ray crystallography showed that the compounds inhibited both VIM-2 and NDM-1 at least partially by stripping the catalytic zinc ions. These inhibitors also displayed a very potent synergistic activity with meropenem (16- to 1000-fold minimum inhibitory concentration (MIC) reduction) against VIM-type- and NDM-1-producing ultraresistant clinical isolates, including Enterobacterales and Pseudomonas aeruginosa. Furthermore, selected compounds exhibited no or moderate toxicity toward HeLa cells, favorable absorption, distribution, metabolism, excretion (ADME) properties, and no or modest inhibition of several mammalian metalloenzymes.

Metallo‐β‐lactamases (MBLs) are increasingly involved as a major mechanism of resistance to carbapenems in relevant opportunistic Gram‐negative pathogens. Unfortunately, clinically efficient MBL inhibitors still represent an unmet medical need. We previously reported several series of compounds based on the 1,2,4‐triazole‐3‐thione scaffold. In particular, Schiff bases formed between diversely 5‐substituted‐4‐amino compounds and 2‐carboxybenzaldehyde were broad‐spectrum inhibitors of VIM‐type, NDM‐1 and IMP‐1 MBLs. Unfortunately, these compounds were unable to restore antibiotic susceptibility of MBL‐producing bacteria, probably because of poor penetration and/or susceptibility to hydrolysis. To improve their microbiological activity, we synthesized and characterized compounds where the hydrazone‐like bond of the Schiff base analogues was replaced by a stable ethyl link. This small change resulted in a narrower inhibition spectrum, as all compounds were poorly or not inhibiting NDM‐1 and IMP‐1, but showed a significantly better activity on VIM‐type enzymes, with Ki values in the μM to sub‐μM range. The resolution of the crystallographic structure of VIM‐2 in complex with one of the best inhibitors yielded valuable information about their binding mode. Interestingly, several compounds were shown to restore the β‐lactam susceptibility of VIM‐type‐producing E. coli laboratory strains and also of K. pneumoniae clinical isolates. In addition, selected compounds were found to be devoid of toxicity toward human cancer cells at high concentration, thus showing promising safety.

Pteridine reductase 1 (PTR1) is a key enzyme of the folate pathway in protozoan parasites of the genera Leishmania and Trypanosoma and is a valuable drug target for tropical diseases. This enzyme is able to catalyze the NADPH-dependent reduction of both conjugated (folate) and unconjugated (biopterin) pterins to their tetrahydro forms, starting from oxidized- or dihydro-state substrates. The currently available X-ray structures of Leishmania major PTR1 (LmPTR1) show the enzyme in its unbound, unconjugated substrate-bound (with biopterin derivatives) and inhibitor-bound forms. However, no structure has yet been determined of LmPTR1 bound to a conjugated substrate. Here, the high-resolution crystal structure of LmPTR1 in complex with folic acid is presented and the intermolecular forces that drive the binding of the substrate in the catalytic pocket are described. By expanding the collection of LmPTR1 structures in complex with process intermediates, additional insights into the active-site rearrangements that occur during the catalytic process are provided. In contrast to previous structures with biopterin derivatives, a small but significant difference in the orientation of Asp181 and Tyr194 of the catalytic triad is found. This feature is shared by PTR1 from T. brucei (TbPTR1) in complex with the same substrate molecule and may be informative in deciphering the importance of such residues at the beginning of the catalytic process.

Heat shock protein 90 (Hsp90) is a ubiquitous molecular chaperone that stabilizes client proteins in a folded and functional state. It is composed of two identical and symmetrical subunits and each monomer consists of three domains, the N-terminal (NTD), the middle (MD), and the C-terminal domain (CTD). Since the chaperone activity requires ATP hydrolysis, molecules able to occupy the ATP-binding pocket in the NTD act as Hsp90 inhibitors, leading to client protein degradation and cell death. Therefore, human Hsp90 represents a validated target for developing new anticancer drugs. Since protozoan parasites use their Hsp90 to trigger important transitions between different stages of their life cycle, this protein also represents a profitable target in anti-parasite drug discovery. Nevertheless, the development of molecules able to selectively target the ATP-binding site of protozoan Hsp90 is challenging due to the high homology with the human Hsp90 NTD (hHsp90-NTD). In a previous work, a series of potent Hsp90 inhibitors based on a 1,4,5-trisubstituted 1,2,3-triazole scaffold was developed. The most promising inhibitor of the series, JMC31, showed potent Hsp90 binding and antiproliferative activity in NCI-H460 cells in the low-nanomolar range. In this work, we present the structural characterization of hHsp90-NTD in complex with JMC31 through X-ray crystallography. In addition, to elucidate the role of residue 112 on the ligand binding and its exploitability for the development of selective inhibitors, we investigated the crystal structures of hHsp90-NTD variants (K112R and K112A) in complex with JMC31.

Drugs that target human thymidylate synthase (hTS), a dimeric enzyme, are widely used in anticancer therapy. However, treatment with classical substrate-site-directed TS inhibitors induces over-expression of this protein and development of drug resistance. We thus pursued an alternative strategy that led us to the discovery of TS-dimer destabilizers. These compounds bind at the monomer-monomer interface and shift the dimerization equilibrium of both the recombinant and the intracellular protein toward the inactive monomers. A structural, spectroscopic, and kinetic investigation has provided evidence and quantitative information on the effects of the interaction of these small molecules with hTS. Focusing on the best among them, E7, we have shown that it inhibits hTS in cancer cells and accelerates its proteasomal degradation, thus causing a decrease in the enzyme intracellular level. E7 also showed a superior anticancer profile to fluorouracil in a mouse model of human pancreatic and ovarian cancer. Thus, over sixty years after the discovery of the first TS prodrug inhibitor, fluorouracil, E7 breaks the link between TS inhibition and enhanced expression in response, providing a strategy to fight drug-resistant cancers.

Metallo-β-lactamases (MBLs) are important contributors of Gram-negative bacteria resistance to β-lactam antibiotics. MBLs are highly worrying because of their carbapenemase activity, their rapid spread in major human opportunistic pathogens while no clinically useful inhibitor is available yet. In this context, we are exploring the potential of compounds based on the 1,2,4-triazole-3-thione scaffold as an original ligand of the di-zinc active sites of MBLs, and diversely substituted at its positions 4 and 5. Here, we present a new series of compounds substituted at the 4-position by a thioether-containing alkyl chain with a carboxylic and/or an aryl group at its extremity. Several compounds showed broad-spectrum inhibition with Ki values in the μM to sub-μM range against VIM-type enzymes, NDM-1 and IMP-1. The presence of the sulfur and of the aryl group was important for the inhibitory activity and the binding mode of a few compounds in VIM-2 was revealed by X-ray crystallography. Importantly, in vitro antibacterial susceptibility assays showed that several inhibitors were able to potentiate the activity of meropenem on Klebsiella pneumoniae clinical isolates producing VIM-1 or VIM-4, with a potentiation effect of up to 16-fold. Finally, a selected compound was found to only moderately inhibit the di-zinc human glyoxalase II, and several showed no or only moderate toxicity toward several human cells, thus favourably completing a promising behaviour.

The use of light-responsive proteins to control both living or synthetic cells, is at the core of the expanding fields of optogenetics and synthetic biology. It is thus apparent that a richer reaction toolbox for the preparation of such systems is of fundamental importance. Here, we provide a proof-of-principle demonstration that Morita-Baylis-Hillman adducts can be employed to perform a facile site-specific, irreversible and diastereoselective click-functionalization of a lysine residue buried into a lipophilic binding pocket and yielding an unnatural chromophore with an extended π-system. In doing so we effectively open the path to the in vitro preparation of a library of synthetic proteins structurally reminiscent of xanthopsin eubacterial photoreceptors. We argue that such a library, made of variable unnatural chromophores inserted in an easy-to-mutate and crystallize retinoic acid transporter, significantly expand the scope of the recently introduced rhodopsin mimics as both optogenetic and "lab-on-a-molecule" tools.

Trypanosoma and Leishmania parasites are the etiological agents of various threatening neglected tropical diseases (NTDs), including human African trypanosomiasis (HAT), Chagas disease, and various types of leishmaniasis. Recently, meaningful progresses in the treatment of HAT, due to Trypanosoma brucei (Tb), have been achieved by the introduction of fexinidazole and the combination therapy eflornithine–nifurtimox. Nevertheless, due to drug resistance issues and the exitance of animal reservoirs, the development of new NTD treatments is still required. For this purpose, we explored the combined targeting of two key folate enzymes, dihydrofolate reductase (DHFR) and pteridine reductase 1 (PTR1). We formerly showed that the TbDHFR inhibitor cycloguanil (CYC) also targets TbPTR1, although with reduced affinity. Here, we explored a small library of CYC analogues to understand how their substitution pattern affects the inhibition of both TbPTR1 and TbDHFR. Some novel structural features responsible for an improved, but preferential, ability of CYC analogues to target TbPTR1 were disclosed. Furthermore, we showed that the known drug pyrimethamine (PYR) effectively targets both enzymes, also unveiling its binding mode to TbPTR1. The structural comparison between PYR and CYC binding modes to TbPTR1 and TbDHFR provided key insights for the future design of dual inhibitors for HAT therapy.

Combining drugs represent an approach to efficiently prevent and overcome drug resistance and to reduce toxicity; yet it is a highly challenging task, particularly if combinations of inhibitors of the same enzyme target are considered. To show that crystallographic and inhibition kinetic information can provide indicators of cancer cell growth inhibition by combinations of two anti-human thymidylate synthase (hTS) drugs, we obtained the X-ray crystal structure of the hTS:raltitrexed:5-fluorodeoxyuridine monophosphate (FdUMP) complex. Its analysis showed a ternary complex with both molecules strongly bound inside the enzyme catalytic cavity. The synergistic inhibition of hTS and its mechanistic rationale were consistent with the structural analysis. When administered in combination to A2780 and A2780/CP ovarian cancer cells, the two drugs inhibited ovarian cancer cell growth additively/synergistically. Together, these results support the idea that X-ray crystallography can provide structural indicators for designing combinations of hTS (or any other target)-directed drugs to accelerate preclinical research for therapeutic application.

Three open-source anti-kinetoplastid chemical boxes derived from a whole-cell phenotypic screening by GlaxoSmithKline (Tres Cantos Anti-Kinetoplastid Screening, TCAKS) were exploited for the discovery of a novel core structure inspiring new treatments of parasitic diseases targeting the trypansosmatidic pteridine reductase 1 (PTR1) and dihydrofolate reductase (DHFR) enzymes. In total, 592 compounds were tested through medium-throughput screening assays. A subset of 14 compounds successfully inhibited the enzyme activity in the low micromolar range of at least one of the enzymes from both Trypanosoma brucei and Lesihmania major parasites (pan-inhibitors), or from both PTR1 and DHFR-TS of the same parasite (dual inhibitors). Molecular docking studies of the protein–ligand interaction focused on new scaffolds not reproducing the well-known antifolate core clearly explaining the experimental data. TCMDC-143249, classified as a benzenesulfonamide derivative by the QikProp descriptor tool, showed selective inhibition of PTR1 and growth inhibition of the kinetoplastid parasites in the 5 μM range. In our work, we enlarged the biological profile of the GSK Kinetobox and identified new core structures inhibiting selectively PTR1, effective against the kinetoplastid infectious protozoans. In perspective, we foresee the development of selective PTR1 and DHFR inhibitors for studies of drug combinations.

Ferritins are nanocage proteins that store iron ions in their central cavity as hydrated ferric oxide biominerals. In mammals, further the L (light) and H (heavy) chains constituting cytoplasmic maxi-ferritins, an additional type of ferritin has been identified, the mitochondrial ferritin (MTF). Human MTF (hMTF) is a functional homopolymeric H-like ferritin performing the ferroxidase activity in its ferroxidase site (FS), in which Fe(II) is oxidized to Fe(III) in the presence of dioxygen. To better investigate its ferroxidase properties, here we performed time-lapse X-ray crystallography analysis of hMTF, providing structural evidence of how iron ions interact with hMTF and of their binding to the FS. Transient iron binding sites, populating the pathway along the cage from the iron entry channel to the catalytic center, were also identified. Furthermore, our kinetic data at variable iron loads indicate that the catalytic iron oxidation reaction occurs via a diferric peroxo intermediate followed by the formation of ferric-oxo species, with significant differences with respect to human H-type ferritin.