Sesquiterpene Lactones with Dual Inhibitory Activity against the Trypanosoma brucei Pteridine Reductase 1 and Dihydrofolate Reductase.

The parasite Trypanosoma brucei (T. brucei) is responsible for human African trypanosomiasis (HAT) and the cattle disease "Nagana" which to this day cause severe medical and socio-economic issues for the affected areas in Africa. So far, most of the available treatment options are accompanied by harmful side effects and are constantly challenged by newly emerging drug resistances. Since trypanosomatids are auxotrophic for folate, their pteridine metabolism provides a promising target for an innovative chemotherapeutic treatment. They are equipped with a unique corresponding enzyme system consisting of the bifunctional dihydrofolate reductase-thymidylate synthase (TbDHFR-TS) and the pteridine reductase 1 (TbPTR1). Previously, gene knockout experiments with PTR1 null mutants have underlined the importance of these enzymes for parasite survival. In a search for new chemical entities with a dual inhibitory activity against the TbPTR1 and TbDHFR, a multi-step in silico procedure was employed to pre-select promising candidates against the targeted enzymes from a natural product database. Among others, the sesquiterpene lactones (STLs) cynaropicrin and cnicin were identified as in silico hits. Consequently, an in-house database of 118 STLs was submitted to an in silico screening yielding 29 further virtual hits. Ten STLs were subsequently tested against the target enzymes in vitro in a spectrophotometric inhibition assay. Five compounds displayed an inhibition over 50% against TbPTR1 as well as three compounds against TbDHFR. Cynaropicrin turned out to be the most interesting hit since it inhibited both TbPTR1 and TbDHFR, reaching IC50 values of 12.4 µM and 7.1 µM, respectively.

In Silico Identification and In Vitro Evaluation of Natural Inhibitors of Leishmania major Pteridine Reductase I.

In a continuation of our computational efforts to find new natural inhibitors of a variety of target enzymes from parasites causing neglected tropical diseases (NTDs), we now report on 15 natural products (NPs) that we have identified as inhibitors of Leishmania major pteridine reductase I (LmPTR1) through a combination of in silico and in vitro investigations. Pteridine reductase (PTR1) is an enzyme of the trypanosomatid parasites' peculiar folate metabolism, and has previously been validated as a drug target. Initially, pharmacophore queries were created based on four 3D structures of LmPTR1 using co-crystallized known inhibitors as templates. Each of the pharmacophore queries was used to virtually screen a database of 1100 commercially available natural products. The resulting hits were submitted to molecular docking analyses in the substrate binding site of the respective protein structures used for the pharmacophore design. This approach led to the in silico identification of a total of 18 NPs with predicted binding affinity to LmPTR1. These compounds were subsequently tested in vitro for inhibitory activity towards recombinant LmPTR1 in a spectrophotometric inhibition assay. Fifteen out of the 18 tested compounds (hit rate = 83%) showed significant inhibitory activity against LmPTR1 when tested at a concentration of 50 µM. The IC50 values were determined for the six NPs that inhibited the target enzyme by more than 50% at 50 µM, with sophoraflavanone G being the most active compound tested (IC50 = 19.2 µM). The NPs identified and evaluated in the present study may represent promising lead structures for the further rational drug design of more potent inhibitors against LmPTR1.

Antitarget, Anti-SARS-CoV-2 Leads, Drugs, and the Drug Discovery-Genetics Alliance Perspective.

The most advanced antiviral molecules addressing major SARS-CoV-2 targets (Main protease, Spike protein, and RNA polymerase), compared with proteins of other human pathogenic coronaviruses, may have a short-lasting clinical efficacy. Accumulating knowledge on the mechanisms underlying the target structural basis, its mutational progression, and the related biological significance to virus replication allows envisaging the development of better-targeted therapies in the context of COVID-19 epidemic and future coronavirus outbreaks. The identification of evolutionary patterns based solely on sequence information analysis for those targets can provide meaningful insights into the molecular basis of host-pathogen interactions and adaptation, leading to drug resistance phenomena. Herein, we will explore how the study of observed and predicted mutations may offer valuable suggestions for the application of the so-called "synthetic lethal" strategy to SARS-CoV-2 Main protease and Spike protein. The synergy between genetics evidence and drug discovery may prioritize the development of novel long-lasting antiviral agents.

Biological evaluation of MR36, a novel non-polyglutamatable thymidylate synthase inhibitor that blocks cell cycle progression in melanoma cell lines.

Melanoma is one of the most common cancers, and its incidence has continued to increase over the past few decades. Chemotherapy resistance and related defects in apoptotic signaling are critical for the high mortality of melanoma. Effective drugs are lacking because apoptosis regulation in this tumor type is not well understood. The folate pathway has been considered an interesting target for anticancer therapies, and approaches targeting this pathway have recently been extended to melanoma treatment. In this study, the intracellular apoptosis signaling pathways of two melanoma cells lines (SK-MEL-2 and SK-MEL-28) were investigated after treatment with a new experimental antifolate substance (MR36) that targets thymidylate synthase. In both melanoma cell lines, apoptosis induction was triggered by a p53-independent mechanism. MR36-induced apoptosis was associated with a loss of both mitochondrial membrane potential and caspase-3 activation. Induction of cell cycle arrest by MR36 was associated with changes in the expression of key cell cycle regulators, such as p21 and cyclin D1, and the hypophosphorylation of pRb. In addition, Fas signaling was also analyzed. These findings suggest that, unlike classical antifolates, MR36 exerted an inhibitory effect on both the enzymatic function and expression of thymidylate synthase, thereby inducing apoptosis through the activation of the extrinsic and intrinsic pathways in the melanoma cell lines. MR36 showed a different mechanism of action from the known antifolates (Nolatrexed and Pemetrexed) that resulted in higher anticancer activity. Therefore, MR36 should be included as a potential new therapeutic treatment in melanoma research.

Multitarget, Selective Compound Design Yields Potent Inhibitors of a Kinetoplastid Pteridine Reductase 1.

The optimization of compounds with multiple targets is a difficult multidimensional problem in the drug discovery cycle. Here, we present a systematic, multidisciplinary approach to the development of selective antiparasitic compounds. Computational fragment-based design of novel pteridine derivatives along with iterations of crystallographic structure determination allowed for the derivation of a structure-activity relationship for multitarget inhibition. The approach yielded compounds showing apparent picomolar inhibition of T. brucei pteridine reductase 1 (PTR1), nanomolar inhibition of L. major PTR1, and selective submicromolar inhibition of parasite dihydrofolate reductase (DHFR) versus human DHFR. Moreover, by combining design for polypharmacology with a property-based on-parasite optimization, we found three compounds that exhibited micromolar EC50 values against T. brucei brucei while 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.

Folic Acid-Peptide Conjugates Combine Selective Cancer Cell Internalization with Thymidylate Synthase Dimer Interface Targeting.

Drug-target interaction, cellular internalization, and target engagement should be addressed to design a lead with high chances of success in further optimization stages. Accordingly, we have designed conjugates of folic acid with anticancer peptides able to bind human thymidylate synthase (hTS) and enter cancer cells through folate receptor α (FRα) highly expressed by several cancer cells. Mechanistic analyses and molecular modeling simulations have shown that these conjugates bind the hTS monomer-monomer interface with affinities over 20 times larger than the enzyme active site. When tested on several cancer cell models, these conjugates exhibited FRα selectivity at nanomolar concentrations. A similar selectivity was observed when the conjugates were delivered in synergistic or additive combinations with anticancer agents. At variance with 5-fluorouracil and other anticancer drugs that target the hTS catalytic pocket, these conjugates do not induce overexpression of this protein and can thus help combating drug resistance associated with high hTS levels.

Spermidine/spermine N1-acetyltranferase modulation by novel folate cycle inhibitors in cisplatin-sensitive and -resistant human ovarian cancer cell lines.

OBJECTIVE: Polyamines have been shown to play a role in the growth and survival of several solid tumors, including ovarian cancer. Intracellular polyamine depletion by the inhibition of biosynthesis enzymes or by the induction of the catabolic pathway leads to antiproliferative effects in many different tumor cell lines. Recent studies showed that the thymidylate synthase inhibitor 5-fluorouracil (5-FU) affects polyamine metabolism in colon carcinoma cells through the induction of the key catabolic enzyme spermidine/spermine N1-acetyltransferase (SSAT). METHODS: We therefore examined whether combinations of novel folate cycle inhibitors with quinoxaline structure and drugs that specifically target polyamine metabolism, such as diethylderivatives of norspermine (DENSPM) or spermine (BESpm), have synergistic effect in killing cisplatin-sensitive and drug-resistant daughter human ovarian cell lines. RESULTS: Our results showed that simultaneous drug combination or quinoxaline pre-treatment synergistically increased SSAT expression, depleted polyamines, increased reactive oxygen species production, and produced synergistic tumor cell killing in both cell lines. Of note, this combined therapy increased the chemosensitivity of cisplatin-resistant cells and cross-resistant to the polyamine analogues. On the contrary, some pre-treatment regimens of Spm analogues were antagonistic. CONCLUSIONS: These results show that SSAT plays an important role in novel folate cycle inhibitors effects and suggest that their combination with analogues has potential for development as therapy for ovarian carcinoma based on SSAT modulation.

Designing Chimeric Molecules for Drug Discovery by Leveraging Chemical Biology.

After the first seed concept introduced in the 18th century, different disciplines have attributed different names to dual-functional molecules depending on their application, including bioconjugates, bifunctional compounds, multitargeting molecules, chimeras, hybrids, engineered compounds. However, these engineered constructs share a general structure: a first component that targets a specific cell and a second component that exerts the pharmacological activity. A stable or cleavable linker connects the two modules of a chimera. Herein, we discuss the recent advances in the rapidly expanding field of chimeric molecules leveraging chemical biology concepts. This Perspective is focused on bifunctional compounds in which one component is a lead compound or a drug. In detail, we discuss chemical features of chimeric molecules and their use for targeted delivery and for target engagement studies.

Proteomic and Bioinformatic Studies for the Characterization of Response to Pemetrexed in Platinum Drug Resistant Ovarian Cancer.

Proteomics and bioinformatics are a useful combined technology for the characterization of protein expression level and modulation associated with the response to a drug and with its mechanism of action. The folate pathway represents an important target in the anticancer drugs therapy. In the present study, a discovery proteomics approach was applied to tissue samples collected from ovarian cancer patients who relapsed after the first-line carboplatin-based chemotherapy and were treated with pemetrexed (PMX), a known folate pathway targeting drug. The aim of the work is to identify the proteomic profile that can be associated to the response to the PMX treatment in pre-treatement tissue. Statistical metrics of the experimental Mass Spectrometry (MS) data were combined with a knowledge-based approach that included bioinformatics and a literature review through ProteinQuest™ tool, to design a protein set of reference (PSR). The PSR provides feedback for the consistency of MS proteomic data because it includes known validated proteins. A panel of 24 proteins with levels that were significantly different in pre-treatment samples of patients who responded to the therapy vs. the non-responder ones, was identified. The differences of the identified proteins were explained for the patients with different outcomes and the known PMX targets were further validated. The protein panel herein identified is ready for further validation in retrospective clinical trials using a targeted proteomic approach. This study may have a general relevant impact on biomarker application for cancer patients therapy selection.

Pharmacokinetics and disposition of miltefosine in healthy mice and hamsters experimentally infected with Leishmania infantum.

Miltefosine is the only currently available oral drug for treatment of leishmaniasis. However, information on the pharmacokinetics (PK) of miltefosine is relatively scarce in animals. PK parameters and disposition of the molecule was determined in healthy NMRI mice and Syrian hamsters infected and treated with different miltefosine doses and regimens. Long half-life of the molecule was confirmed and differential pattern of accumulation of the drug was observed in analyzed organs in mice and hamster. Long treatment schedules produced miltefosine levels over IC50 value against L. infantum intracellular amastigotes for at least 24 days in spleen and liver of infected hamsters. The observed differential pattern of organ accumulation of the drug in mice and hamster supports the relevance of both species for translational research on chemotherapy of leishmaniasis.

Label-free fiber optic optrode for the detection of class C ß-lactamases expressed by drug resistant bacteria.

This paper reports the experimental assessment of an automated optical assay based on label free optical fiber optrodes for the fast detection of class C ß-lactamases (AmpC BLs), actually considered as one of the most important sources of resistance to ß-lactams antibiotics expressed by resistant bacteria. Reflection-type long period fiber gratings (RT-LPG) have been used as highly sensitive label free optrodes, while a higher affine boronic acid-based ligand was here selected to enhance the overall assay performances compared to those obtained in our first demonstration. In order to prove the feasibility analysis towards a fully automated optical assay, an engineered system was developed to simultaneously manipulate and interrogate multiple fiber optic optrodes in the different phases of the assay. The automated system tested in AmpC solutions at increasing concentrations demonstrated a limit of detection (LOD) of 6 nM, three times better when compared with the results obtained in our previous work. Moreover, the real effectiveness of the proposed optical assay has been also confirmed in complex matrices as the case of lysates of Escherichia coli overexpressing AmpC.

Exploiting the 2-Amino-1,3,4-thiadiazole Scaffold To Inhibit Trypanosoma brucei Pteridine Reductase in Support of Early-Stage Drug Discovery.

Pteridine reductase-1 (PTR1) is a promising drug target for the treatment of trypanosomiasis. We investigated the potential of a previously identified class of thiadiazole inhibitors of Leishmania major PTR1 for activity against Trypanosoma brucei (Tb). We solved crystal structures of several TbPTR1-inhibitor complexes to guide the structure-based design of new thiadiazole derivatives. Subsequent synthesis and enzyme- and cell-based assays confirm new, mid-micromolar inhibitors of TbPTR1 with low toxicity. In particular, compound 4m, a biphenyl-thiadiazole-2,5-diamine with IC50 = 16 µM, was able to potentiate the antitrypanosomal activity of the dihydrofolate reductase inhibitor methotrexate (MTX) with a 4.1-fold decrease of the EC50 value. In addition, the antiparasitic activity of the combination of 4m and MTX was reversed by addition of folic acid. By adopting an efficient hit discovery platform, we demonstrate, using the 2-amino-1,3,4-thiadiazole scaffold, how a promising tool for the development of anti-T. brucei agents can be obtained.

Decoding the Structural Basis For Carbapenem Hydrolysis By Class A ß-lactamases: Fishing For A Pharmacophore.

Nowadays clinical therapy witnesses a challenging bacterial resistance limiting the available armament of antibiotics. Over the decades strains resistant to all antibiotics have been selected while medicinal chemists were not able to develop agents capable of destroying them or to prevent their extension. In particular, carbapenem-resistant Enterobacteriaceae (CRE), representing one of the most common human pathogens, have been reported with increased frequency since their first identification twenty years ago. The enterobacterial carbapenemases differ from the extended spectrum ß-lactamases (ESBL) in their ability to hydrolyze ß-lactams, cephalosporins and most importantly monobactams and carbapenems. They are progressively spreading throughout the world, therefore leaving no effective ß-lactam to cure bacterial infections. Several BLs-carbapenemase Xray structures have been determined making these enzymes attractive targets for structure-based drug design studies. However, very little has been done so far to powerfully address the inhibitor design issues for this emerging type of BLs. Here, we focus on the structural basis for molecular recognition and for broad spectrum activity of class A carbapenemases: based on available 3-dimensional structural information we identify a theoretical pharmacophoric model as a starting point for the development of needed carbapenemases inhibitors.

Alanine mutants of the interface residues of human thymidylate synthase decode key features of the binding mode of allosteric anticancer peptides.

Allosteric peptide inhibitors of thymidylate synthase (hTS) bind to the dimer interface and stabilize the inactive form of the protein. Four interface residues were mutated to alanine, and interaction studies were employed to decode the key role of these residues in the peptide molecular recognition. This led to the identification of three crucial interface residues F59, L198, and Y202 that impart activity to the peptide inhibitors and suggest the binding area for further inhibitor design.

Characterization of 2,4-Diamino-6-oxo-1,6-dihydropyrimidin-5-yl Ureido Based Inhibitors of Trypanosoma brucei FolD and Testing for Antiparasitic Activity.

The bifunctional enzyme N(5),N(10)-methylenetetrahydrofolate dehydrogenase/cyclo hydrolase (FolD) is essential for growth in Trypanosomatidae. We sought to develop inhibitors of Trypanosoma brucei FolD (TbFolD) as potential antiparasitic agents. Compound 2 was synthesized, and the molecular structure was unequivocally assigned through X-ray crystallography of the intermediate compound 3. Compound 2 showed an IC50 of 2.2 µM, against TbFolD and displayed antiparasitic activity against T. brucei (IC50 49 µM). Using compound 2, we were able to obtain the first X-ray structure of TbFolD in the presence of NADP(+) and the inhibitor, which then guided the rational design of a new series of potent TbFolD inhibitors.

Permeation through the cell membrane of a boron-based ß-lactamase inhibitor.

Bacteria express beta-lactamases to counteract the beneficial action of antibiotics. Benzo[b]-thiophene-2-boronic acid (BZB) derivatives are ß-lactamase inhibitors and, as such, promising compounds to be associated with ß-lactam antibacterial therapies. The uncharged form of BZB, in particular, is suggested to diffuse through the outer membrane of gram negative bacteria. In this study, through the combination of electrophysiological experiments across reconstituted PC/n-decane bilayers and metadynamics-based free energy calculations, we investigate the permeation mechanism of boronic compounds. Our experimental data establish that BZB passes through the membrane, while computer simulations provide hints for the existence of an aqueous, water-filled monomolecular channel. These findings provide new perspectives for the design of boronic acid derivatives with high membrane permeability.