Structure-Property Optimization of a Series of Imidazopyridines for Visceral Leishmaniasis

Leishmaniasis is a collection of diseases caused by more than 20 Leishmania parasite species that manifest as either visceral, cutaneous, or mucocutaneous leishmaniasis. Despite the significant mortality and morbidity associated with leishmaniasis, it remains a neglected tropical disease. Existing treatments have variable efficacy, significant toxicity, rising resistance, and limited oral bioavailability, which necessitates the development of novel and affordable therapeutics. Here, we report on the continued optimization of a series of imidazopyridines for visceral leishmaniasis and a scaffold hop to a series of substituted 2-(pyridin-2-yl)-6,7-dihydro-5H-pyrrolo[1,2-a]imidazoles with improved absorption, distribution, metabolism, and elimination properties

Accelerating Drug Discovery Efforts for Trypanosomatidic Infections Using an Integrated Transnational Academic Drug Discovery Platform

According to the World Health Organization, more than 1 billion people are at risk of or are affected by neglected tropical diseases. Examples of such diseases include trypanosomiasis, which causes sleeping sickness; leishmaniasis; and Chagas disease, all of which are prevalent in Africa, South America, and India. Our aim within the New Medicines for Trypanosomatidic Infections project was to use (1) synthetic and natural product libraries, (2) screening, and (3) a preclinical absorption, distribution, metabolism, and excretion\u2013toxicity (ADME-Tox) profiling platform to identify compounds that can enter the trypanosomatidic drug discovery value chain. The synthetic compound libraries originated from multiple scaffolds with known antiparasitic activity and natural products from the Hypha Discovery MycoDiverse natural products library. Our focus was first to employ target-based screening to identify inhibitors of the protozoan Trypanosoma brucei pteridine reductase 1 (TbPTR1) and second to use a Trypanosoma brucei phenotypic assay that made use of the T. brucei brucei parasite to identify compounds that inhibited cell growth and caused death. Some of the compounds underwent structure-activity relationship expansion and, when appropriate, were evaluated in a preclinical ADME-Tox assay panel. This preclinical platform has led to the identification of lead-like compounds as well as validated hits in the trypanosomatidic drug discovery value chain

Inibitori dell’interazione proteina-proteina per superare la resistenza farmacologica in diversi tipi di cancro: ottimizzazione del lead E7, un inbitore dissociativo della Timidilato sintasi umana

La timidilato sintasi umana (hTS) svolge un ruolo fondamentale nella sintesi del DNA, essenziale per la sopravvivenza cellulare. L’hTS è coinvolta nel percorso dei folati, nello specifico nella biosintesi ex novo delle pirimidine. La struttura della proteina e le sue funzioni sono strettamente correlate per l’esplicazione dell’attività enzimatica. Nonostante il quantitativo di lavori riportati in letteratura, le caratteristiche delle relazioni struttura-funzione dell’ hTS non sono ancora chiare, in particolare le diverse conformazioni e le forme di equilibrio monomero/dimero di questa proteina omodimerica obbligata e il suo ruolo nella regolazione dell’espressione genica. Il concetto espresso precedentemente è stato ampiamente investigato durante il progetto AIRC intitolato “Inibitori dell’interazione proteina-proteina della timidilato sintasi contro il cancro al colon retto”, che è stato parte del mio progetto di dottorato. Inoltre, il mio progetto è incluso nel programma del PhD regionale ONCOPENTA1. hTS è tra i bersagli più studiati per la progettazione di farmaci antitumorali. Oltre 3300 studi clinici sono stati condotti dall'inizio degli anni sessanta, alcuni dei quali hanno portato alla scoperta di farmaci anti-TS come il Fluorouracile (FU), Pemetrexed e Raltitrexed che sono utilizzati in diversi tipi di tumori come quello del colon-retto (CRC), e recentemente, dell’ovarico (OC). I farmaci citati, dopo un uso prolungato determinano resistenza farmacologica rendendoli inefficaci. Inoltre nell’ OC è presente la resistenza incrociata per l’utilizzo di farmaci al platino. Uno dei principali meccanismi di tale resistenza è la sovraespressione dell’hTS. Un risultato importante è l’ottenimento di nuovi farmaci abili di superare l’effetto della resistenza farmacologica indotta da elevati livelli della proteina TS. Pertanto, l'inibizione proteica rispetto alla sua regolazione TSmRNA richiede una strategia alternativa e la scoperta di un nuovo meccanismo di inibizione. Attualmente, i farmaci anti-TS sono inibitori che si legano nella tasca catalitica, mimando i substrati. Gli inibitori allosterici (non substrati analoghi), legandosi all’interfaccia dimerica della proteina, possono alterare l’equilibrio dimero\monomero con l’incremento della forma monomerica, ed innescando così la modulazione delle vie cellulari, ad ora inesplorate. E7 è stato il primo composto recentemente individuato con tali caratteristica ma necessita di un lavoro di ottimizzazione per migliorare le proprietà farmacologiche. Lo scopo principale della tesi è la progettazione e lo sviluppo di nuovi inibitori, analoghi di E7, che alterano l’equilibrio dimerico della proteina. I nuovi composti dovrebbero indurre una perturbazione dell’ equilibrio dimero monomero in favore della forma monomerica, inibendo la funzione catalitica, preservando l’attività regolatoria e conseguentemente la riduzione della resistenza farmacologica. Per raggiungere tali obiettivi, ho sintetizzato più di 40 composti che sono stati testati ed analizzati con studi di inibizione enzimatica e di FRET, la quale rileva la capacità di questi composti di ridurre la forma dimerica della TS. Per stabilire se i nostri composti determinano la separazione del dimero a livello cellulare, ho progettato e sintetizzato composti legati a sonde fluorescenti. Inoltre, ho sviluppato una nuova sintesi per la produzione del composto in grande quantità e la risoluzione enantiomerica tramite HPLC preparativa chirale per ottenere i dati in vivo del nostro composto migliore. Il mio lavoro di ottimizzazione ha portato all’ottenimento del composto AIC-C37 che presenta una migliore attività biologica e solubilità (rispetto ad E7 e FU) ed un interessante punto di collegamento molecolare per studiare il meccanismo in vivo.Human thymidylate synthase (hTS) plays a fundamental role in the synthesis of DNA, essential for cell survival. hTS is involved in the folate pathways, specifically in the de novo pyrimidine biosynthesis. Protein structure and functions are intimately correlated for the explication of the enzymatic activity. Despite the enormous amount of work reported in the literature, hTS structure-function relationships features are still unclear, especially the different conformations and monomers/dimer equilibrium of this obligate homodimeric protein and its role in protein expression regulation. The above-mentioned concept was largely investigated during the execution of the AIRC project entitled “Protein-protein interaction inhibitors of thymidylate synthase against colorectal cancer” that was also in part the topic of my PhD thesis. Additionally, my project was included in the PhD regional program dedicated to ONCOPENTA1. hTS is the most renowned anticancer drug target. Over 3300 clinical trials have been performed since the early sixties, some of them providing anti-TS clinical drugs as Fluorouracil (FU), Pemetrexed and Raltitrexed that are adopted in different cancer types such as colorectal cancer (CRC) and recently in ovarian cancer (OC). All the mentioned drugs, after prolonged use, cause drug resistance that makes the drug ineffective. In OC, cross-resistance with platinum drugs is also observed. One of the main resistance mechanisms is the overexpression of hTS. The development of new drugs able to overcome drug resistance effects due to high TS protein level would be an important achievement. This requires a change of strategy and the discovery of new mechanisms of inhibition that should unpair the protein inhibition with respect to its TSmRNA regulation. Currently, TS-targeting drugs bind at the catalytic-pocket and mostly resemble the protein substrates. Allosteric inhibitors (non-substrate analogues) binding at the protein dimeric interface may alter the dimer/monomer equilibrium by increasing the monomer form amount thus triggering cellular pathways modulation previously unexplored. The first compound with the mentioned features (E7) was recently identified. However a lead optimization work is necessary to optimize the compound properties. The specific aim of my thesis work is the design and development of novel dissociative inhibitors stemming from the E7 lead. The new compounds should induce a perturbation in the dimer-monomer equilibrium in favour of the monomeric form thus, inhibiting the catalytic function, preserving the regulatory activity and consequently reduce the development of drug resistance. To achieve this aim, I have synthesized more than 40 compounds that were tested and analyzed using enzyme kinetic inhibition studies and fluorescence resonance energy transfer (FRET) assays that revealed the capability of these compounds to disrupt the dimeric form of hTS. To gain evidence that our compounds determine hTS dimer disrupters engagement in cells, I have designed and synthesized compounds with fluorescence probe that we resorted in fluorescence microscopy. Moreover, I have developed a scale-up methodology for chemical processes and preparative chiral HPLC resolution to obtain the in vivo data. My lead optimization work provided a compound, AIC-C37, that showed higher solubility and efficacy with respect to E7 and Fluorouracil and interesting anchoring point for in vitro and in vivo mechanism of action studies. 1 Progetto (ONCOPENTA): Oncologia di Precisione e Nuove Terapie Antitumorali. Sviluppo di inibitori dell’interazione protein-proteina contro la farmaco-resistenza nei sarcomi e carcinom

An Improved Synthesis of CENTA, a Chromogenic Substrate for β-Lactamases

7-β-Thien-2-yl-acetamido-3-[(4-nitro-3-carboxyphenyl) thiomethyl]-3-cephem-4-carboxylic acid (CENTA) is a yellow chromogenic β-lactamases (BL) substrate. It hydrolyses readily in the presence of all BL and is therefore suitable for kinetic studies, the detection of BL enzymes in crude extracts and chromatographic fractions. CENTA is commercially available at a high price because of the cumbersome synthetic protocol, the only currently available for its preparation. Here we describe a new efficient and improved process for the preparation of CENTA. Starting from the easily available 7-aminocephalosporanic acid (7-ACA) through a three-step synthesis, CENTA was obtained with a 75% overall yield. The newly developed process proceeds through a pivotal intermediate in cephalosporin chemistry, which may be used as starting compound for the development of new cephalosporin derivatives

Human Thymidylate Synthase Inhibitors Halting Ovarian Cancer Growth

Human thymidylate synthase (hTS) has an important role in DNA biosynthesis, thus it is essential for cell survival. TS is involved in the folate pathways, specifically in the de novo pyrimidine biosynthesis. Structure and functions are intimately correlated, account for cellular activity and, in a broader view, with in vivo mechanisms. hTS is a target for anticancer agents, some of which are clinical drugs. The understanding of the detailed mechanism of TS inhibition by currently used drugs and of the interaction with the mechanism of action of other anticancer agents can suggest new perspective of TS inhibition able to improve the anticancer effect and to overcome drug resistance. TS-targeting drugs in therapy today are inhibitors that bind at the active site and that mostly resemble the substrates. Nonsubstrate analogs offer an opportunity for allosteric binding and novel mode of inhibition in the cancer cells. This chapter illustrates the relationship among the large number of hTS actions at molecular and clinical levels, its role as a target for ovarian cancer therapy, in particular in cases of overexpression of hTS and other folate proteins such as those induced by platinum drug treatments, and address the potential combination of TS inhibitors with other suitable anticancer agents

X-ray-crystallography deciphers the activity of broad spectrum boronic acid \u3b2-Lactamases inhibitors

Recent decades have witnessed a dramatic increase of multidrug resistant (MDR) bacteria, compromising the efficacy of available antibiotics, and a continual decline in the discovery of novel antibacterials. We recently reported the first library of benzo[b]thiophen-2-ylboronic acid inhibitors sharing broad spectrum activity against \uf062-Lactamases (BLs). The ability of these compounds to inhibit structurally and mechanistically different types of \uf062-Lactamases has been here structurally investigated. An extensive x-ray crystallographic analysis of boronic acids (BAs) binding to proteins representative of serine BLs (SBLs) and metallo \uf062-Lactamases (MBLs) have been conducted to depict the role played by the boronic group in driving molecular recogni-tion, especially in the interaction with MBLs. Our derivatives are the first case of non-cyclic boronic acids active against MBLs and represent a productive route toward potent broad-spectrum inhibitors

First virtual screening and experimental validation of inhibitors targeting GES-5 carbapenemase

The worldwide spread of beta-lactamases with hydrolytic activity extended to last resort carbapenems is aggravating the antibiotic resistance problem and endangers the successful antimicrobial treatment of clinically relevant pathogens. As recently highlighted by the World Health Organization, new strategies to contain antimicrobial resistance are urgently needed. Class A carbapenemases include members of the KPC, GES and SFC families. These enzymes have the ability to hydrolyse penicillins, cephalosporins and carbapenems, while also being less susceptible to available beta-lactam inhibitors, such as clavulanic acid. The KPC family is the most prevalent. It is mostly found on plasmids in Klebsiella pneumoniae, meaning that great amounts of attention, in terms of inhibitor design and structural biology, have been dedicated to it, whereas no efforts have yet been dedicated to GES-type enzymes, despite their ability to rapidly and horizontally disseminate. We herein report the first in silico screening against GES-5, which is the most dangerous GES-type beta-lactamase, using a library of 800K commercially available candidates that all share drug-like properties, such as their MW, logP, rotatable bonds and HBA/HBD atoms. The best screening results were filtered to enrich the number of different chemotypes, and then submitted to molecular docking. The 34 most promising candidates were selected for in vitro validation in biochemical assays against recombinant GES-5. Six hits acted as inhibitors, in the high micromolar range, towards GES-5 and led to the identification of the first, novel chemotypes with inhibitory activity against this clinically relevant carbapenemase

Diastereoselective Synthesis of (1,3-Dioxan-4-yl)pyrimidine and Purin Nucleoside Analogues

(1,3-Dioxan-4-yl)-substituted nucleoside analogues, higher homologues of antiviral and anticancer 1,3-dioxolanes, were prepared from the key intermediate (4-acetoxy-1,3-dioxan-2-yl)methyl benzoate and silylated bases. Glycosylation, carried out under Vorbrüggen conditions in the presence of trimethylsilyltrifluoromethanesulfonate (TMSOTf) as a catalyst, afforded the desired compounds with high stereoselectivity and regioselectivity, with only the desired β-anomeric N-1 pyrimidine and N-9 purin nucleosides being obtained. 1H NMR experiments established that the β-anomers were diequatorial, and this assignment was confirmed by singlecrystal X-ray diffraction. Despite their structural similarities with natural nucleosides, none of the synthesized nucleosides showed antiviral activity

Comparative mapping of on-targets and off-targets for the discovery of anti-trypanosomatid folate pathway inhibitors

Background Multi-target approaches are necessary to properly analyze or modify the function of a biochemical pathway or a protein family. An example of such a problem is the repurposing of the known human anti-cancer drugs, antifolates, as selective anti-parasitic agents. This requires considering a set of experimentally validated protein targets in the folate pathway of major pathogenic trypanosomatid parasites and humans: (i) the primary parasite on-targets: pteridine reductase 1 (PTR1) (absent in humans) and bifunctional dihydrofolate reductase-thymidylate synthase (DHFRâTS), (ii) the primary off-targets: human DHFR and TS, and (iii) the secondary on-target: human folate receptor β, a folate/antifolate transporter. Methods We computationally compared the structural, dynamic and physico-chemical properties of the targets. We based our analysis on available inhibitory activity and crystallographic data, including a crystal structure of the bifunctional T. cruzi DHFRâTS with tetrahydrofolate bound determined in this work. Due to the low sequence and structural similarity of the targets analyzed, we employed a mapping of binding pockets based on the known common ligands, folate and methotrexate. Results Our analysis provides a set of practical strategies for the design of selective trypanosomatid folate pathway inhibitors, which are supported by enzyme inhibition measurements and crystallographic structures. Conclusions The ligand-based comparative computational mapping of protein binding pockets provides a basis for repurposing of anti-folates and the design of new anti-trypanosmatid agents. General significance Apart from the target-based discovery of selective compounds, our approach may be also applied for protein engineering or analyzing evolutionary relationships in protein families