Inhibitor Specificity via Protein Dynamics Insights from the Design of Antibacterial Agents Targeted Against Thymidylate Synthase

AbstractStructure-based drug design of species-specific inhibitors generally exploits structural differences in proteins from different organisms. Here, we demonstrate how achieving specificity can be aided by targeting differences in the dynamics of proteins. Thymidylate synthase (TS) is a good target for anticancer agents and a potential target for antibacterial agents. Most inhibitors are folate-analogs that bind at the folate binding site and are not species specific. In contrast, α156 is not a folate-analog and is specific for bacterial TS; it has been shown crystallographically to bind in a nonconserved binding site. Docking calculations and crystal structure-based estimation of the essential dynamics of TSs from five different species show that differences in the dynamics of TSs make the active site more accessible to α156 in the prokaryotic than in the eukaryotic TSs and thereby enhance the specificity of α156

Nuovi derivati chinossalinici inibitori della Diidrofolato Reduttasi (DHFR) e della Timidilato Sintetasi (TS) della <i>Leishmania major</i>: risultati preliminari

L’incidenza delle malattie protozoarie imputate al genere Leishmania è aumentata enormemente con la diffusione dell’AIDS. In particolare la forma viscerale della Leishmaniosi sta emergendo come malattia nuova e sempre più frequente. Oltre gli antimoniali altri farmaci importanti sono le pentammidine e l’amfotericina B. Un approccio abbondantemente esplorato in passato è stato lo sviluppo di farmaci antifolici antiprotozoari che bloccano il ciclo dell’acido folico, che, previa attivazione metabolica di enzimi folato dipendenti, attraverso il trasporto di unità carboniose, è responsabile della sintesi de novo degli acidi nucleici e degli amminoacidi. In questo senso sono stati usati il trimetoprim e la pirimetamina che sono attivi contro i plasmodi e i toxoplasmi, ma non contro le Leishmanie e i tripanosomi. Questi due protozoi appartenenti alla classe dei Kinetoplastidae sono sensibili agli antifolici antitumorali come il metotrexato (MTX), ma sviluppano immediatamente una chemioresistenza in quanto riattivano l’autoproduzione di acido folico mediante una via di salvataggio che impiega l’enzima Pteridina riduttasi, recentemente scoperto, appunto, nelle Leishmanie major e nel tripanosoma cruzi. Sulla base di queste premesse ci è sembrato che le nostre molecole a struttura chinossalinica, analoghe al metotrexato, potessero essere impiegate come inibitori enzimatici di questa famiglia di folato

Structure-based discovery and in-parallel optimization of novelcompetitive inhibitors of thymidylate synthase

AbstractBackground:The substrate sites of enzymes are attractive targets for structurebased inhibitor design. Two difficulties hinder efforts to discover and elaborate new (nonsubstrate-like) inhibitors for these sites. First, novel inhibitors often bind at nonsubstrate sites. Second, a novel scaffold introduces chemistry that is frequently unfamiliar, making synthetic elaboration challenging.Results:In an effort to discover and elaborate a novel scaffold for a substrate site, we combined structure-based screening with in-parallel synthetic elaboration. These techniques were used to find new inhibitors that bound to the folate site of Lactobacillus casei thymidylate synthase (LcTS), an enzyme that is a potential target for proliferative diseases, and is highly studied. The available chemicals directory was screened, using a molecular-docking computer program, for molecules that complemented the three-dimensional structure of this site. Five high-ranking compounds were selected for testing. Activity and clocking studies led to a derivative of one of these, dansyltyrosine (Ki 65 μM. Using solid-phase in-parallel techniques 33 derivatives of this lead were synthesized and tested. These analogs are dissimilar to the substrate but bind competitively with it. The most active analog had a Ki of 1.3 μM. The tighter binding inhibitors were also the most specific for LcTS versus related enzymes.Conclusions:TS can recognize inhibitors that are dissimilar to, but that bind competitively with, the folate substrate. Combining structure-based discovery with in-parallel synthetic techniques allowed the rapid elaboration of this series of compounds. More automated versions of this approach can be envisaged

5,7-dinitro e 5,7-diammino chinossaline analoghe dell'antifolico classico metotressato e non classico trimetrexato. Sintesi e valutazione dell'attività antifolica e antitumorale in vitro

Nell’ambito di un esteso progetto di ricerca sulle potenzialità farmacobiologiche dei derivati chinossalinici come analoghi degli antifolici classici, metotressato, e non classici, trimetressato, sono state sintetizzate numerose molecole che hanno mostrato di possedere in vitro attività antitumorale ed antifolica. Di questi composti verranno descritti la sintesi ed i risultati biologici relativi alla loro attività antitumorale e a quella antifolica

Analoghi chinossalinici omologhi del thymitaq e 2-(ariltio)chinossaline analoghe del trimetrexato e del metotrexato

Abbiamo progettato una nuove serie di chinossaline le quali possono comportarsi come bioisosteri delle pteridine e delle chinazoline dotate di attività antifolica, apparse recentemente in letteratura

Chroman-4-One Derivatives Targeting Pteridine Reductase 1 and Showing Anti-Parasitic Activity

Flavonoids have previously been identified as antiparasitic agents and pteridine reductase 1 (PTR1) inhibitors. Herein, we focus our attention on the chroman-4-one scaffold. Three chroman-4-one analogues (1-3) of previously published chromen-4-one derivatives were synthesized and biologically evaluated against parasitic enzymes (Trypanosoma brucei PTR1-TbPTR1 and Leishmania major-LmPTR1) and parasites (Trypanosoma brucei and Leishmania infantum). A crystal structure of TbPTR1 in complex with compound 1 and the first crystal structures of LmPTR1-flavanone complexes (compounds 1 and 3) were solved. The inhibitory activity of the chroman-4-one and chromen-4-one derivatives was explained by comparison of observed and predicted binding modes of the compounds. Compound 1 showed activity both against the targeted enzymes and the parasites with a selectivity index greater than 7 and a low toxicity. Our results provide a basis for further scaffold optimization and structure-based drug design aimed at the identification of potent anti-trypanosomatidic compounds targeting multiple PTR1 variants

Profiling of Flavonol Derivatives for the Development of Antitrypanosomatidic Drugs

Flavonoids represent a potential source of new antitrypanosomatidic leads. Starting from a library of natural products, we combined target-based screening on pteridine reductase 1 with phenotypic screening on Trypanosoma brucei for hit identification. Flavonols were identified as hits, and a library of 16 derivatives was synthesized. Twelve compounds showed EC50 values against T. brucei below 10 \u3bcM. Four X-ray crystal structures and docking studies explained the observed structure-activity relationships. Compound 2 (3,6-dihydroxy-2-(3-hydroxyphenyl)-4H-chromen-4-one) was selected for pharmacokinetic studies. Encapsulation of compound 2 in PLGA nanoparticles or cyclodextrins resulted in lower in vitro toxicity when compared to the free compound. Combination studies with methotrexate revealed that compound 13 (3-hydroxy-6-methoxy-2-(4-methoxyphenyl)-4H-chromen-4-one) has the highest synergistic effect at concentration of 1.3 \u3bcM, 11.7-fold dose reduction index and no toxicity toward host cells. Our results provide the basis for further chemical modifications aimed at identifying novel antitrypanosomatidic agents showing higher potency toward PTR1 and increased metabolic stability

Targeting Class A and C Serine \u3b2-Lactamases with a Broad-Spectrum Boronic Acid Derivative

Production of \u3b2-lactamases (BLs) is the most widespread resistance mechanism adopted by bacteria to fight \u3b2-lactam antibiotics. The substrate spectrum of BLs has become increasingly broad, posing a serious health problem. Thus, there is an urgent need for novel BL inhibitors. Boronic acid transition-state analogues are able to reverse the resistance conferred by class A and C BLs. We describe a boronic acid analogue possessing interesting and potent broad-spectrum activity vs class A and C serine-based BLs. Starting from benzo(b)thiophene-2-boronic acid (BZBTH2B), a nanomolar non-\u3b2-lactam inhibitor of AmpC that can potentiate the activity of a third-generation cephalosporin against AmpC-producing resistant bacteria, we designed a novel broad-spectrum nanomolar inhibitor of class A and C BLs. Structure-based drug design (SBDD), synthesis, enzymology data, and X-ray crystallography results are discussed. We clarified the inhibitor binding geometry responsible for broad-spectrum activity vs serine-active BLs using double mutant thermodynamic cycle studies

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