Towards the generation and screening of a three-million linear peptide library

reservedLe nuove terapie farmacologiche dovrebbero trattare le esigenze mediche ancora insoddisfatte con un’elevata efficacia e senza effetti collaterali. Al fine di raggiungere tale obiettivo, si ricercano molecole che abbiano una potente attività contro le proteine implicate nelle diverse malattie. Per ampliare la quantità di possibili bersagli terapeutici, includendo anche le proteine intracellulari, sono necessari nuovi metodi per la scoperta di molecole che presentino una potente attività e che siano altamente permeabili alle membrane. Il gruppo di ricerca de LPPT ha recentemente sviluppato dei metodi per la sintesi chimica high throughput su micropiastre di decine di migliaia di piccoli composti peptidici che siano permeabili alle membrane e che presentino proprietà che ne permettano lo sviluppo come terapie somministrabili per via orale. In questo elaborato viene riportato lo studio effettuato per trovare un metodo che permetta la sintesi di quasi tre milioni di composti peptidici in un’unica libreria. Proponiamo la generazione di una libreria così ampia con un approccio mix-and-split: in seguito alla sintesi di 1.536 peptidi in parallelo sul sintetizzatore automatico di peptidi, la resina nei pozzetti della micropiastra sarebbe rimossa, mescolata e quindi ridistribuita nelle micropiastre per ottenere miscele di 96 peptidi in ogni pozzetto. A questo punto, le miscele verrebbero accoppiate ad un primo acido carbossilico su resina, consentendo quindi di ottenere 96 prodotti amidici per pozzetto. I prodotti verrebbero poi rilasciati dalla resina e le soluzioni verrebbero quindi distribuite in altre micropiastre per essere accoppiate a un secondo acido carbossilico in soluzione, nuovamente portando alla formazione di 96 amidi per ogni pozzetto. In totale, questo approccio permetterebbe la sintesi di 2.949.120 composti con un solo round di sintesi. Per confermare la validità di questo approccio sintetico, è stato innanzitutto necessario verificare che l’attività di un composto hit fosse rilevabile a concentrazioni ridotte: utilizzando la manipolazione acustica dei liquidi per creare miscele simili a quelle previste dall’approccio mix-and-split, è stata dimostrato che l’attività del composto attivo di controllo (IC50 di 100 nM) non purificato è facilmente rilevabile in una miscela equi-molare di 96 composti. In secondo luogo, sono stati sintetizzati 384 peptidi di prova mediante l’approccio mix-and-split, per confermare l’ortogonalità dei due gruppi protettori scelti per le ammine e dimostrare che il composto attivo di controllo potesse essere correttamente deconvoluto in un ciclo di sintesi parallelo. Sfortunatamente, in seguito alla validazione di questi prerequisiti e all’ottimizzazione delle condizioni di sintesi, l’attività del composto attivo non è stata rilevata nello screening funzionale delle miscele di peptidi di prova. L’assenza di attività potrebbe essere causata da una diminuzione della concentrazione dei composti nelle miscele, la quale potrebbe derivare da imprecisione nella procedura di mix-and-split. Per assicurare che l’esito negativo dello screening non dipenda da possibili problemi nella sintesi, un’analisi HRMS verrà effettuata per determinare la presenza del composto attivo nelle miscele e compararne la concentrazione nelle miscele e nei pozzetti individuali. Nonostante il fallimento di questa tecnica, la possibilità di creare librerie così ampie ha il potenziale di rivoluzionare la ricerca di nuovi possibili farmaci, dunque gli sforzi verso questo obiettivo continueranno.Novel therapeutics should treat unmet medical needs with high efficacy and no side effects. Towards this goal, molecules with potent activity against proteins implicated in diseases are sought. To expand the scope of druggable protein targets to include intracellular proteins, new methods are required for the discovery of molecules which are both highly potent and membrane permeable. The LPPT group has recently developed methods for the high-throughput, microplate-based chemical synthesis of tens of thousands of small, peptidic compounds with properties suitable for development as membrane permeable and orally bioavailable therapeutics. Here, we describe the investigation made towards a new method for the synthesis of nearly three million of such compounds in one library. We propose the generation of such a large library via a mix-and-split approach. After 1,536 peptides are synthesised in parallel on an automated peptide synthesiser, the resin in each well of the microplate would be removed, mixed, and then redistributed to microplates to give mixtures of 96 compounds per well. At this point, the mixtures would be coupled to a first carboxylic acid on resin, resulting in 96 amide products per well. The products would then be released from resin, and the resulting liquid stocks distributed into microplates to be coupled to a second carboxylic acid in solution, again giving 96 amide products per well. In total, this approach would enable the synthesis of 2,949,120 compounds with one round of synthesis. To confirm the validity of this approach, it was first verified that the activity of a hit compound is detected at the lowered concentrations by using acoustic liquid handling to create mixtures similar to those envisioned via the mix-and-split approach, showing that the activity of an active, crude control compound (IC50 of 100 nM) can be easily detected in an equimolar mixture of 96 compounds. Secondly, 384 test peptides were synthesised via the mix-and-split approach to confirm that the two chosen amine protecting groups were orthogonal and to demonstrate that the active control structure could be correctly deconvoluted in a parallel round of synthesis. Unfortunately, after the validation of these prerequisites and the optimisation of the synthesis conditions to achieve the desired products, the activity of the active compound couldn’t be detected in the screening of the test peptide mixtures. The lack of activity could stem from a decrease in compound concentration that may be caused by imprecision in the mix-and-split procedure. To ensure that the failed outcome isn’t caused by potential synthesis issues, an HRMS analysis will be conducted to determine the presence of the active compound in the mixtures and compare its concentration in the mixtures and in the individual wells. Despite the failure of this technique, the possibility to create such vast peptide libraries has the potential to revolutionise the search for novel drug candidates, therefore the efforts towards this objective will continue

Screening for Tuberculosis Infection among Migrants: A Cost-Effectiveness Analysis in the Italian Context

Background: Screening of tuberculosis infection (TBI) among migrants from high-incidence countries is a cornerstone of tuberculosis control in low-incidence countries. However, the optimal screening strategy has not been defined yet. Methods: A quasi-experimental study involving migrants residing in the province of Brescia was carried out that aimed at assessing the completion rate, time to completion, preventive treatment initiation rate, and cost-effectiveness of two strategies for TBI screening. They underwent TBI screening with the IGRA-only strategy (arm 1) or with the sequential strategy (tuberculin skin test, TST, followed by IGRA in case of a positive result—arm 2). The two strategies were compared in terms of screening completion, time to complete the screening process, therapy initiation, and cost-effectiveness. Results: Between May 2019 and May 2022, 657 migrants were evaluated, and 599 subjects were included in the study, with 358 assigned to arm 1 and 237 to arm 2. Screening strategy was the only factor associated with screening completion in a multivariable analysis, with the subjects assigned to the IGRA-only strategy more likely to complete the screening cascade (n = 328, 91.6% vs. n = 202, 85.2%, IRR 1.08, 95% CI (1.01–1.14), p = 0.019). The time to complete the screening process was significantly longer for patients assigned to the sequential strategy arm (74 days vs. 46 days, p = 0.002). Therapy initiation did not significantly differ between the two arms, and cost-effectiveness was higher for the sequential strategy. Conclusion: Sequential strategy implementation for TBI screening among migrants may be justified by its higher cost-effectiveness in spite of the lower completion of the screening cascade

Tocilizumab for patients with COVID-19 pneumonia. The single-arm TOCIVID-19 prospective trial

BackgroundTocilizumab blocks pro-inflammatory activity of interleukin-6 (IL-6), involved in pathogenesis of pneumonia the most frequent cause of death in COVID-19 patients.MethodsA multicenter, single-arm, hypothesis-driven trial was planned, according to a phase 2 design, to study the effect of tocilizumab on lethality rates at 14 and 30 days (co-primary endpoints, a priori expected rates being 20 and 35%, respectively). A further prospective cohort of patients, consecutively enrolled after the first cohort was accomplished, was used as a secondary validation dataset. The two cohorts were evaluated jointly in an exploratory multivariable logistic regression model to assess prognostic variables on survival.ResultsIn the primary intention-to-treat (ITT) phase 2 population, 180/301 (59.8%) subjects received tocilizumab, and 67 deaths were observed overall. Lethality rates were equal to 18.4% (97.5% CI: 13.6-24.0, P=0.52) and 22.4% (97.5% CI: 17.2-28.3, P<0.001) at 14 and 30 days, respectively. Lethality rates were lower in the validation dataset, that included 920 patients. No signal of specific drug toxicity was reported. In the exploratory multivariable logistic regression analysis, older age and lower PaO2/FiO2 ratio negatively affected survival, while the concurrent use of steroids was associated with greater survival. A statistically significant interaction was found between tocilizumab and respiratory support, suggesting that tocilizumab might be more effective in patients not requiring mechanical respiratory support at baseline.ConclusionsTocilizumab reduced lethality rate at 30 days compared with null hypothesis, without significant toxicity. Possibly, this effect could be limited to patients not requiring mechanical respiratory support at baseline.Registration EudraCT (2020-001110-38); clinicaltrials.gov (NCT04317092)