IT202000019708A1 - Angiotensin-converting enzyme 2 (ACE2) receptor-binding peptides and their medical uses - Google Patents
Angiotensin-converting enzyme 2 (ACE2) receptor-binding peptides and their medical uses Download PDFInfo
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
- C07K7/04—Linear peptides containing only normal peptide links
- C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
- C07K7/04—Linear peptides containing only normal peptide links
- C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
- C12Y304/17—Metallocarboxypeptidases (3.4.17)
- C12Y304/17023—Angiotensin-converting enzyme 2 (3.4.17.23)
Description
PEPTIDI IN GRADO DI LEGARE IL RECETTORE ENZIMA DI CONVERSIONE DELL?ANGIOTENSINA 2 (ACE2) E LORO USI ANGIOTENSIN-CONVERTING ENzyme 2 (ACE2) RECEPTOR-BINDING PEPTIDES AND THEIR USES
MEDICI DOCTORS
La presente invenzione riguarda peptidi in grado di legare il recettore enzima di conversione dell?angiotensina 2 (ACE2) e loro usi medici. The present invention relates to peptides capable of binding the angiotensin converting enzyme 2 receptor (ACE2) and their medical uses.
In particolare, la presente invenzione riguarda peptidi capaci di legare il recettore ACE2 e loro usi medici, nel trattamento e prevenzione di infezioni virali causate da virus che sfruttano un?interazione con il recettore ACE2 per entrare nelle cellule ospite, come il SARS-CoV-2, e delle malattie causate da suddette infezioni. In particular, the present invention relates to peptides capable of binding the ACE2 receptor and their medical uses, in the treatment and prevention of viral infections caused by viruses that exploit an interaction with the ACE2 receptor to enter host cells, such as SARS-CoV- 2, and diseases caused by the above infections.
La sindrome acuta respiratoria grave da coronavirus 2 (SARS-CoV-2) fu per prima identificata in Cina nel Dicembre 2019 ed in pochi mesi divenne una minaccia mondiale che condizion? pesantemente la salute pubblica, la vita sociale e l?economia delle nazioni. Nei pazienti affetti in modo pi? severo, l?infezione ? caratterizzata da una devastante risposta di citochine, trombosi e disfunzione d?organo (1-2). Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was first identified in China in December 2019 and within months it became a global threat affecting heavily public health, social life and the economy of nations. In patients affected more severe, the infection? characterized by a devastating cytokine response, thrombosis and organ dysfunction (1-2).
SARS-CoV, il predecessore del SARS-CoV-2, fu anch?esso per prima riconosciuto in Cina, nel 2002, come responsabile della sindrome acuta respiratoria grave (SARS). In pochi mesi si diffuse in altre 30 nazioni (principalmente asiatiche, per esempio, Hong Kong, Singapore, Taiwan, Vietnam etc, con molti pochi casi in Canada, Stati Uniti ed Europa) uccidendo circa il 10% del totale di 8000 persone infette. SARS-CoV, the predecessor of SARS-CoV-2, was also first recognized in China in 2002 as responsible for severe acute respiratory syndrome (SARS). In a few months it spread to 30 other countries (mainly Asian, for example, Hong Kong, Singapore, Taiwan, Vietnam etc, with very few cases in Canada, the United States and Europe) killing about 10% of the total 8,000 infected people.
L?eradicazione della SARS (gli ultimi casi riportati furono identificati nel 2004) ? ritenuta essere stata il risultato di misure essenzialmente identiche a quelle applicate durante le epidemie medioevali, quali l?isolamento dei pazienti infetti e la quarantena di chiunque fosse noto o sospettato essere stato in contatto con persone infette. Nessun farmaco ? stato messo a punto durante l?epidemia SARS-CoV neanche negli anni successivi, e tutti i tentativi di produrre vaccini contro la SARS e la sindrome respiratoria mediorientale (MERS) fallirono. Studi su potenziali vaccini sollevarono problemi di sicurezza dopo l?osservazione che incrementano il rischio di immunopatologie polmonari (3-6).Inoltre, i vaccini non garantiscono immunizzazione contro i coronavirus dal momento che questi virus sono caratterizzati da alte velocit? di mutazione. The eradication of SARS (the last reported cases were identified in 2004) ? believed to have been the result of measures essentially the same as those applied during medieval epidemics, such as the isolation of infected patients and the quarantine of anyone known or suspected to have been in contact with infected people. No drugs ? was developed during the SARS-CoV epidemic even in the following years, and all attempts to produce vaccines against SARS and Middle East respiratory syndrome (MERS) failed. Studies of potential vaccines raised safety concerns after the observation that they increase the risk of pulmonary immunopathologies (3-6). Furthermore, vaccines do not guarantee immunization against coronaviruses since these viruses are characterized by high rates of diffusion. of mutation.
Riguardo il SARS-CoV-2, molte sperimentazioni cliniche sono in corso per il trattamento della malattia da coronavirus 2019 (COVID-19). Fra questi, la plasmaterapia (anche chiamata plasma convalescente), che fa uso del plasma ottenuto da donatori guariti, e precedentemente usato con successo nel trattamento della SARS, MERS, e la pandemia H1N1 nel 2009, present? risultati eccellenti su pazienti gravemente malati di covid-19. Molti trattamenti farmacologici sono anche sotto investigazione per fasi differenti della malattia, i quali implicano farmaci antivirali, e farmaci o anticorpi capaci di ridurre le anomale eccessive reazioni immunologiche responsabili dei danni d?organo (8). Questi trattamenti consistono di sostanze riposizionate da altre malattie, che possono velocizzare la ricerca di opzioni terapeutiche considerato che i loro meccanismi di azione e profili di tossicit? sono almeno in parte noti. Regarding SARS-CoV-2, many clinical trials are ongoing for the treatment of coronavirus disease 2019 (COVID-19). Among them, plasma therapy (also called convalescent plasma), which uses plasma obtained from recovered donors, and previously used successfully in the treatment of SARS, MERS, and the H1N1 pandemic in 2009, is present. excellent results on patients seriously ill with covid-19. Many pharmacological treatments are also under investigation for different stages of the disease, which involve antiviral drugs, and drugs or antibodies capable of reducing the abnormal excessive immunological reactions responsible for organ damage (8). These treatments consist of substances repositioned from other diseases, which may expedite the search for treatment options given that their mechanisms of action and toxicity profiles are different. are at least partially known.
Riguardo gli antivirali, un comune svantaggio di questi farmaci ? rappresentato dagli effetti collaterali severi e dal fatto che possono indurre resistenza ai virus. In particolare, considerate le caratteristiche di elevate velocit? di mutazione ed infezione del SARS-CoV-2, si pu? prevedere che l?utilit? clinica di approcci terapeutici basati su farmaci che colpiscono specificamente i componenti molecolari del SARS-CoV-2 potrebbe essere ridotta dalla rapida emergenza di ceppi resistenti. Inoltre, c?? da considerare la difficolt? intrinseca di produrre antivirali nuovi ed efficienti. Ci? ? anche evidente con il fatto che alla data attuale nessun nuovo farmaco antivirale ? stato ottenuto per la specifica neutralizzazione del SARS-CoV-2 o del suo predecessore, SARS-CoV, nonostante siano trascorse quasi due decadi dalla prima comparsa del pi? vecchio dei due virus. Dopo tutto, il vecchio ed il nuovo coronavirus presentano lo stesso meccanismo di infezione. Regarding antivirals, a common disadvantage of these drugs? represented by severe side effects and the fact that they can induce resistance to viruses. In particular, consider the characteristics of high speeds? of mutation and infection of SARS-CoV-2, is it possible? to foresee that the? utility? clinical trials of drug-based therapeutic approaches that specifically target the molecular components of SARS-CoV-2 could be curtailed by the rapid emergence of resistant strains. Also, there?? to consider the difficulty? intrinsic ability to produce new and efficient antivirals. There? ? also evident with the fact that to date no new antiviral drug ? been obtained for the specific neutralization of SARS-CoV-2 or its predecessor, SARS-CoV, despite the fact that almost two decades have passed since the first appearance of the most? old of the two viruses. After all, the old and new coronaviruses have the same infection mechanism.
Il genoma del SARS-CoV-2 condivide circa l?80% di identit? di sequenza con il SARS-CoV. In aggiunta, la glicoproteina spike, posizionata sulla superficie virale, ? sfruttata da entrambi i virus per legarsi allo stesso recettore cellulare dell?ospite che ? l?enzima di conversione dell?angiotensina 2 (ACE2) (910), e per mediare la fusione delle membrane virali e cellulari (11-12). Quindi, l?interazione tra le proteine spike ed ACE2 ? essenziale per il virus per conseguire l?accesso nelle cellule ed iniziare la sua replicazione. Does the SARS-CoV-2 genome share about 80% identity? sequence with SARS-CoV. In addition, the spike glycoprotein, located on the viral surface, is exploited by both viruses to bind to the same cellular receptor of the host which is angiotensin-converting enzyme 2 (ACE2) (910), and to mediate the fusion of viral and cellular membranes (11-12). So, the interaction between spike proteins and ACE2? essential for the virus to gain entry into cells and begin its replication.
Anche se la pandemia da SARS-CoV-2 cesser? in modo naturale, in assenza di farmaci che bloccano specificamente questo tipo di virus resteremo quasi impreparati se un?altra variante pericolosa del virus emerger? in futuro. Perci?, sar? molto utile aprire la strada a strategie farmacologiche che colpiscono efficientemente e specificamente il SARS-CoV-2 ed altri membri della famiglia dei beta coronavirus. Even if the SARS-CoV-2 pandemic ends? naturally, in the absence of drugs that specifically block this type of virus we will remain almost unprepared if another dangerous variant of the virus emerges? in the future. Therefore?, will? very useful to pave the way for pharmacological strategies that efficiently and specifically target SARS-CoV-2 and other members of the beta coronavirus family.
Alla luce di quanto sopra, ? quindi chiara la necessit? di rendere disponibili nuovi composti per il trattamento e la prevenzione di infezioni e malattie causate da coronavirus, in particolare dal SARS-CoV-2, che siano capaci di superare gli svantaggi delle terapie conosciute. In light of the above, so clear the need? to make available new compounds for the treatment and prevention of infections and diseases caused by coronaviruses, in particular by SARS-CoV-2, which are capable of overcoming the disadvantages of known therapies.
In base alla presente invenzione, sono stati ora trovati piccoli peptidi, i quali sono consistenti di specifici motivi di amminoacidi e sono capaci di legare l?enzima di conversione dell?angiotensina 2 (ACE2) con alta affinit? e di antagonizzare l?interazione di questa proteina con le proteine spike di virus. In particolare, i piccoli peptidi della presente invenzione legano ACE2 con alta affinit? ad un sito che si sovrappone con la regione di ACE2 che interagisce con la proteina spike. Based on the present invention, small peptides have now been found, which consist of specific amino acid motifs and are capable of binding angiotensin-converting enzyme 2 (ACE2) with high affinity and to antagonize the interaction of this protein with virus spike proteins. In particular, the small peptides of the present invention bind ACE2 with high affinity to a site that overlaps with the region of ACE2 that interacts with the spike protein.
Quindi, i peptidi relativi alla presente invenzione sono capaci di bloccare l?interazione di SARS-CoV-2, SARS-CoV e coronavirus umano NL63 (HCoV-NL63), dato che peptidi e virus legano regioni sovrapponibili nel recettore ACE2. Inoltre, i peptidi della presente invenzione possono anche bloccare altri virus che sfruttano il recettore ACE2 per entrare nelle cellule ospite. Thus, the peptides related to the present invention are capable of blocking the interaction of SARS-CoV-2, SARS-CoV and human coronavirus NL63 (HCoV-NL63), since peptides and viruses bind overlapping regions in the ACE2 receptor. Furthermore, the peptides of the present invention can also block other viruses that exploit the ACE2 receptor to enter host cells.
Sulla base di quanto sopra, i peptidi relativi alla presente invenzione rappresentano un trattamento farmacologico contro il SARS-CoV-2 ed altri virus che sfruttano le interazioni con il recettore ACE2 per entrare nelle cellule ospiti. Based on the above, the peptides related to the present invention represent a pharmacological treatment against SARS-CoV-2 and other viruses which exploit the interactions with the ACE2 receptor to enter host cells.
I peptidi in accordo alla presente invenzione sono vantaggiosamente capaci di bloccare l?infezione nella fase iniziale, prevenendo l?ingresso dei virus nelle cellule. In aggiunta, i peptidi in accordo alla presente invenzione svolgono la loro funzione antivirale extracellularmente. Quindi, basse concentrazioni di peptidi possono essere usate ed i peptidi non necessitano di penetrare nelle cellule. Ci? implica che i peptidi in accordo alla presente invenzione presentano profili di tossicit? bassi. The peptides according to the present invention are advantageously capable of blocking the infection in the initial phase, preventing the entry of the viruses into the cells. In addition, the peptides according to the present invention perform their antiviral function extracellularly. Thus, low concentrations of peptides can be used and the peptides do not need to penetrate the cells. There? implies that the peptides according to the present invention exhibit toxicity profiles? bass.
Come menzionato sopra, i peptidi in accordo alla presente invenzione sono capaci di legare ACE2, in aggiunta al bloccare qualsiasi virus che sfrutta una interazione con il recettore ACE2 per entrare nelle cellule ospiti (alla data attuale, SARS-CoV, SARS-CoV-2, e coronavirus NL63 umano (HCoV-NL63)). As mentioned above, the peptides according to the present invention are capable of binding ACE2, in addition to blocking any virus that exploits an interaction with the ACE2 receptor to enter host cells (currently, SARS-CoV, SARS-CoV-2 , and human coronavirus NL63 (HCoV-NL63)).
Inoltre, i peptidi in accordo alla presente invenzione possono essere usati come farmaci antivirali contro varianti virali (sia vecchi virus e sia nuovi virus) che legano il recettore ACE2 dentro o vicino la regione di sovrapposizione menzionata sopra. Quindi, a differenza dei trattamenti convenzionali basati su farmaci antivirali o vaccini, i peptidi della presente invenzione possono svolgere il loro effetto antivirale anche contro varianti di virus derivanti da mutazione (o mutazioni) del virus. Furthermore, the peptides according to the present invention can be used as antiviral drugs against viral variants (both old viruses and new viruses) which bind the ACE2 receptor in or near the overlapping region mentioned above. Therefore, unlike conventional treatments based on antiviral drugs or vaccines, the peptides of the present invention can perform their antiviral effect also against virus variants deriving from mutation (or mutations) of the virus.
Un ulteriore vantaggio dei peptidi concernenti la presente invenzione ? che essi sono disegnati in modo da permettere un facile inserimento di vari gruppi chimici alle loro estremit? (specialmente all?N-termine) senza influenzare la loro capacit? di legare ACE2. A further advantage of the peptides relating to the present invention ? that they are designed to allow easy insertion of various chemical groups at their ends? (especially at? N-term) without affecting their ability? to bind ACE2.
In aggiunta, i peptidi dell?invenzione sono caratterizzati da un disolfuro intramolecolare, il quale comporta la formazione di peptidi ciclici e conferisce ai peptidi una conformazione adatta per l?interazione con ACE2. Ciclizzazioni analoghe a quelle ottenute con un disolfuro intramolecolare possono essere ottenute sfruttando altre adeguate coniugazioni chimiche tra amminoacidi, le quali sono note dallo stato dell?arte. In addition, the peptides of the invention are characterized by an intramolecular disulfide, which leads to the formation of cyclic peptides and gives the peptides a conformation suitable for interaction with ACE2. Cyclizations analogous to those obtained with an intramolecular disulfide can be obtained by exploiting other suitable chemical conjugations between aminoacids, which are known from the state of the art.
Inoltre, l?efficienza di questi peptidi nell?antagonizzare l?interazione spike-ACE2 pu? essere modificata coniugando chimicamente adeguati gruppi voluminosi (come noto dallo stato dell?arte) per produrre il desiderato ingombro sterico al di dentro o vicino la regione di ACE2 adibita a bersaglio per le interazioni di legame da parte delle proteine virali spike. Furthermore, the efficiency of these peptides in antagonizing the spike-ACE2 interaction can be modified by chemically conjugating appropriate bulk groups (as known from the state of the art) to produce the desired steric hindrance in or near the region of ACE2 targeted for binding interactions by viral spike proteins.
In aggiunta, la modalit? con cui questi peptidi sono somministrati (orale, sistemica, aerosolizzati nelle vie respiratorie, etc) o stabilizzati (per conseguire resistenza o suscettibilit? all?attivit? di proteasi o a modifiche da parte di altri enzimi) o rilasciati nei tessuti desiderati pu? essere modificata inserendo adeguati gruppi chimici (come noto dallo stato dell?arte). In addition, the mode with which these peptides are administered (orally, systemically, aerosolized into the respiratory tract, etc) or stabilized (to achieve resistance or susceptibility to protease activity or modifications by other enzymes) or released into the desired tissues can? be modified by inserting suitable chemical groups (as known from the state of the art).
I peptidi della presente invenzione sono disegnati per legarsi ad una particolare regione di ACE2 che ? relativamente piccola, piatta e circondata da diversi siti di glicosilazione. Queste caratteristiche hanno finora reso difficile bersagliare farmacologicamente questa regione di ACE2 con farmaci per finalit? antivirali e di altro tipo. The peptides of the present invention are designed to bind to a particular region of ACE2 which ? relatively small, flat, and surrounded by several glycosylation sites. These features have so far made it difficult to pharmacologically target this region of ACE2 with targeted drugs. antivirals and others.
La possibilit? di avere farmaci che si legano su questa nuova regione apre nuove prospettive anche per la regolazione dell?attivit? di ACE2. Perci?, in aggiunta agli usi antivirali, la capacit? di questi peptidi di legare fisicamente ACE2 potrebbe anche essere usata per proteggere/stabilizzare farmacologicamente questa proteina in modo da sostenere pi? alta attivit? enzimatica di ACE2 dal momento che questo ? desiderabile in alcune condizioni. Infatti, un?incrementata attivit? di ACE2 ? stata dimostrata essere protettiva in malattie cardiovascolari (14-17), diabete (18), danni a fegato e reni (19-20), ed insufficienza polmonare (21-22). Gli effetti benefici di un incremento indotto dell?attivit? di ACE2 sono stati dimostrati per diverse patologie impiegando enzima ACE2 ricombinante e piccole molecole attivanti (23), ed un enzima simile ad ACE2 derivato da batteri (24). The possibility? to have drugs that bind on this new region opens up new perspectives also for the regulation of? by ACE2. Therefore, in addition to the antiviral uses, the ability of these peptides to physically bind ACE2 could also be used to pharmacologically protect/stabilize this protein in order to support more? high activity enzyme of ACE2 since this ? desirable under some conditions. In fact, a? Increased activity? of ACE2 ? has been shown to be protective in cardiovascular disease (14-17), diabetes (18), liver and kidney damage (19-20), and pulmonary failure (21-22). The beneficial effects of an induced increase in activity? of ACE2 have been demonstrated for various pathologies using recombinant ACE2 enzyme and small activating molecules (23), and an ACE2-like enzyme derived from bacteria (24).
Forma pertanto oggetto specifico della presente invenzione un peptide in grado di legare il recettore enzima di conversione dell?angiotensina 2, detto peptide comprendendo o essendo consistente nei seguenti motivi di amminoacidi: Therefore, a specific object of the present invention is a peptide capable of binding the angiotensin-converting enzyme 2 receptor, said peptide comprising or consisting of the following amino acid motifs:
Motivo I Reason I
Secondo la presente invenzione, i peptidi possono comprendere o consistere in uno dei seguenti motivi di amminoacidi: According to the present invention, the peptides may comprise or consist of any of the following amino acid motifs:
oppure or
Motivo III Reason III
I motivi sono scritti secondo la sintassi di Prosite (25). Specificamente, la parentesi quadra indica che la posizione ? occupata da uno degli amminoacidi elencati nella stessa parentesi, "x" rappresenta un qualsiasi amminoacido, e la parentesi alla destra di un amminoacido indica quante volte l?amminoacido ? ripetuto. La possibile forma stereoisomera L oppure D ? indicata sotto il motivo; ?L/D" significa che il particolare amminoacido pu? essere in forma L oppure D. The motifs are written according to the Prosite syntax (25). Specifically, the square bracket indicates that the position ? occupied by one of the amino acids listed in the same parenthesis, "x" represents any amino acid, and the parenthesis to the right of an amino acid indicates how many times the amino acid ? repeated. The possible stereoisomeric form L or D ? indicated below the reason; "L/D" means that the particular amino acid can be in L or D form.
In particolare, il peptide secondo la presente invenzione ? in grado di legare sulla regione dei residui Asp30, Asn33, His34, Glu37, Gly319, Leu320, Pro321, Thr324, Lys353, Gly354, Phe356, Met383, Ala384, Ala386, Ala387, Gln388, Pro389, Arg393, Phe555, Arg559 del recettore enzima di conversione dell?angiotensina 2. In particular, the peptide according to the present invention ? able to bind on the Asp30, Asn33, His34, Glu37, Gly319, Leu320, Pro321, Thr324, Lys353, Gly354, Phe356, Met383, Ala384, Ala386, Ala387, Gln388, Pro389, Arg393, Phe555, Arg559 residue region of the enzyme receptor of angiotensin conversion 2.
Secondo una specifica forma di realizzazione della presente invenzione, il peptide pu? comprendere o consistere in una sequenza scelta dal gruppo consistente in Gln-Cys-Tyr-Met-Cys-D-Ser-D-Val-Tyr (SEQ ID NO:1), Lys-Cys-Tyr-Met-Cys-D-Ser-Val-Tyr (SEQ ID NO:2), Lys-Cys-Tyr-Met-Cys-D-Glu-Val-Tyr (SEQ ID NO:3) e Lys-Cys-Tyr-Leu-Cys-D-Glu-Ala-Tyr-Gly-Val (SEQ ID NO:4), preferibilmente SEQ ID NO:1, oppure loro combinazioni. According to a specific embodiment of the present invention, the peptide can comprise or consist of a sequence selected from the group consisting of Gln-Cys-Tyr-Met-Cys-D-Ser-D-Val-Tyr (SEQ ID NO:1), Lys-Cys-Tyr-Met-Cys-D- Ser-Val-Tyr (SEQ ID NO:2), Lys-Cys-Tyr-Met-Cys-D-Glu-Val-Tyr (SEQ ID NO:3) and Lys-Cys-Tyr-Leu-Cys-D- Glu-Ala-Tyr-Gly-Val (SEQ ID NO:4), preferably SEQ ID NO:1, or combinations thereof.
I peptidi di sequenza SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3 e SEQ ID NO:4 sono qui anche chiamati SG2, SG1, SG3 e SG4, rispettivamente. The peptides of sequence SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3 and SEQ ID NO:4 are also referred to herein as SG2, SG1, SG3 and SG4, respectively.
Essi possono essere rappresentati come segue: They can be represented as follows:
Come menzionato sopra, i piccoli peptidi SG1, SG2, SG3, e SG4 hanno alta affinit? di legame all?ACE2 umana in siti di questo recettore sovrapponibili con le regioni di legame sfruttate da SARS-CoV, SARS-CoV-2, e HCoV-NL63 nella loro interazione con le cellule ospite. Le linee che connettono le due cisteine rappresentano un legame disolfuro intramolecolare (S-S). As mentioned above, the small peptides SG1, SG2, SG3, and SG4 have high affinity of binding to human ACE2 in sites of this receptor that overlap with the binding regions exploited by SARS-CoV, SARS-CoV-2, and HCoV-NL63 in their interaction with host cells. The lines connecting the two cysteines represent an intramolecular disulfide bond (S-S).
Il legame disolfuro intramolecolare pu? essere formato tra Cys2 e Cys5 dei peptidi. Questo disolfuro conferisce ai peptidi una conformazione adatta per il legame con ACE2. Inoltre, il legame disolfuro intramolecolare causa ciclizzazione molecolare, la quale incrementa la stabilit? dei peptidi. I legami disolfuro possono essere formati sia prima dell?uso dei peptidi, o anche durante il loro uso. Infatti, i legami disolfuro si formano spontaneamente in condizioni redox ossidanti, per esempio nel plasma del sangue (13). Quindi, la formazione del legame disolfuro intramolecolare in questi peptidi pu? avvenire anche dopo la somministrazione dei peptidi ai pazienti. ? particolarmente possibile che questa reazione avvenga perch? i peptidi contengono due cisteine prossimali e sono esposti alle condizioni ossidative nel microambiente extracellulare dove ACE2, l?obiettivo designato dei peptidi, ? localizzato. The intramolecular disulfide bond can be formed between Cys2 and Cys5 of the peptides. This disulfide gives the peptides a conformation suitable for binding to ACE2. Furthermore, the intramolecular disulfide bond causes molecular cyclization, which increases the stability of the molecule. of the peptides. Disulfide bonds can be formed either before the use of the peptides, or even during their use. In fact, disulphide bonds are formed spontaneously under oxidizing redox conditions, for example in blood plasma (13). Thus, intramolecular disulfide bond formation in these peptides can occur even after administration of the peptides to patients. ? particularly possible that this reaction occurs because? the peptides contain two proximal cysteines and are exposed to oxidative conditions in the extracellular microenvironment where ACE2, the intended target of the peptides, ? localized.
I peptidi sono progettati per legare ACE2 nella regione mostrata in Figura 1. The peptides are designed to bind ACE2 in the region shown in figure 1.
Secondo la presente invenzione, le combinazioni dei peptidi di sequenza SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3 e SEQ ID NO:4 possono essere per esempio: SEQ ID NO:1 e SEQ ID NO:2; SEQ ID NO:1 e SEQ ID NO:3; SEQ ID NO:1 e SEQ ID NO:4; SEQ ID NO:2 e SEQ ID NO:3; SEQ ID NO:2 e SEQ ID NO:4; SEQ ID NO:3 e SEQ ID NO:4; SEQ ID NO:1, SEQ ID NO:2 e SEQ ID NO:3; SEQ ID NO:1, SEQ ID NO:3 e SEQ ID NO:4; SEQ ID NO:1, SEQ ID NO:2 e SEQ ID NO:4; SEQ ID NO:2, SEQ ID NO:3 e SEQ ID NO:4; SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3 e SEQ ID NO:4. I peptidi menzionati sopra SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3 e SEQ ID NO:4 sono identificate dal motivo I e II. According to the present invention, the combinations of the peptides of the sequence SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3 and SEQ ID NO:4 can be for example: SEQ ID NO:1 and SEQ ID NO:2 ; SEQ ID NO:1 and SEQ ID NO:3; SEQ ID NO:1 and SEQ ID NO:4; SEQ ID NO:2 and SEQ ID NO:3; SEQ ID NO:2 and SEQ ID NO:4; SEQ ID NO:3 and SEQ ID NO:4; SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3; SEQ ID NO:1, SEQ ID NO:3 and SEQ ID NO:4; SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:4; SEQ ID NO:2, SEQ ID NO:3 and SEQ ID NO:4; SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3 and SEQ ID NO:4. The peptides mentioned above SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3 and SEQ ID NO:4 are identified by motif I and II.
Secondo ulteriori forme di realizzazione della presente invenzione, il peptide pu? comprendere o consistere in una sequenza scelta dal gruppo consistente in According to further embodiments of the present invention, the peptide can comprise or consist of a sequence chosen from the group consisting of
Gln-Glu-His-Cys-Tyr-Met-Cys-Ala-Glu-Leu-Met-Gln (SEQ ID NO:5), Gln-Glu-His-Cys-Tyr-Met-Cys-Ala-Glu-Leu-Met-Gln (SEQ ID NO:5),
D-Tyr-Glu-His-Cys-Tyr-Met-Cys-Ser-Asp-D-Met-Tyr (SEQ ID NO:6), D-Tyr-Glu-His-Cys-Tyr-Met-Cys-Ser-Asp-D-Met-Tyr (SEQ ID NO:6),
D-Tyr-Gly-Asp-His-Cys-Tyr-Met-Cys-D-Asp-Val-D-Tyr (SEQ ID NO:7), D-Tyr-Gly-Asp-His-Cys-Tyr-Met-Cys-D-Asp-Val-D-Tyr (SEQ ID NO:7),
D-Tyr-Gly-Asp-His-Cys-Tyr-Met-Cys-D-Asp-Val-Tyr(SEQ ID NO:8) , D-Tyr-Gly-Asp-His-Cys-Tyr-Met-Cys-D-Asp-Val-Tyr(SEQ ID NO:8) ,
Gln-Cys-Tyr-Gly-Cys-D-Ala-D-Ala-Tyr (SEQ ID NO:9), Gln-Cys-Tyr-Gly-Cys-D-Val-D-Val-Tyr (SEQ ID NO:10), Gln-Cys-Tyr-Gly-Cys-D-Ala-D-Ala-Tyr (SEQ ID NO:9), Gln-Cys-Tyr-Gly-Cys-D-Val-D-Val-Tyr (SEQ ID NO:9 :10),
Gln-Cys-Tyr-Met-Cys-D-Ser-Val-Tyr (SEQ ID NO:11), Glu-His-Cys-Tyr-Met-Cys-Ser-Asp-Ala-Tyr (SEQ ID NO:12), Gln-Cys-Tyr-Met-Cys-D-Ser-Val-Tyr (SEQ ID NO:11), Glu-His-Cys-Tyr-Met-Cys-Ser-Asp-Ala-Tyr (SEQ ID NO:12 ),
Glu-His-Cys-Tyr-Met-Cys-Ser-Glu-Val-Tyr (SEQ ID NO:13), Glu-His-Cys-Tyr-Met-Cys-Ser-Glu-Val-Tyr (SEQ ID NO:13),
Gly-Gly-Gly-Glu-His-Cys-Tyr-Met-Cys-Ser-Asp-D-Met-Tyr (SEQ ID NO:14), Gly-Gly-Gly-Glu-His-Cys-Tyr-Met-Cys-Ser-Asp-D-Met-Tyr (SEQ ID NO:14),
His-Cys-Tyr-Met-Cys-D-Ser-Ile-Tyr (SEQ ID NO:15), His-Cys-Tyr-Met-Cys-D-Ser-Met-Tyr (SEQ ID NO:16), His-Cys-Tyr-Met-Cys-D-Ser-Val-Tyr (SEQ ID NO:17), His-Cys-Tyr-Met-Cys-Ser-Tyr-Ile (SEQ ID NO:18), Lys-Cys-D-Tyr-Tyr-Cys-D-Ser-Leu-Met (SEQ ID NO:19), His-Cys-Tyr-Met-Cys-D-Ser-Ile-Tyr (SEQ ID NO:15), His-Cys-Tyr-Met-Cys-D-Ser-Met-Tyr (SEQ ID NO:16), His-Cys-Tyr-Met-Cys-D-Ser-Val-Tyr (SEQ ID NO:17), His-Cys-Tyr-Met-Cys-Ser-Tyr-Ile (SEQ ID NO:18), Lys- Cys-D-Tyr-Tyr-Cys-D-Ser-Leu-Met (SEQ ID NO:19),
Lys-Cys-Tyr-Asp-Cys-D-Ser-Val-Tyr (SEQ ID NO:20), Lys-Cys-Tyr-Gln-Cys-Gln-Tyr-Tyr (SEQ ID NO:21), Lys-Cys-Tyr-Glu-Cys-D-Ser-Val-Tyr (SEQ ID NO:22), Lys-Cys-Tyr-Leu-Cys-D-Glu-Ala-Tyr-D-Ala-Ile (SEQ ID NO:23), Lys-Cys-Tyr-Asp-Cys-D-Ser-Val-Tyr (SEQ ID NO:20), Lys-Cys-Tyr-Gln-Cys-Gln-Tyr-Tyr (SEQ ID NO:21), Lys- Cys-Tyr-Glu-Cys-D-Ser-Val-Tyr (SEQ ID NO:22), Lys-Cys-Tyr-Leu-Cys-D-Glu-Ala-Tyr-D-Ala-Ile (SEQ ID NO :23),
Lys-Cys-Tyr-Leu-Cys-D-Glu-Ala-Tyr-D-Ala-Val (SEQ ID NO:24), Lys-Cys-Tyr-Leu-Cys-D-Glu-Ala-Tyr-D-Ala-Val (SEQ ID NO:24),
Lys-Cys-Tyr-Leu-Cys-D-Glu-Val-Tyr-Gly-Val (SEQ ID NO:25), Lys-Cys-Tyr-Leu-Cys-D-Glu-Val-Tyr-Gly-Val (SEQ ID NO:25),
Lys-Cys-Tyr-Leu-Cys-D-Glu-Val-Tyr-Ile (SEQ ID NO:26), Lys-Cys-Tyr-Leu-Cys-D-Glu-Val-Tyr-Ile (SEQ ID NO:26),
Lys-Cys-Tyr-Leu-Cys-D-Ser-Leu-Tyr (SEQ ID NO:27), Lys-Cys-Tyr-Met-Cys-D-Glu-D-Val-Tyr (SEQ ID NO:28), Lys-Cys-Tyr-Leu-Cys-D-Ser-Leu-Tyr (SEQ ID NO:27), Lys-Cys-Tyr-Met-Cys-D-Glu-D-Val-Tyr (SEQ ID NO:28 ),
Lys-Cys-Tyr-Met-Cys-D-Ser-Leu-D-Met (SEQ ID NO:29), Lys-Cys-Tyr-Met-Cys-D-Ser-Leu-D-Met (SEQ ID NO:29),
Lys-Cys-Tyr-Met-Cys-D-Ser-Leu-Tyr (SEQ ID NO:30), Ser-Ser-His-Cys-Tyr-Met-Cys-Gln-Glu-Leu-Gln-Tyr (SEQ ID NO:31), Lys-Cys-Tyr-Met-Cys-D-Ser-Leu-Tyr (SEQ ID NO:30), Ser-Ser-His-Cys-Tyr-Met-Cys-Gln-Glu-Leu-Gln-Tyr (SEQ ID NO:31),
Ser-Ser-His-Cys-Tyr-Met-Cys-Gln-Glu-Val-Gln-Tyr (SEQ ID NO:32), Ser-Ser-His-Cys-Tyr-Met-Cys-Gln-Glu-Val-Gln-Tyr (SEQ ID NO:32),
Ser-Ser-His-Cys-Tyr-Met-Cys-Gln-Glu-Val-Tyr (SEQ ID NO:33), Ser-Ser-His-Cys-Tyr-Met-Cys-Gln-Glu-Val-Tyr (SEQ ID NO:33),
Tyr-Asp-His-Cys-Tyr-Met-Cys-D-Asp-Ala-D-Tyr (SEQ ID NO:34), Tyr-Asp-His-Cys-Tyr-Met-Cys-D-Asp-Ala-D-Tyr (SEQ ID NO:34),
Tyr-Asp-His-Cys-Tyr-Met-Cys-D-Asp-Ala-Tyr (SEQ ID NO:35), Tyr-Asp-His-Cys-Tyr-Met-Cys-D-Asp-Ala-Tyr (SEQ ID NO:35),
Tyr-Asp-His-Cys-Tyr-Met-Cys-D-Asp-D-Met-D-Tyr (SEQ ID NO:36), Tyr-Asp-His-Cys-Tyr-Met-Cys-D-Asp-D-Met-D-Tyr (SEQ ID NO:36),
Tyr-Asp-His-Cys-Tyr-Met-Cys-D-Asp-D-Met-Tyr (SEQ ID NO:37), Tyr-Asp-His-Cys-Tyr-Met-Cys-D-Asp-D-Met-Tyr (SEQ ID NO:37),
Tyr-Asp-His-Cys-Tyr-Met-Cys-D-Asp-Met-Tyr (SEQ ID NO:38), Tyr-Asp-His-Cys-Tyr-Met-Cys-D-Asp-Met-Tyr (SEQ ID NO:38),
Tyr-Asp-His-Cys-Tyr-Met-Cys-D-Ser-Met-Tyr (SEQ ID NO:39), Tyr-Asp-His-Cys-Tyr-Met-Cys-D-Ser-Met-Tyr (SEQ ID NO:39),
Tyr-Glu-His-Cys-Tyr-Met-Cys-Asp-D-Met-Tyr (SEQ ID NO:40), Tyr-Glu-His-Cys-Tyr-Met-Cys-Asp-D-Met-Tyr (SEQ ID NO:40),
Tyr-Glu-His-Cys-Tyr-Met-Cys-Glu-Thr-D-Tyr (SEQ ID NO:41), Tyr-Glu-His-Cys-Tyr-Met-Cys-Glu-Thr-D-Tyr (SEQ ID NO:41),
Tyr-Glu-His-Cys-Tyr-Thr-Cys-D-Ala-Ile-D-Tyr (SEQ ID NO:42), Tyr-Glu-His-Cys-Tyr-Thr-Cys-D-Ala-Ile-D-Tyr (SEQ ID NO:42),
Tyr-Glu-His-Cys-Tyr-Thr-Cys-Glu-Ile-D-Tyr (SEQ ID NO:43), Tyr-Glu-His-Cys-Tyr-Thr-Cys-Glu-Ile-D-Tyr (SEQ ID NO:43),
Tyr-Glu-His-Cys-Tyr-Val-Cys-Glu-Thr-D-Tyr (SEQ ID NO:44), Tyr-Glu-His-Cys-Tyr-Val-Cys-Glu-Thr-D-Tyr (SEQ ID NO:44),
Tyr-Gly-Asp-His-Cys-Tyr-Met-Cys-D-Ser-Met-Tyr (SEQ ID NO:45), Tyr-Gly-Asp-His-Cys-Tyr-Met-Cys-D-Ser-Met-Tyr (SEQ ID NO:45),
Tyr-Gly-Glu-His-Cys-Tyr-Met-Cys-Asp-Val-Tyr (SEQ ID NO:46), o loro combinazioni. Tyr-Gly-Glu-His-Cys-Tyr-Met-Cys-Asp-Val-Tyr (SEQ ID NO:46), or combinations thereof.
Secondo una forma di realizzazione della presente invenzione, i piccoli peptidi leganti ACE2 possono essere modificati inserendo linker e gruppi chimici (come dallo stato dell?arte) cos? da modificare importanti propriet? e funzioni dei peptidi (stabilit?, localizzazione, capacit? di interferire nel legame delle proteine virali spike con ACE2, capacit? di modulare la funzione di ACE2, etc). Per esempio i peptidi della presente invenzione possono essere legati ad adeguati gruppi voluminosi per produrre il desiderato ingombro sterico all?interno o vicino la regione di ACE2 utilizzata come sito di interazione dalle proteine virali spike, per esempio glicole polietilenico (PEG), o qualsiasi gruppo o anche sequenza peptidica che pu? fornire ingombro sterico o FITC; gruppi chimici per modi di rilascio, per aumentare la solubilit? in vivo (per esempio PEG o sequenze peptidiche idrofile), stabilit? (per esempio PEG, albumina, oppure peptidi leganti albumina ed in quest?ultimo caso i peptidi della presente invenzione possono legarsi spontaneamente ed in vivo all?albumina dei pazienti dopo che essi siano stati somministrati), per raggiungere specifici siti (per esempio l?albumina ? risaputa accumularsi preferenzialmente ai siti di infiammazione, che ? una condizione caratterizzante l?infezione SARS-CoV-2), e per aggiungere una capacit? di modulare la funzione di ACE2. Quindi, i peptidi secondo la presente invenzione possono essere coniugati, preferibilmente all?N-termine, ad uno o pi? gruppi chimici funzionali scelti dai gruppi consistenti in glicole polietilenico (PEG), FITC, albumina o peptidi leganti albumina. According to one embodiment of the present invention, the small ACE2 binding peptides can be modified by inserting linkers and chemical groups (as per the state of the art) as well as to change important properties? and peptide functions (stability, localization, ability to interfere in the binding of viral spike proteins with ACE2, ability to modulate ACE2 function, etc). For example, the peptides of the present invention can be linked to suitable bulk groups to produce the desired steric hindrance in or near the region of ACE2 used as an interaction site by viral spike proteins, e.g. polyethylene glycol (PEG), or any group or even a peptide sequence that can? provide steric hindrance or FITC; chemical groups for ways of release, to increase the solubility? in vivo (for example PEG or hydrophilic peptide sequences), stability? (for example PEG, albumin, or albumin binding peptides and in the latter case the peptides of the present invention can bind spontaneously and in vivo to the albumin of patients after they have been administered), to reach specific sites (for example the? albumin is known to preferentially accumulate at sites of inflammation, which is a hallmark condition of SARS-CoV-2 infection), and to add an ability to to modulate the function of ACE2. Thus, the peptides according to the present invention can be conjugated, preferably at the N-terminus, to one or more? functional chemical groups selected from the groups consisting of polyethylene glycol (PEG), FITC, albumin or albumin binding peptides.
Secondo la presente invenzione, il peptide pu? essere coniugato a detti uno o pi? gruppi chimici funzionali mediante uno o pi? linker, come According to the present invention, the peptide can be conjugated to said one or more? functional chemical groups by one or more? linkers, like
- Ahx-Lys-Gly-Gly-Gly oppure Lys-Gly-Gly-Gly (SEQ ID NO:47), - Ahx-Lys-Gly-Gly-Gly or Lys-Gly-Gly-Gly (SEQ ID NO:47),
- (Gly)n dove n pu? essere un numero variabile tra 1 e 10, - (Gly)n where n pu? be a variable number between 1 and 10,
- (Gly-Ala)n dove n pu? essere un numero variabile tra 1 e 5 e il residuo alanina pu? essere nella forma stereochimica L oppure D, - (Gly-Ala)n where n pu? be a variable number between 1 and 5 and the residual alanine pu? be in the stereochemical form L or D,
- (Gly-Ser)n dove n pu? essere un numero variabile tra 1 e 5 ed il residuo serina pu? essere nella forma stereochimica L oppure D, - (Gly-Ser)n where n pu? be a variable number between 1 and 5 and the residual serine pu? be in the stereochemical form L or D,
- una sequenza tagliabile dal dominio peptidasi di ACE2 come quelle che soddisfano il motivo consenso Pro-Xaa(1-3)-Pro+(idrofobo/basico), dove Xaa(1-3) pu? essere qualsiasi stringa di amminoacidi (con lunghezza da 1 a 3 amminoacidi), il segno ?+? rappresenta il legame tagliabile, ed il C-termine pu? essere un residuo idrofobo oppure un residuo basico. Sequenze identificabili da tale motivo e sequenze aggiuntive note essere tagliabili da ACE2 sono per esempio Asp-Arg-Val-Tyr-Ile-His-Pro-Phe-His+Leu (SEQ ID NO:48), Asp-Arg-Val-Tyr-Ile-His-Pro+Phe (SEQ ID NO:49), Gln-Arg-Pro-Arg-Leu-Ser-His-Lys-Gly-Pro-Met-Pro+Phe (SEQ ID NO:50), Tyr-Pro-Phe-Val-Glu-Pro+Ile (SEQ ID NO:51), Pro-Pro-Gly-Phe-Ser-Pro-Phe+Arg (SEQ ID NO:52), Tyr-Gly-Gly-Phe-Leu-Arg-Arg-Ile-Arg-Pro-Lys-Leu+Lys (SEQ ID NO:53), Ala-Pro+Lys (SEQ ID NO:54), Tyr-Val-Ala-Asp-Ala-Pro+Lys (SEQ ID NO:55), Gln-Leu-Tyr-Glu-Asn-Lys-Pro+Arg (SEQ ID NO:56), Arg-Pro-Pro-Gly-Phe-Ser-Pro+Phe (SEQ ID NO:57), Lys-Arg-Pro-Pro-Gly-Phe-Ser-Pro+Phe (SEQ ID NO:58). L?utilit? di aggiungere un linker tagliabile da parte di ACE2 consiste nel fatto che la coniugazione dei peptidi con altri gruppi pu? essere di aiuto per motivi di stabilizzazione o localizzazione ma nello stesso tempo essa potrebbe far decrescere la loro affinit? per ACE2. In tali casi, sarebbe preferibile che la porzione dei peptidi adibita al legame con ACE2 diventi pi? esposta solo dopo che i peptidi coniugati incontrano le cellule esprimenti ACE2, che sono i siti dove questi peptidi diventano pi? necessari. Ci? pu? essere ottenuto sfruttando linker tagliabili dal recettore ACE2. - a cleavable sequence from the peptidase domain of ACE2 such as those satisfying the consensus motif Pro-Xaa(1-3)-Pro+(hydrophobic/basic), where Xaa(1-3) can? be any string of amino acids (1 to 3 amino acids long), the ?+? represents the bond that can be cut, and the C-term pu? be a hydrophobic residue or a basic residue. Sequences identifiable by this motif and additional sequences known to be cuttable by ACE2 are for example Asp-Arg-Val-Tyr-Ile-His-Pro-Phe-His+Leu (SEQ ID NO:48), Asp-Arg-Val-Tyr -Ile-His-Pro+Phe (SEQ ID NO:49), Gln-Arg-Pro-Arg-Leu-Ser-His-Lys-Gly-Pro-Met-Pro+Phe (SEQ ID NO:50), Tyr -Pro-Phe-Val-Glu-Pro+Ile (SEQ ID NO:51), Pro-Pro-Gly-Phe-Ser-Pro-Phe+Arg (SEQ ID NO:52), Tyr-Gly-Gly-Phe -Leu-Arg-Arg-Ile-Arg-Pro-Lys-Leu+Lys (SEQ ID NO:53), Ala-Pro+Lys (SEQ ID NO:54), Tyr-Val-Ala-Asp-Ala-Pro +Lys (SEQ ID NO:55), Gln-Leu-Tyr-Glu-Asn-Lys-Pro+Arg (SEQ ID NO:56), Arg-Pro-Pro-Gly-Phe-Ser-Pro+Phe (SEQ ID NO:57), Lys-Arg-Pro-Pro-Gly-Phe-Ser-Pro+Phe (SEQ ID NO:58). The utility to add a linker cuttable by ACE2 consists in the fact that the conjugation of the peptides with other groups can? be of help for reasons of stabilization or localization but at the same time it could decrease their affinity? for ACE2. In such cases, it would be preferable that the portion of the peptides used to bind to ACE2 become smaller. exposed only after the conjugated peptides encounter ACE2-expressing cells, which are the sites where these peptides become more? necessary. There? can? be achieved by exploiting cleavable linkers from the ACE2 receptor.
La presente invenzione concerne anche una composizione farmaceutica comprendente o consistente in un peptide come definito sopra, in combinazione con uno o pi? eccipienti e/o adiuvanti farmacologicamente accettabili. The present invention also relates to a pharmaceutical composition comprising or consisting of a peptide as defined above, in combination with one or more? pharmacologically acceptable excipients and/or adjuvants.
La composizione farmaceutica secondo la presente invenzione pu? ulteriormente comprendere uno o pi? di un farmaco scelti dal gruppo consistente in un farmaco antinfiammatorio, come Tocilizumab (anticorpo monoclonale che agisce come un'antagonista del recettore dell?interleuchina 6 (IL-6)) e analoghi come Sarilumab (anticorpo monoclonale che agisce inibendo l?interleuchina 6 (IL-6)), Anakinra (inibitore dell?interleuchina 1), Baricitinib (inibitore della chinasi Giano), Eculizumab (anticorpo monoclonale contro complemento C5), Emapalumab (anticorpo monoclonale contro interferone gamma); un farmaco antimalarico, come Idrossiclorochina (che ? antinfiammatorio ed antimalarico); un farmaco antibiotico, come Azitromicina; un farmaco antivirale, come Remdesivir (pro farmaco di un analogo nucleotidico con attivit? antivirale ad ampio spettro), Lopinavir e Ritonavir (inibitori di proteasi virali che sono usati in combinazione), Interferoni (citochine con attivit? antivirale); un farmaco anticoagulante, come Eparina. The pharmaceutical composition according to the present invention can further understand one or more? of a drug selected from the group consisting of an anti-inflammatory drug, such as Tocilizumab (monoclonal antibody that acts as an interleukin 6 (IL-6) receptor antagonist) and analogues such as Sarilumab (monoclonal antibody that acts by inhibiting interleukin 6 (IL-6) IL-6)), Anakinra (interleukin 1 inhibitor), Baricitinib (janus kinase inhibitor), Eculizumab (monoclonal antibody against complement C5), Emapalumab (monoclonal antibody against interferon gamma); an antimalarial drug, such as hydroxychloroquine (which is anti-inflammatory and antimalarial); an antibiotic drug, such as Azithromycin; an antiviral drug, such as Remdesivir (prodrug of a nucleotide analog with broad spectrum antiviral activity), Lopinavir and Ritonavir (viral protease inhibitors which are used in combination), Interferons (cytokines with antiviral activity); an anticoagulant drug, such as Heparin.
In aggiunta, la presente invenzione concerne un peptide come definito sopra, o composizione farmaceutica come definita sopra per uso medico. In addition, the present invention relates to a peptide as defined above, or a pharmaceutical composition as defined above for medical use.
La presente invenzione concerne anche un peptide come definito sopra o composizione farmaceutica come definita sopra per l?uso nel trattamento e nella prevenzione di un?infezione e/o di una malattia virali, le quali sono causate da un virus in grado di entrare nelle cellule ospiti per mezzo del recettore ACE2. The present invention also relates to a peptide as defined above or a pharmaceutical composition as defined above for use in the treatment and prevention of a viral infection and/or disease, which is caused by a virus capable of entering cells hosts via the ACE2 receptor.
Detto virus pu? essere un coronavirus, quale per esempio un alfacoronavirus, come HCoV-NL63, o un betacoronavirus, come SARS-CoV-2 or SARS-CoV, preferibilmente un betacoronavirus, pi? preferibilmente SARS-CoV-2. Secondo la presente invenzione, la malattia menzionata sopra pu? essere la sindrome acuta respiratoria grave, preferibilmente COVID-19. Said virus can be a coronavirus, such as an alphacoronavirus, such as HCoV-NL63, or a betacoronavirus, such as SARS-CoV-2 or SARS-CoV, preferably a betacoronavirus, plus? preferably SARS-CoV-2. According to the present invention, the disease mentioned above can be severe acute respiratory syndrome, preferably COVID-19.
Il peptide o la composizione farmaceutica secondo la presente invenzione possono essere somministrati oralmente, sistematicamente, o spruzzati nelle vie respiratorie. The peptide or the pharmaceutical composition according to the present invention can be administered orally, systematically, or sprayed into the respiratory tract.
Secondo un?ulteriore forma di realizzazione, la presente invenzione concerne il peptide o la composizione farmaceutica come definiti sopra per l?uso come attivatore allosterico di ACE2 per la prevenzione ed il trattamento di malattie in cui si ha bisogno di tale attivazione. Per esempio, tale malattia pu? essere una malattia cardiovascolare, diabete, danni a fegato e reni o/e insufficienza respiratoria. Gli attivatori di ACE2, legandosi alla superficie della proteina esternamente al sito attivo dell?enzima ed influenzando i mutui movimenti rigidi dei due sub-domini catalitici rispetto ad un cardine, bloccano la proteina in una conformazione attiva. According to a further embodiment, the present invention relates to the peptide or pharmaceutical composition as defined above for use as an allosteric activator of ACE2 for the prevention and treatment of diseases in which such activation is needed. For example, this disease can be cardiovascular disease, diabetes, liver and kidney damage, or/and respiratory failure. ACE2 activators, by binding to the protein surface external to the enzyme?s active site and by influencing the mutual rigid movements of the two catalytic subdomains relative to a hinge, lock the protein in an active conformation.
La presente invenzione concerne anche una combinazione di un peptide come definito sopra con uno o pi? farmaci per l?uso separato o sequenziale nel trattamento e nella prevenzione di una infezione e/o una malattia virali, le quali sono causate da un virus in grado di entrare nelle cellule ospiti mediante il recettore ACE2, in cui detto farmaco ? scelto dal gruppo consistente in un farmaco antinfiammatorio, come Tocilizumab (anticorpo monoclonale che agisce come un?antagonista del recettore dell?interleuchina 6 (IL-6)) ed analoghi come Sarilumab (anticorpo monoclonale che agisce inibendo l?interleuchina-6 (IL-6)), Anakinra (inibitore dell?interleuchina-1), Baricitinib (inibitore della chinasi Giano), Eculizumab (anticorpo monoclonale contro complemento C5), Emapalumab (anticorpo monoclonale contro l?interferone gamma); un farmaco antimalarico, come Idrossiclorochina (che ? un antinfiammatorio ed antimalarico); un farmaco antibiotico, come Azitromicina; un farmaco antivirale, come Remdesivir (pro farmaco di un analogo nucleotidico con attivit? antivirale ad ampio spettro), Lopinavir e Ritonavir (inibitori di proteasi virali che sono usati in combinazione), Interferoni (citochine con attivit? antivirale); un farmaco anticoagulante, come Eparina. The present invention also relates to a combination of a peptide as defined above with one or more? drugs for separate or sequential use in the treatment and prevention of a viral infection and/or disease, which is caused by a virus capable of entering host cells via the ACE2 receptor, in which said drug is? chosen from the group consisting of an anti-inflammatory drug, such as Tocilizumab (monoclonal antibody acting as an interleukin 6 (IL-6) receptor antagonist) and analogues such as Sarilumab (monoclonal antibody acting as an interleukin-6 (IL-6) 6)), Anakinra (interleukin-1 inhibitor), Baricitinib (janus kinase inhibitor), Eculizumab (monoclonal antibody against complement C5), Emapalumab (monoclonal antibody against interferon-gamma); an antimalarial drug, such as hydroxychloroquine (which is an anti-inflammatory and antimalarial); an antibiotic drug, such as Azithromycin; an antiviral drug, such as Remdesivir (prodrug of a nucleotide analog with broad spectrum antiviral activity), Lopinavir and Ritonavir (viral protease inhibitors which are used in combination), Interferons (cytokines with antiviral activity); an anticoagulant drug, such as Heparin.
Secondo la presente invenzione, ?uso separato? ? inteso significare la somministrazione, nello stesso tempo, dei due composti della composizione secondo l?invenzione in forme farmaceutiche distinte. ?Uso sequenziale? ? inteso significare la somministrazione successiva dei due composti della composizione secondo l?invenzione, ciascuno in una forma farmaceutica distinta. According to the present invention, ?separate use? ? understood to mean the administration, at the same time, of the two compounds of the composition according to the invention in distinct pharmaceutical forms. ?Sequential use? ? understood to mean the successive administration of the two compounds of the composition according to the invention, each in a distinct pharmaceutical form.
Secondo la presente invenzione, il virus pu? essere un coronavirus, quale per esempio un alfacoronavirus, come HCoV-NL63, o un betacoronavirus, quale SARS-CoV-2 o SARS-CoV, preferibilmente un betacoronavirus, pi? preferibilmente SARS-CoV-2. La malattia menzionata sopra pu? essere la sindrome acuta respiratoria grave, preferibilmente COVID-19. According to the present invention, the virus can be a coronavirus, such as an alphacoronavirus, such as HCoV-NL63, or a betacoronavirus, such as SARS-CoV-2 or SARS-CoV, preferably a betacoronavirus, plus? preferably SARS-CoV-2. The disease mentioned above can be severe acute respiratory syndrome, preferably COVID-19.
La presente invenzione sar? ora descritta in modo illustrativo, ma non limitativo, in accordo a sue forme di realizzazione preferite, con particolare riferimento agli esempi e ai disegni allegati, in cui: The present invention will be now described in an illustrative, but non-limiting manner, according to preferred embodiments thereof, with particular reference to the examples and accompanying drawings, in which:
Figura 1 mostra il modo di legame designato dei piccolo peptidi (indicati come SG1, SG2 in Figura 1A e SG3, SG4 in Figura 1B; l?N- ed il C-termine dei peptidi sono indicati) sulla proteina ACE2. La struttura della proteina ACE2 ? stata ottenuta dal Protein Data Bank (PDB) con codice di accesso 1R42, e sottoposta a simulazioni MD insieme a ciascun peptide complessato e per tutti i complessi ACE2/peptide. In Figura 1C ? mostrata la struttura cristallografica del complesso formato dal dominio di legame del recettore di spike SARS-CoV-2 ed ACE2 (PDB 6M0J). Tutti i complessi sono orientati in modo da presentare la stessa prospettiva relativamente alla proteina ACE2. Si pu? vedere che le regioni di legame designate di ciascun peptide si sovrappongono con la regione di legame della proteina spike. Figure 1 shows the designated binding mode of the small peptides (indicated as SG1, SG2 in Figure 1A and SG3, SG4 in Figure 1B ; the N- and C-terminus of the peptides are indicated) on the ACE2 protein. The structure of the ACE2 protein? was obtained from the Protein Data Bank (PDB) with access code 1R42, and subjected to MD simulations together with each complexed peptide and for all ACE2/peptide complexes. In Figure 1C ? shown the crystallographic structure of the complex formed by the binding domain of the SARS-CoV-2 spike receptor and ACE2 (PDB 6M0J). All complexes are oriented to present the same perspective with respect to the ACE2 protein. Can you? see that the designated binding regions of each peptide overlap with the binding region of the spike protein.
Figura 2 mostra i risultati degli esperimenti di microscopia di fluorescenza per determinare la colocalizzazione dei peptidi SG1, SG2, SG3, e SG4 con ACE2 in due differenti cellule (Caco-2 e HepG2). Per monitorare la loro localizzazione, questi peptidi sono stati etichettati con FITC (fluorescenza verde) mediante un linker nel seguente modo: FITC-Ahx-Lys-GlyGly-Gly-SG1, FITC-Ahx-Lys-Gly-Gly-Gly-SG2, FITC-Ahx-Lys-Gly-Gly-Gly-SG3, e FITC-Ahx-Lys-Gly-Gly-Gly-SG4 (Lys-Gly-Gly-Gly SEQ ID NO:47). La localizzazione di ACE2 ? stata monitorata misurando la fluorescenza dell?anticorpo primario contro ACE2 coniugato con Alexa Fluor 546 (fluorescenza rossa). (A) Risultati della localizzazione di ACE2 ed i peptidi in cellule Caco-2. (B) Risultati della localizzazione di ACE2 ed i peptidi in cellule HepG2. La riga in basso mostra le immagini di fluorescenza emessa dall?anticorpo, la riga centrale mostra le immagini di fluorescenza emesse dai peptidi, e la riga in alto mostra le immagini combinate (la fluorescenza combinata emessa da ciascun peptide e l?anticorpo)(le frecce indicano il colore di fusione arancio risultante dalla combinazione delle immagini di fluorescenza, implicando co-localizzazione dei peptidi con ACE2). Le immagini delle cellule trattate con PBS da solo sono mostrate nella prima colonna (NT) e quelle trattate con ciascun peptide nelle altre colonne (il peptide usato ? indicato sulla sommit? di ciascuna colonna). Figure 2 shows the results of fluorescence microscopy experiments to determine the colocalization of the peptides SG1, SG2, SG3, and SG4 with ACE2 in two different cells (Caco-2 and HepG2). To monitor their localization, these peptides were labeled with FITC (green fluorescence) by a linker as follows: FITC-Ahx-Lys-GlyGly-Gly-SG1, FITC-Ahx-Lys-Gly-Gly-Gly-SG2, FITC-Ahx-Lys-Gly-Gly-Gly-SG3, and FITC-Ahx-Lys-Gly-Gly-Gly-SG4 (Lys-Gly-Gly-Gly SEQ ID NO:47). The localization of ACE2 ? was monitored by measuring the fluorescence of the primary antibody against Alexa Fluor 546-conjugated ACE2 (red fluorescence). (A) Results of the localization of ACE2 and the peptides in Caco-2 cells. (B) Results of the localization of ACE2 and the peptides in HepG2 cells. The bottom row shows the fluorescence images emitted by the antibody, the middle row shows the fluorescence images emitted by the peptides, and the top row shows the combined images (the combined fluorescence emitted by each peptide and the antibody) (the arrows indicate the orange fusion color resulting from the combination of the fluorescence images, implying co-localization of the peptides with ACE2). Images of cells treated with PBS alone are shown in the first column (NT) and those treated with each peptide in the other columns (the peptide used is indicated at the top of each column).
Figura 3 mostra la posizione dei residui catalitici nell?ACE2 non complessata (PDB 1R42), ACE2 complessata con il dominio di legame del recettore di spike SARS-CoV-2 (PDB 6M0J), ed ACE2 complessata con i peptidi (SG1, SG2, SG3, ed SG4) derivata dai conformeri delle simulazioni MD registrate dopo l?equilibratura della MD). I residui catalitici sono etichettati sulla struttura di ACE2 non legata. Per paia selezionati di residui catalitici le loro distanze in ? sono riportate. Si pu? vedere che i residui catalitici contribuiti dai due sub-domini di ACE2 sono pi? vicini nell?ACE2 complessata al dominio di legame del recettore di spike SARS-CoV-2, ed anche in ACE2 complessata con i peptidi SG1, SG2, SG3, ed SG4, rispetto alla struttura di ACE2 non complessata. Figure 3 shows the location of catalytic residues in uncomplexed ACE2 (PDB 1R42), ACE2 complexed with the SARS-CoV-2 spike receptor binding domain (PDB 6M0J), and ACE2 complexed with peptides (SG1, SG2, SG3, and SG4) derived from the conformers of the MD simulations recorded after the MD balancing). The catalytic residues are labeled on the unbound ACE2 structure. For selected pairs of catalytic residues their distances in ? are reported. Can you? see that the catalytic residues contributed by the two subdomains of ACE2 are more? neighbors in ACE2 complexed to the SARS-CoV-2 spike receptor binding domain, and also in ACE2 complexed with the peptides SG1, SG2, SG3, and SG4, relative to the structure of uncomplexed ACE2.
Esempio 1: Studio della capacit? dei peptidi SG1, SG2, SG3 ed SG4 di interagire con la proteina ACE2 Example 1: Study of the ability? of the peptides SG1, SG2, SG3 and SG4 to interact with the protein ACE2
Materiali e Metodi Materials and methods
Peptidi Peptides
I peptidi furono sintetizzati con inclusa una etichetta fluorescente per esperimenti di microscopia di fluorescenza confocale. A tale fine, i peptidi SG1, SG2, SG3, ed SG4 furono coniugati all?N-termine con il gruppo fluorescente fluoresceina isothiocianato (FITC) attraverso l?acido 6-ammino exanoic (NH2-CH2-CH2-CH2-CH2-CH2-COOH) come spaziatore (Ahx) ed un?addizionale linker tetrapeptidico (Lys-Gly-Gly-Gly SEQ ID NO:47) per incrementare la distanza del fluoroforo dai residui del peptide che legano ACE2 (per impedire possibili interferenze con il legame). In ciascun peptide un legame disolfuro intramolecolare tra le 2 cisteine fu formato. Questi peptide modificati furono ottenuti dal servizio di sintesi personalizzata della D.B.A. Italia/GenScript. La purezza dei peptidi ? stata determinata con HPLC e certificata dal fornitore come segue: FITC-Ahx-Lys-Gly-Gly-Gly-SG1, 98.8%; FITC-Ahx-Lys-Gly-Gly-Gly-SG2, 90.7%, FITC-Ahx-Lys-Gly-Gly-Gly-SG3, 99.1%; FITC-Ahx-Lys-Gly-Gly-Gly-SG4, 99.3%. Peptides were synthesized including a fluorescent label for confocal fluorescence microscopy experiments. To this end, the peptides SG1, SG2, SG3, and SG4 were N-term conjugated with the fluorescein isothiocyanate (FITC) fluorescent group via 6-amino hexanoic acid (NH2-CH2-CH2-CH2-CH2-CH2 -COOH) as a spacer (Ahx) and an additional tetrapeptide linker (Lys-Gly-Gly-Gly SEQ ID NO:47) to increase the distance of the fluorophore from the ACE2-binding peptide residues (to prevent possible binding interference) . In each peptide an intramolecular disulfide bond between the 2 cysteines was formed. These modified peptides were obtained from the D.B.A.'s custom synthesis service. Italy/GenScript. The purity of the peptides ? been determined by HPLC and certified by the supplier as follows: FITC-Ahx-Lys-Gly-Gly-Gly-SG1, 98.8%; FITC-Ahx-Lys-Gly-Gly-Gly-SG2, 90.7%, FITC-Ahx-Lys-Gly-Gly-Gly-SG3, 99.1%; FITC-Ahx-Lys-Gly-Gly-Gly-SG4, 99.3%.
Determinazione della localizzazione cellulare del recettore ACE2, dei peptidi, e colocalizzazione dei peptidi con ACE2 mediante immunofluorescneza Determination of cellular localization of ACE2 receptor, peptides, and colocalization of peptides with ACE2 by immunofluorescence
Le cellule Caco-2 cells da adenocarcinoma colorettale umano e le cellule HepG2 da epatocarcinoma umano impiegate in questo esperimento furono acquistate da ATCC (American Tissue Culture Collection). Le cellule sono state coltivate come indicato da ATCC. In particolare, le cellule Caco-2 furono mantenute in mezzo di coltura DMEM (Dulbecco's Modified Eagle Medium)/F12 addizionato con Siero Bovino Fetale (FBS) al 10% e con antibiotici (miscele di streptomicina e penicillina); le cellule HepG2 furono mantenute in coltura con terreno DMEM addizionato con FBS al 10% e antibiotici (miscela di streptomicina e penicillina). I mezzi e complementi utilizzati furono tutti acquistati da Gibco (Thermofisher Scientific Italia). Entrambi i tipi cellulari furono amplificati per gli esperimenti in condizioni di coltura standard (incubati a 37?C, 5% di CO2, con umidit? di ca. 95%). Caco-2 cells from human colorectal adenocarcinoma and HepG2 cells from human hepatocellular carcinoma used in this experiment were purchased from ATCC (American Tissue Culture Collection). Cells were cultured as directed by ATCC. In particular, Caco-2 cells were maintained in DMEM (Dulbecco's Modified Eagle Medium)/F12 medium supplemented with 10% Fetal Bovine Serum (FBS) and with antibiotics (mixtures of streptomycin and penicillin); HepG2 cells were maintained in culture with DMEM medium supplemented with 10% FBS and antibiotics (mixture of streptomycin and penicillin). The means and accessories used were all purchased from Gibco (Thermofisher Scientific Italia). Both cell types were amplified for experiments under standard culture conditions (incubated at 37°C, 5% CO2, with ca. 95% humidity).
Per gli esperimenti, le cellule furono seminate alla densit? di 10000 cellule/pozzetto in vetrino con camera ad 8 pozzetti e fissati in acetone freddo per 10 minuti a temperatura ambiente. Successivamente, i vetrini furono soggetti a due lavaggi con PBS (tampone fosfato salino) acquistato da Gibco (Thermofisher Scientific Italia), e successivamente esposti per 20 minuti a vari trattamenti a temperatura ambiente. I trattamenti inclusero PBS da solo (NT), e, separatamente, ciascuno dei seguenti peptidi etichettati con FITC (ciascuno a concentrazione 5?M in PBS): For the experiments, the cells were seeded at the density of 10,000 cells/well in an 8-well chambered slide and fixed in cold acetone for 10 minutes at room temperature. Subsequently, the slides were subjected to two washings with PBS (phosphate saline buffer) purchased from Gibco (Thermofisher Scientific Italia), and subsequently exposed for 20 minutes to various treatments at room temperature. Treatments included PBS alone (NT), and, separately, each of the following FITC-labeled peptides (each at 5µM concentration in PBS):
FITC-Ahx-Lys-Gly-Gly-Gly-SG1, FITC-Ahx-Lys-Gly-Gly-Gly-SG1,
FITC-Ahx-Lys-Gly-Gly-Gly-SG2, FITC-Ahx-Lys-Gly-Gly-Gly-SG2,
FITC-Ahx-Lys-Gly-Gly-Gly-SG3, FITC-Ahx-Lys-Gly-Gly-Gly-SG3,
FITC-Ahx-Lys-Gly-Gly-Gly-SG4, FITC-Ahx-Lys-Gly-Gly-Gly-SG4,
dove FITC ? un fluoroforo, il quale pu? essere sostituito da qualsiasi altro gruppo chimico desiderato; Ahx-Lys-Gly-Gly-Gly ? uno spaziatore o linker (Lys-Gly-Gly-Gly SEQ ID NO:47); SG1, SG2, SG3 e SG4 sono i sopra menzionati peptidi delle sequenze SEQ ID NO:2, SEQ ID NO:1, SEQ ID NO:3 e SEQ ID NO:4, rispettivamente. SG1, SG2, SG3 e SG4 rappresentano la regione che lega ACE2. where FITC ? a fluorophore, which pu? be replaced by any other desired chemical group; Ahx-Lys-Gly-Gly-Gly ? a spacer or linker (Lys-Gly-Gly-Gly SEQ ID NO:47); SG1, SG2, SG3 and SG4 are the aforementioned peptides of the sequences SEQ ID NO:2, SEQ ID NO:1, SEQ ID NO:3 and SEQ ID NO:4, respectively. SG1, SG2, SG3 and SG4 represent the ACE2 binding region.
I sopra menzionati peptidi etichettati con FITC possono essere rappresentati come segue: The aforementioned FITC-tagged peptides can be represented as follows:
(Lys-Gly-Gly-Gly-Lys-Cys-Tyr-Met-Cys-D-Ser-Val-Tyr (SEQ ID NO:2); Lys-Gly-Gly-Gly-Gln-Cys-Tyr-Met-Cys-D-Ser-D-Val-Tyr (SEQ ID NO:1); Lys-Gly-Gly-Gly-Lys-Cys-Tyr-Met-Cys-D-Glu-Val-Tyr (SEQ ID NO:3); Lys-Gly-Gly-Gly-Lys-Cys-Tyr-Leu-Cys-D-Glu-Ala-Tyr-Gly-Val (SEQ ID NO:4)). (Lys-Gly-Gly-Gly-Lys-Cys-Tyr-Met-Cys-D-Ser-Val-Tyr (SEQ ID NO:2); Lys-Gly-Gly-Gly-Gln-Cys-Tyr-Met- Cys-D-Ser-D-Val-Tyr (SEQ ID NO:1); Lys-Gly-Gly-Gly-Lys-Cys-Tyr-Met-Cys-D-Glu-Val-Tyr (SEQ ID NO:3 ); Lys-Gly-Gly-Gly-Lys-Cys-Tyr-Leu-Cys-D-Glu-Ala-Tyr-Gly-Val (SEQ ID NO:4)).
Alla fine dei 20 minuti, 3 lavaggi consecutivi in PBS di 5 minuti ciascuno. Alla fine di questa serie di lavaggi, i vetrini furono incubati per 1 ora a temperature ambiente con un anticorpo primario contro ACE2 (enzima di conversione dell?angiotensina 2). L?anticorpo coniugato con Alexa-Fluor546, fu acquistato dalla Santa Cruz Biotechnology e diluito 1:100 in PBS e Albumina del Siero Bovino (BSA, dalla Gibco) al 0.5%. Alla fine dell?esposizione, i vetrini furono sottoposti ad un?altra serie di 3 lavaggi con PBS (ciascun lavaggio per 5 minuti) e finalmente sottoposti a colorazione dei nuclei con DAPI (4?,6-diamidino-2-fenilindolo) diluito 1:5000 in PBS. Alla fine della colorazione dei nuclei i vetrini furono lavati in PBS e montati con una miscela di PBS e glicerolo in rapporto 1:1. Le immagini di microscopia di fluorescenza furono acquisite ed analizzate con il microscopio confocale Olympus FluoView FV1000 con obiettivo 60x e software FV10-ASW (versione 2.0). At the end of the 20 minutes, 3 consecutive washes in PBS for 5 minutes each. At the end of this series of washes, the slides were incubated for 1 hour at room temperature with a primary antibody against ACE2 (angiotensin converting enzyme 2). The Alexa-Fluor546 conjugated antibody was purchased from Santa Cruz Biotechnology and diluted 1:100 in PBS and 0.5% Bovine Serum Albumin (BSA, from Gibco). At the end of the exposure, the slides were subjected to another series of 3 washes with PBS (each wash for 5 minutes) and finally subjected to nuclei staining with DAPI (4?,6-diamidino-2-phenylindole) diluted 1 :5000 at PBS. At the end of the staining of the nuclei, the slides were washed in PBS and mounted with a mixture of PBS and glycerol in a 1:1 ratio. Fluorescence microscopy images were acquired and analyzed with the Olympus FluoView FV1000 confocal microscope with 60x objective and FV10-ASW software (version 2.0).
Simulazioni di dinamica molecolare e determinazione dell?affinit? di binding Molecular dynamics simulations and affinity determination? of bindings
Per determinare l'affinit? di binding dei peptidi verso ACE2, la seguente procedura ? stata eseguita. I modelli di struttura dei peptidi furono individualmente posizionati sulla struttura della proteina ACE2 (ACE2 dal Protein Data Bank, PDB, con codice di accesso 1R42) impiegando un modo di legare simile al docking atteso cos? come esso fu concepito grazie alla progettazione degli stessi peptidi (ricordando che i peptidi furono progettati appositamente con il fine di soddisfare caratteristiche di affinit?, basate su geometria e tipo di atomi interagenti, per la regione di ACE2 scelta come target). I peptidi furono inizialmente posizionati senza causare alcuna collisione atomica con ACE2 per evitare possibile produzione di artefatti nella stabilit? di struttura che potrebbero viziare il campionamento conformazionale durante le simulazioni MD. Cos?, nelle loro pose iniziali, i peptidi furono messi ad interagire solo labilmente con ACE2 cos? che durante le simulazioni essi potevano o legarsi pi? stabilmente ad ACE2 (compatibilmente con le affinit? e correttezza del docking pensato) oppure dissociare da questo recettore. La dissociazione era particolarmente possibile perch? la regione di ACE2 che i peptidi furono programmati a riconoscere come target ? sulla superficie della proteina e presenta una forma pressoch? piatta (Figura 1) (cio?, la regione bersaglio di legame su ACE2 non consiste di una cavit? proteica all'interno della quale i peptidi potrebbero rimanere stabilmente intrappolati meramente perch? non trovano il percorso per sfuggire verso l'esterno). To determine the affinity? of binding of the peptides towards ACE2, the following procedure ? been performed. The peptide structure models were individually placed on the ACE2 protein structure (ACE2 from the Protein Data Bank, PDB, access code 1R42) using a binding mode similar to the expected docking so how it was conceived thanks to the design of the peptides themselves (remembering that the peptides were specifically designed with the aim of satisfying affinity characteristics, based on geometry and type of interacting atoms, for the ACE2 region chosen as target). The peptides were initially positioned without causing any atomic collisions with ACE2 to avoid possible production of stability artifacts. of structure that could bias the conformational sampling during MD simulations. Thus, in their initial poses, the peptides were placed to interact only lazily with ACE2 so? that during the simulations they could either bind more? stably to ACE2 (compatibly with the affinities and correctness of the thought docking) or to dissociate from this receptor. The dissociation was particularly possible because the region of ACE2 that the peptides were programmed to target? on the surface of the protein and has a shape almost? flat (Figure 1) (that is, the target binding region on ACE2 does not consist of a protein cavity within which the peptides could become stably trapped merely because they do not find a pathway to escape outward).
Le simulazioni di Dinamica Molecolare (MD) furono eseguite usando NAMD (v2.13) a temperatura di 310.15 K in solvente acquoso esplicito a forza ionica 0.1 (mediante aggiunta di ioni Na<+ >e Cl-) con il campo di forza Charmm36 inclusivo di parametri per proteine e zuccheri (gli originali gruppi N-glicosilati, lo ione Zn<2+>, e le molecole di acqua cristallografiche come presenti nella struttura cristallografica PDB di ACE2 furono tutti inclusi nelle simulazioni) utilizzando un passo temporale di 1.0 fs/passo e legami flessibili, senza restrizioni di posizione per alcun atomo. Prima delle esecuzioni delle simulazioni MD, due cicli di minimizzazione furono effettuati (nel primo ciclo, gli scheletri di entrambi la proteina ed il peptide furono mantenuti fissi, lasciando muovere solo le catene laterali della proteina ACE2 e del peptide, le molecole di acqua, e ioni; nel secondo ciclo tutti gli atomi furono minimizzati). Dopo aver raggiunto l'equilibratura, i complessi ACE2/peptide ottenuti da conformeri MD a 10 ns furono usati per determinare l'affinit? di legame dei peptidi verso ACE2. I complessi ACE2/peptide furono impiegati come direttamente generati durante le simulazioni a 310.15 K (temperatura fisiologica) senza sottoporre le strutture ad ottimizzazioni geometriche. L'affinit? di legame dei peptidi ed ACE2 fu determinata con il metodo di valutazione di Wang et al. (26). Molecular Dynamics (MD) simulations were performed using NAMD (v2.13) at 310.15 K temperature in explicit aqueous solvent at 0.1 ionic strength (by addition of Na<+ >and Cl- ions) with the inclusive Charmm36 force field of parameters for proteins and sugars (the original N-glycosylated groups, Zn<2+> ion, and crystallographic water molecules as present in the PDB crystal structure of ACE2 were all included in the simulations) using a time step of 1.0 fs/ step and flexible bonds, with no position restrictions for any atom. Before the runs of the MD simulations, two rounds of minimization were performed (in the first round, the skeletons of both the protein and the peptide were kept fixed, leaving only the side chains of the ACE2 protein and peptide, water molecules, and ions; in the second cycle all atoms were minimized). After equilibration was achieved, the ACE2/peptide complexes obtained from 10 ns MD conformers were used to determine the affinity? binding of peptides to ACE2. The ACE2/peptide complexes were employed as directly generated during the simulations at 310.15 K (physiological temperature) without subjecting the structures to geometric optimizations. The affinity? of peptide binding and ACE2 was determined by the evaluation method of Wang et al. (26).
Risultati Results
Per verificare che le cellule Caco-2 e HepG2 esprimevano il recettore ACE2 e per determinare se i peptidi colocalizzano con esso (che ? un'indicazione che un'interazione fisica avviene tra ACE2 ed i peptidi), le cellule furono trattate come sopra, cio? con PBS solo (NT) o con i singoli peptidi colorati in verde (FITC-Ahx-Lys-Gly-Gly-Gly-SG1, FITC-Ahx-Lys-Gly-Gly-Gly-SG2, FITC-Ahx-Lys-Gly-Gly-Gly-SG3, FITC-Ahx-Lys-Gly-Gly-Gly-SG4). Come mostrato in Figura 2-A, le cellule Caco-2 esprimono il recettore ACE2 (in rosso), il quale ? legato da tutti i 4 peptidi (come pu? essere visto con il colore arancione (vedi frecce) di fusione risultante dalla combinazione delle immagini di fluorescenza) ed, in particolare, pi? quantitativamente dal peptide FITC-Ahx-Lys-Gly-Gly-Gly-SG2. Come mostrato in Figura 2-B, anche le cellule HepG2 esprimono il recettore ACE2 (in rosso), il quale ? legato da tutti i 4 peptidi (colore di fusione arancio risultante dalla combinazione delle immagini di fluorescenza) (vedi frecce), ed in particolare pi? quantitativamente dai peptidi FITC-Ahx-Lys-Gly-Gly-Gly-SG2 e FITC-Ahx-Lys-Gly-Gly-Gly-SG3. To verify that Caco-2 and HepG2 cells expressed the ACE2 receptor and to determine whether the peptides colocalize with it (which is an indication that a physical interaction occurs between ACE2 and the peptides), the cells were treated as above, i.e. ? with PBS alone (NT) or with single peptides stained green (FITC-Ahx-Lys-Gly-Gly-Gly-SG1, FITC-Ahx-Lys-Gly-Gly-Gly-SG2, FITC-Ahx-Lys-Gly -Gly-Gly-SG3, FITC-Ahx-Lys-Gly-Gly-Gly-SG4). As shown in Figure 2-A, Caco-2 cells express the ACE2 receptor (in red), which is bound by all 4 peptides (as can be seen with the orange color (see arrows) of fusion resulting from the combination of the fluorescence images) and, in particular, more? quantitatively from the FITC-Ahx-Lys-Gly-Gly-Gly-SG2 peptide. As shown in Figure 2-B, HepG2 cells also express the ACE2 receptor (in red), which is bound by all 4 peptides (orange fusion color resulting from the combination of fluorescence images) (see arrows), and in particular pi? quantitatively from the peptides FITC-Ahx-Lys-Gly-Gly-Gly-SG2 and FITC-Ahx-Lys-Gly-Gly-Gly-SG3.
L'affinit? di legame, calcolata come logaritmo negativo della costante di dissociazione (pKd) su ciascun complesso ACE2/peptide ottenuto da conformeri MD produsse come risultati 9.74, 8.75, 10.60, e 8.55, rispettivamente per i peptidi peptides SG1, SG2, SG3, ed SG4. The affinity? of binding, calculated as the negative logarithm of the dissociation constant (pKd) on each ACE2/peptide complex obtained from MD conformers yielded results of 9.74, 8.75, 10.60, and 8.55, for the peptides SG1, SG2, SG3, and SG4, respectively.
EXAMPLE 2: Studio della capacit? dei peptidi della presente invenzione di competere con l'interazione tra SARS-CoV-2 Spike ed ACE2 EXAMPLE 2: Study of the ability? of the peptides of the present invention to compete with the interaction between SARS-CoV-2 Spike and ACE2
Per determinare la capacit? dei peptidi di competere con l'interazione tra SARS-CoV-2 Spike ed ACE2, il COVID-19 Spike-ACE2 Binding Assay Kit dalla RayBiotech fu usato (codice prodotto CoV-SACE2-1). Questo kit permette di determinare la quota di interazione ACE2/spike e come essa cambia per aggiunta di potenziali competitori misurando variazioni nell'assorbanza ottica. I saggi furono fatti usando i peptidi etichettati con FITC (FITC-Ahx-Lys-Gly-Gly-Gly-SG1, FITC-Ahx-Lys-Gly-Gly-Gly-SG2, FITC-Ahx-Lys-Gly-Gly-Gly-SG3, ed FITC-Ahx-Lys-Gly-Gly-Gly-SG4) uno per volta a concentrazione di 10 micromolare. I risultati (espressi come percentuale di interazione della proteina spike con ACE2 in conseguenza dell'aggiunta dei peptidi) furono i seguenti: nessun peptide aggiunto, 100% di interazione ACE2/spike; FITC-Ahx-Lys-Gly-Gly-Gly-SG1, 99.7% di interazione ACE2/spike, FITC-Ahx-Lys-Gly-Gly-Gly-SG2, 39.5% di interazione ACE2/spike; FITC-Ahx-Lys-Gly-Gly-Gly-SG3, 91.0% di interazione ACE2/spike; FITC-Ahx-Lys-Gly-Gly-Gly-SG4, 88.60% di interazione ACE2/spike. Quindi, gli ultimi tre peptidi, e specialmente FITC-Ahx-Lys-Gly-Gly-Gly-SG2, causarono una sensibile riduzione nella capacit? della proteina spike di legare ACE2. To determine the capacity of peptides to compete with the interaction between SARS-CoV-2 Spike and ACE2, the COVID-19 Spike-ACE2 Binding Assay Kit from RayBiotech was used (product code CoV-SACE2-1). This kit allows to determine the amount of ACE2/spike interaction and how it changes due to the addition of potential competitors by measuring changes in optical absorbance. Assays were done using the FITC-labelled peptides (FITC-Ahx-Lys-Gly-Gly-Gly-SG1, FITC-Ahx-Lys-Gly-Gly-Gly-SG2, FITC-Ahx-Lys-Gly-Gly-Gly -SG3, and FITC-Ahx-Lys-Gly-Gly-Gly-SG4) one at a time at a concentration of 10 micromolar. The results (expressed as the percentage of spike protein interaction with ACE2 as a consequence of the addition of the peptides) were as follows: no peptide added, 100% ACE2/spike interaction; FITC-Ahx-Lys-Gly-Gly-Gly-SG1, 99.7% ACE2/spike interaction, FITC-Ahx-Lys-Gly-Gly-Gly-SG2, 39.5% ACE2/spike interaction; FITC-Ahx-Lys-Gly-Gly-Gly-SG3, 91.0% ACE2/spike interaction; FITC-Ahx-Lys-Gly-Gly-Gly-SG4, 88.60% ACE2/spike interaction. Thus, the last three peptides, and especially FITC-Ahx-Lys-Gly-Gly-Gly-SG2, caused a marked reduction in the ability to of the spike protein to bind ACE2.
ESEMPIO 3: Studio della capacit? dei peptidi della presente invenzione nell'incrementare l'attivit? enzimatica del recettore ACE2. EXAMPLE 3: Study of the ability? of the peptides of the present invention in increasing the activity? ACE2 receptor enzyme.
E' stato recentemente osservato che il legame con il SARS-CoV-2 incrementa l'attivit? enzimatica del recettore ACE2 (26). Riguardo il meccanismo che permette tale incremento di attivit? enzimatica, attraverso allineamenti di struttura di proteine gli autori hanno evidenziato che l'interazione del dominio di legame del recettore della proteina spike del SARS-CoV-2 induce un reciproco movimento dei due sub-domini catalitici di ACE2 intorno ad un cardine, il quale dispone i residui catalitici mutualmente pi? vicini. Questo cambio strutturale ? stato proposto facilitare energeticamente la proteolisi dei substrati da parte di ACE2. Gli autori valutarono tale movimento relativo dei sub-domini misurando l'angolo formato da Asn137 sul bordo di un sub-dominio nella struttura di ACE2 nativa (PDB 1R42), l'atomo di zinco nel centro catalitico, e Asn137 della sovrapposta ACE2 legata al dominio RBD del SARS-CoV-2 (PDB 6M0J), riscontrando un incremento dell'angolo di 5?. Eseguendo tale analisi sui conformeri delle simulazioni MD (ottenuti dopo equilibratura) della proteina ACE2 complessata con i peptidi SG1, SG2, SG3, ed SG4, ? stato trovato che il suddetto angolo, rispetto alla struttura nativa di ACE2 (PDB 1R42) anche in questo caso increment?: SG1, 3.73?; SG2, 7.6? ; SG3, 11.7?; ed SG4, 7.9?. Questo movimento dei sub-domini ? anche suggerito dalle seguenti osservazioni. Le strutture cristallografiche mostrano che i residui catalitici forniti dai due sub-domini di ACE2 sono pi? vicini nel complesso del recettore con la proteina spike del SARS-CoV-2 in confronto ad ACE2 non complessata, ed i residui catalitici diventano anche pi? vicini in seguito al legame dei peptidi della presente invenzione (SG1, SG2, SG3, ed SG4) su ACE2 come osservato con le simulazioni MD (Figura 3). Questi risultati indicano che l'incrementata prossimit? dei due sub-domini catalitici pu? anche essere indotta dall'interazione con questi peptidi. E' perci? plausibile che i peptidi della presente invenzione possono stimolare l'attivit? enzimatica di ACE2 in analogia con quanto ? stato sperimentalmente osservato sull'interazione della proteina SARS-CoV-2 spike con ACE2. Ci? pu? essere basato su un effetto in comune dei ligandi considerato che i peptidi sono progettati per per legare una regione di ACE2 che si sovrappone alla regione presa come target dalla proteina spike. It has recently been observed that binding with SARS-CoV-2 increases the activity enzyme of the ACE2 receptor (26). About the mechanism that allows this increase in activity? through protein structure alignments, the authors showed that the interaction of the receptor binding domain of the SARS-CoV-2 spike protein induces a reciprocal movement of the two catalytic sub-domains of ACE2 around a hinge, which has the catalytic residues mutually pi? neighbors. This structural change? It has been proposed to energetically facilitate the proteolysis of substrates by ACE2. The authors assessed this relative motion of the subdomains by measuring the angle formed by Asn137 at the edge of a subdomain in the structure of native ACE2 (PDB 1R42), the zinc atom in the catalytic center, and Asn137 of the superimposed ACE2 bound to RBD domain of SARS-CoV-2 (PDB 6M0J), finding an angle increase of 5?. By performing this analysis on the conformers of the MD simulations (obtained after equilibration) of the ACE2 protein complexed with the peptides SG1, SG2, SG3, and SG4, ? it was found that the above angle, with respect to the native structure of ACE2 (PDB 1R42) also in this case increment?: SG1, 3.73?; SG2, 7.6? ; SG3, 11.7?; and SG4, 7.9?. This movement of sub-domains ? also suggested by the following observations. The crystallographic structures show that the catalytic residues contributed by the two subdomains of ACE2 are more? neighbors in receptor complex with the SARS-CoV-2 spike protein compared to uncomplexed ACE2, and the catalytic residues become even more? neighbors upon binding of the peptides of the present invention (SG1, SG2, SG3, and SG4) to ACE2 as observed with MD simulations ( Figure 3 ). These results indicate that the increased proximity? of the two catalytic sub-domains pu? also be induced by interaction with these peptides. Is it therefore? plausible that the peptides of the present invention can stimulate the activity? enzyme analysis of ACE2 in analogy with how much ? been experimentally observed on the interaction of SARS-CoV-2 spike protein with ACE2. There? can? be based on a common effect of the ligands given that the peptides are designed to bind a region of ACE2 that overlaps the region targeted by the spike protein.
L'innalzamento farmacologico dell'attivit? enzimatica di ACE2 ? desiderabile perch? procura una funzione protettiva in una variet? di malattie (14-22). The pharmacological increase of the activity? enzyme of ACE2 ? desirable why? does it provide a protective function in a variety? of diseases (14-22).
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| PCT/IT2021/050235 WO2022029810A1 (en) | 2020-08-07 | 2021-07-29 | Peptides able to bind angiotensin-converting enzyme 2 (ace2) and medical uses thereof |
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