In silico prediction of drug resistance: from cancer targeted therapy to cancer targeted prevention

2012/2013Le recenti scoperte sulla natura dei processi genetici, implicati nelle trasformazioni neoplastiche, ci hanno permesso di identificare le principali lesioni cellulari che portano alla formazione del tumore, individuando in questo modo target selettivi sulla base dei quali progettare nuovi agenti terapeutici efficaci. La terapia bersaglio ha rappresentato una rivoluzione permettendo di superare l’aspecificità del trattamento antitumorale e colpendo, nello specifico, la causa biomolecolare del processo patologico neoplastico. I recenti sviluppi della modellistica molecolare si sono resi indispensabili per la ricerca oncologica. Questi, infatti, aprono delle opportunità per la scoperta di nuovi farmaci attraverso la comprensione delle basi molecolari delle malattie; si tratta di un metodo conveniente per identificare dei composti che siano in grado di bloccare l’azione di molte proteine, suggerite come fattori fondamentali in diversi tipi di neoplasie. In questo lavoro di tesi verrà adoperata un’ampia gamma di tecniche di simulazione molecolare al fine di studiare differenti problemi che possono comparire durante il trattamento chemioterapico. La scoperta di nuovi dettagli nei meccanismi che conducono alla carcinogenesi ha permesso di identificare proteine target innovative e più precise per affrontare la terapia senza effetti collaterali per le cellule sane. In particolare, saranno riportati in dettaglio gli studi che coinvolgono tre nuovi differenti target: il recettore σ1, la proteina -catenina e lo sviluppo di dendrimeri per una terapia di silenziamento genico. Tuttavia, nonostante i successi clinici della terapia bersaglio, molti pazienti che inizialmente hanno risposto positivamente manifestano una recidiva, come risultato di una resistenza acquisita; questo tipo di risposta può manifestarsi dopo un periodo variabile di trattamento cronico. L’approccio delle tecniche in silico può essere utilizzato per predire l’avvento di mutazioni secondarie attivanti e al contempo resistenti alla terapia e quindi aiutare nello sviluppo di una strategia di prevenzione focalizzata su una terapia multi-farmaco. In questo lavoro, verranno discusse in particolare delle mutazioni che coinvolgono i recettori c-Kit e Smoothened, pre e post trattamento. L’ampia serie di esempi illustrati e discussi qui enfatizza il ruolo e le potenzialità del modeling molecolare nello sviluppo della terapia bersaglio anti tumorale. Una valutazione in silico permette di prendere in considerazione la specificità molecolare del problema e ridurre drasticamente i tempi e i costi richiesti per formulare nuovi farmaci e strategie di intervento.XXVI Ciclo198

Smoothened (SMO) receptor mutations dictate resistance to\ua0vismodegib in basal cell carcinoma

Basal cell carcinomas (BCCs) and a subset of medulloblastomas are characterized by loss- of-function mutations in the tumor suppressor gene, PTCH1. PTCH1 normally functions by repressing the activity of the Smoothened (SMO) receptor. Inactivating PTCH1 mutations result in constitutive Hedgehog pathway activity through uncontrolled SMO signaling. Tar- geting this pathway with vismodegib, a novel SMO inhibitor, results in impressive tumor regression in patients harboring genetic defects in this pathway. However, a secondary mutation in SMO has been reported in medulloblastoma patients following relapse on vis- modegib to date. This mutation preserves pathway activity, but appears to confer resis- tance by interfering with drug binding. Here we report for the first time on the molecular mechanisms of resistance to vismodegib in two BCC cases. The first case, showing progression after 2 months of continuous vismo- degib (primary resistance), exhibited the new SMO G497W mutation. The second case, showing a complete clinical response after 5 months of treatment and a subsequent pro- gression after 11 months on vismodegib (secondary resistance), exhibited a PTCH1 nonsense mutation in both the pre- and the post-treatment specimens, and the SMO D473Y mutation in the post-treatment specimens only. In silico analysis demonstrated that SMOG497W undergoes a conformational rearrangement resulting in a partial obstruc- tion of the protein drug entry site, whereas the SMO D473Y mutation induces a direct effect on the binding site geometry leading to a total disruption of a stabilizing hydrogen bond network. Thus, the G497W and D473Y SMO mutations may represent two different mech- anisms leading to primary and secondary resistance to vismodegib, respectively

Clinical features and outcomes of elderly hospitalised patients with chronic obstructive pulmonary disease, heart failure or both

Background and objective: Chronic obstructive pulmonary disease (COPD) and heart failure (HF) mutually increase the risk of being present in the same patient, especially if older. Whether or not this coexistence may be associated with a worse prognosis is debated. Therefore, employing data derived from the REPOSI register, we evaluated the clinical features and outcomes in a population of elderly patients admitted to internal medicine wards and having COPD, HF or COPD + HF. Methods: We measured socio-demographic and anthropometric characteristics, severity and prevalence of comorbidities, clinical and laboratory features during hospitalization, mood disorders, functional independence, drug prescriptions and discharge destination. The primary study outcome was the risk of death. Results: We considered 2,343 elderly hospitalized patients (median age 81 years), of whom 1,154 (49%) had COPD, 813 (35%) HF, and 376 (16%) COPD + HF. Patients with COPD + HF had different characteristics than those with COPD or HF, such as a higher prevalence of previous hospitalizations, comorbidities (especially chronic kidney disease), higher respiratory rate at admission and number of prescribed drugs. Patients with COPD + HF (hazard ratio HR 1.74, 95% confidence intervals CI 1.16-2.61) and patients with dementia (HR 1.75, 95% CI 1.06-2.90) had a higher risk of death at one year. The Kaplan-Meier curves showed a higher mortality risk in the group of patients with COPD + HF for all causes (p = 0.010), respiratory causes (p = 0.006), cardiovascular causes (p = 0.046) and respiratory plus cardiovascular causes (p = 0.009). Conclusion: In this real-life cohort of hospitalized elderly patients, the coexistence of COPD and HF significantly worsened prognosis at one year. This finding may help to better define the care needs of this population

Antidiabetic Drug Prescription Pattern in Hospitalized Older Patients with Diabetes

Objective: To describe the prescription pattern of antidiabetic and cardiovascular drugs in a cohort of hospitalized older patients with diabetes. Methods: Patients with diabetes aged 65 years or older hospitalized in internal medicine and/or geriatric wards throughout Italy and enrolled in the REPOSI (REgistro POliterapuie SIMI—Società Italiana di Medicina Interna) registry from 2010 to 2019 and discharged alive were included. Results: Among 1703 patients with diabetes, 1433 (84.2%) were on treatment with at least one antidiabetic drug at hospital admission, mainly prescribed as monotherapy with insulin (28.3%) or metformin (19.2%). The proportion of treated patients decreased at discharge (N = 1309, 76.9%), with a significant reduction over time. Among those prescribed, the proportion of those with insulin alone increased over time (p = 0.0066), while the proportion of those prescribed sulfonylureas decreased (p < 0.0001). Among patients receiving antidiabetic therapy at discharge, 1063 (81.2%) were also prescribed cardiovascular drugs, mainly with an antihypertensive drug alone or in combination (N = 777, 73.1%). Conclusion: The management of older patients with diabetes in a hospital setting is often sub-optimal, as shown by the increasing trend in insulin at discharge, even if an overall improvement has been highlighted by the prevalent decrease in sulfonylureas prescription

The “Diabetes Comorbidome”: A Different Way for Health Professionals to Approach the Comorbidity Burden of Diabetes

(1) Background: The disease burden related to diabetes is increasing greatly, particularly in older subjects. A more comprehensive approach towards the assessment and management of diabetes’ comorbidities is necessary. The aim of this study was to implement our previous data identifying and representing the prevalence of the comorbidities, their association with mortality, and the strength of their relationship in hospitalized elderly patients with diabetes, developing, at the same time, a new graphic representation model of the comorbidome called “Diabetes Comorbidome”. (2) Methods: Data were collected from the RePoSi register. Comorbidities, socio-demographic data, severity and comorbidity indexes (Cumulative Illness rating Scale CIRS-SI and CIRS-CI), and functional status (Barthel Index), were recorded. Mortality rates were assessed in hospital and 3 and 12 months after discharge. (3) Results: Of the 4714 hospitalized elderly patients, 1378 had diabetes. The comorbidities distribution showed that arterial hypertension (57.1%), ischemic heart disease (31.4%), chronic renal failure (28.8%), atrial fibrillation (25.6%), and COPD (22.7%), were the more frequent in subjects with diabetes. The graphic comorbidome showed that the strongest predictors of death at in hospital and at the 3-month follow-up were dementia and cancer. At the 1-year follow-up, cancer was the first comorbidity independently associated with mortality. (4) Conclusions: The “Diabetes Comorbidome” represents the perfect instrument for determining the prevalence of comorbidities and the strength of their relationship with risk of death, as well as the need for an effective treatment for improving clinical outcomes

Beta-Blocker Use in Older Hospitalized Patients Affected by Heart Failure and Chronic Obstructive Pulmonary Disease: An Italian Survey From the REPOSI Register

Beta (β)-blockers (BB) are useful in reducing morbidity and mortality in patients with heart failure (HF) and concomitant chronic obstructive pulmonary disease (COPD). Nevertheless, the use of BBs could induce bronchoconstriction due to β2-blockade. For this reason, both the ESC and GOLD guidelines strongly suggest the use of selective β1-BB in patients with HF and COPD. However, low adherence to guidelines was observed in multiple clinical settings. The aim of the study was to investigate the BBs use in older patients affected by HF and COPD, recorded in the REPOSI register. Of 942 patients affected by HF, 47.1% were treated with BBs. The use of BBs was significantly lower in patients with HF and COPD than in patients affected by HF alone, both at admission and at discharge (admission, 36.9% vs. 51.3%; discharge, 38.0% vs. 51.7%). In addition, no further BB users were found at discharge. The probability to being treated with a BB was significantly lower in patients with HF also affected by COPD (adj. OR, 95% CI: 0.50, 0.37-0.67), while the diagnosis of COPD was not associated with the choice of selective β1-BB (adj. OR, 95% CI: 1.33, 0.76-2.34). Despite clear recommendations by clinical guidelines, a significant underuse of BBs was also observed after hospital discharge. In COPD affected patients, physicians unreasonably reject BBs use, rather than choosing a β1-BB. The expected improvement of the BB prescriptions after hospitalization was not observed. A multidisciplinary approach among hospital physicians, general practitioners, and pharmacologists should be carried out for better drug management and adherence to guideline recommendations

Clinical features and outcomes of elderly hospitalised patients with chronic obstructive pulmonary disease, heart failure or both

Background and objective: Chronic obstructive pulmonary disease (COPD) and heart failure (HF) mutually increase the risk of being present in the same patient, especially if older. Whether or not this coexistence may be associated with a worse prognosis is debated. Therefore, employing data derived from the REPOSI register, we evaluated the clinical features and outcomes in a population of elderly patients admitted to internal medicine wards and having COPD, HF or COPD + HF. Methods: We measured socio-demographic and anthropometric characteristics, severity and prevalence of comorbidities, clinical and laboratory features during hospitalization, mood disorders, functional independence, drug prescriptions and discharge destination. The primary study outcome was the risk of death. Results: We considered 2,343 elderly hospitalized patients (median age 81 years), of whom 1,154 (49%) had COPD, 813 (35%) HF, and 376 (16%) COPD + HF. Patients with COPD + HF had different characteristics than those with COPD or HF, such as a higher prevalence of previous hospitalizations, comorbidities (especially chronic kidney disease), higher respiratory rate at admission and number of prescribed drugs. Patients with COPD + HF (hazard ratio HR 1.74, 95% confidence intervals CI 1.16-2.61) and patients with dementia (HR 1.75, 95% CI 1.06-2.90) had a higher risk of death at one year. The Kaplan-Meier curves showed a higher mortality risk in the group of patients with COPD + HF for all causes (p = 0.010), respiratory causes (p = 0.006), cardiovascular causes (p = 0.046) and respiratory plus cardiovascular causes (p = 0.009). Conclusion: In this real-life cohort of hospitalized elderly patients, the coexistence of COPD and HF significantly worsened prognosis at one year. This finding may help to better define the care needs of this population

SIRT1 Signaling Is Involved in the Vascular Improvement Induced by Moringa Oleifera Seeds during Aging

Vascular aging is linked to reduce NO bioavailability, endothelial dysfunction, oxidative stress, and inflammation. We previously showed that a 4-week treatment of middle-aged Wistar rats (MAWRs, 46 weeks old) with Moringa oleifera seed powder (MOI, 750 mg/kg/day) improved vascular function. Here, we investigated the involvement of SIRT1 in MOI-induced vascular improvement. MAWRs were treated with a standard or MOI-containing diet. Young rats (YWR, 16 weeks old) were the controls and received a standard diet. The hearts and aortas were harvested to evaluate SIRT1 and FOXO1 expression via Western blot and/or immunostaining, SIRT1 activity via a fluorometric assay, and oxidative stress using the DHE fluorescent probe. In the hearts and aortas, SIRT1 expression, reduced in MAWRs compared to YWRs, was enhanced in MOI MAWRs. In the hearts, SIRT1 activity did not differ between YWRs and MAWRs, whereas it was increased in MOI MAWRs compared with them. In the aortas, SIRT1 activity decreased in MAWRs, and it was similar in the MOI MAWRs and YWRs. FOXO1 expression increased in the nuclei of MAWR aortas compared to YWR and was reversed in MOI MAWRs. Interestingly, MOI treatment normalized oxidative stress enhanced in MAWRs, in both the heart and aorta. These results demonstrate the protective role of MOI against cardiovascular dysfunction due to aging via enhanced SIRT1 function and subsequently reduced oxidative stress

Another Brick in the Wall. Validation of the \u3c31Receptor 3D Model by Computer-Assisted Design, Synthesis, and Activity of New \u3c31Ligands

Originally considered an enigmatic polypeptide, the \u3c31 receptor has recently been identified as a unique ligand- regulated protein. Many studies have shown the potential of \u3c31 receptor ligands for the treatment of various diseases of the central nervous system (CNS); nevertheless, almost no information about the 3D structure of the receptor and/or the possible modes of interaction of the \u3c31 protein with its ligands have been unveiled so far. With the present work we validated our \u3c31 3D homology model and assessed its reliability as a platform for \u3c31 ligand structure-based drug design. To this purpose, the 3D \u3c31 model was exploited in the design of 33 new \u3c31 ligands and in their ranking for receptor affinity by extensive molecular dynamics simulation-based free energy calculations. Also, the main interactions involved in receptor/ligand binding were analyzed by applying a per residue free energy deconvolution and in silico alanine scanning mutagenesis calculations. Subsequently, all compounds were synthesized in our laboratory and tested for \u3c31 binding activity in vitro. The agreement between in silico and in vitro results confirms the reliability of the proposed \u3c31 3D model in the a priori prediction of the affinity of new \u3c31 ligands. Moreover, it also supports and corroborates the currently available biochemical data concerning the \u3c31 protein residues considered essential for \u3c31 ligand binding and activity

Synthesis, pharmacological evaluation and \u3c31 receptor interaction analysis of hydroxyethyl substituted piperazines

Starting from (S)- or (R)-aspartate, three synthetic strategies were explored to prepare hydroxyethyl substituted piperazines with different substituents at the N-atoms. \u3c3 receptor affinity was recorded using receptor material from both animal and human origin. \u3c31 affinities determined with guinea pig brain and human RPMI 8226 tumor cell lines differed slightly but showed the same tendency. (S)-2-[4-(Cyclohexylmethyl)-1-(naphthalene-2-ylmethyl)piperazin-2-yl]ethanol (7c) revealed the highest affinity at human \u3c31 receptors (Ki = 6.8 nM). The potent \u3c31 receptor ligand 7c was able to inhibit selectively the growth of three human tumor cell lines with IC50 values in the low micromolar range. The reduced growth of the RPMI-8226 cell line was caused by apoptosis. The interaction of 7c with the \u3c31 receptor was analyzed in detail using the 3D homology model of the \u3c31 receptor. The calculated free binding energies of all hydroxyethylpiperazines nicely correlate with their recorded affinities toward the human \u3c31 receptor