academy of scientifc research and technology · vii and molecular docking studies. eur. j. med....

Academy of Scientifc Research and Technology

أكاديمية البحث العلمى والتكنولوجيا

Part A: Short CVs of the Egyptian PI, &Egyptian teamwork and CVs of foreign

counterpart PI, & teamwork (One page maximum per person)

1. Basic Information

Full Name in Arabic:

الدهنه محمد محمد وجدي

Full name in English:(As you write it in Int. publications, underline family name): Wagdy M. Eldehna

Date of Birth: 10 / 4 / 1983

National ID 28304101604734

Last University Degree: PhD

Faculty, University, Country: Faculty of Pharmacy, Cairo University, Egypt

Graduation Date: Nov.-2015

Title: Lecturer Field of specialization: Medicinal Chemistry

Affiliation: Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Kafrelsheikh University, Egypt

Current Position: Lecturer

Contact Information: Mobile Phone: 0106 883 7640 E-mail: [email protected] and [email protected]

2. Scientific Achievements

h index 7

Citations 117

Total no. of Int. publications in Scopus 23

Last three recent relevant publications Authors (underline your name), year, title, Journal, vol. and pages

1

W. M. Eldehna, M.F. Abo-Ashour, A. Nocentini, P. Gratteri, I.H. Eissa, M. Fares, H.A. Ghabbour, M.M. Elaasser, H.A. Abdel-Aziz, C.T. Supuran (2017) Novel 4/3-((4-oxo-5-(2-oxoindolin-3-ylidene)thiazolidin-2-ylidene)amino) benzenesulfonamides: Synthesis, carbonic anhydrase inhibitory activity, anticancer activity and molecular modelling studies. Eur. J. Med. Chem. Accepted Manuscript

2 W. M. Eldehna, G. H. Al-Ansary, S. Bua, A. Nocentini, P. Gratteri, A. Altoukhy, H. Ghabbour, H. Y. Ahmed, and C.T. Supuran, (2017) Novel indolin-2-one-based sulfonamides as carbonic anhydrase inhibitors: Synthesis, in vitro biological evaluation against carbonic anhydrases isoforms I, II, IV and VII and molecular docking studies. Eur. J. Med. Chem. 127, 521-530.

3 W. M. Eldehna, M. Fares, M. Ceruso, H. A. Ghabbour, S. M. Abou-Seri, H. A. Abdel-Aziz, D. A. El Ella and C.T.Supuran, (2016) Amido/ureidosubstituted benzenesulfonamides-isatin conjugates as low nanomolar/ subnanomolar inhibitors of the tumor-associated carbonic anhydrase isoform XII, Eur. J. Med. Chem. 110, 259-266.

Last two recent granted and submitted Patents NA

Granted Patent: NA Title of the patent: NA No. of the patent: NA

Submitted patent: NA Title of the patent: NA No. of the patent: NA

If applicable, state other tangible scientific and technological achievements such scientific prizes, research grants, membership of Int. Scientifc Boards, Unions, Networks, Alliances…etc. Referee for many highly-reputable international journals.

Please list if you have any proposals funded by ASRT showing the current status of: NA

mailto:[email protected]




حاتم عبدالقادر عبدالعزيز محمد

Full name in English:(As you write it in Int. publications, underline family name): Hatem A. Abdel-Aziz

Date of Birth: 5 / 4 / 1973

National ID 27304052102171


Faculty, University, Country: Faculty of Science, Cairo University, Egypt

Graduation Date: Dec.-2005

Title: Professor Field of specialization: Applied Organic Chemistry

Affiliation: Applied Organic Chemistry Department, National Research Centre, Cairo, Egypt

Current Position: Professor

Contact Information: Mobile Phone: 01009511215 E-mail: [email protected]


h index 22

Citations 1400



1

M. Fares, R. A. Eladwy, A. Nocentini, S. R. Abd El Hadi, H. A. Ghabbour, A. Abdel-Megeed, W. M. Eldehna, H. A. Abdel-Aziz, (2017), Synthesis of bulky-tailed sulfonamides incorporating pyrido[2,3-d][1,2,4]triazolo[4,3-a]pyrimidin-1(5H)-yl) moieties and evaluation of their carbonic anhydrases I, II, IV and IX inhibitory effects, Bioorg. Med. Chem., 25(7), 2210-2217.

2 Eldehna WM, Almahli H, Al-Ansary GH, Ghabbour HA, Aly MH, Ismael OE, Al-Dhfyan A, Abdel-Aziz HA (2017), Synthesis and in vitro anti-proliferative activity of some novel isatins conjugated with quinazoline/phthalazine hydrazines against triple-negative breast cancer MDA-MB-231 cells as apoptosis-inducing agents. J Enzyme Inhib. Med. Chem. 2017 Dec;32(1):600-613

3 Hany S. Ibrahim, Sahar M. Abou-Seri, Hatem A. Abdel-Aziz, (2016), Hydrazinoindolin-2-one derivatives: Chemical classification and investigation of their targets as anticancer agents. Eur J. Med. Chem. Oct 21;122:366-81.








Full Name in Arabic: Full name in English:(As you write it in Int. publications, underline family name): Capasso Clemente

Date of Birth:

National ID AR 0997379

Last University Degree: Doctor in Biology

Faculty, University, Country: University of Naples, Federico II

Graduation Date: December 1992

Title: Researcher Field of specialization: Protein Biochemistry

Affiliation: CNR-Institute of Bioscience and Bioresource (CNR-IBBR)

Current Position: Researcher

Contact Information: Mobile Phone: +39 3456278896 E-mail: [email protected]


h index 39

Citations 4357



1

Supuran, C.T. Inhibition of bacterial carbonic anhydrases as a novel approach to escape .;Capasso, Cdrug resistance. Curr Top Med Chem 2017, 17, 1237-1248.

2 .; Supuran, C.T.; Sharma, P.K. Capasso, CKumar, R.; Bua, S.; Ram, S.; Del Prete, S.; Benzenesulfonamide bearing imidazothiadiazole and thiazolotriazole scaffolds as potent tumor

associated human carbonic anhydrase ix and xii inhibitors. Bioorg Med Chem 2017, 25, 1286-1293.

3 Supuran, C.T.; Capasso, C. Carbonic anhydrase from porphyromonas gingivalis as a drug target. Pathogens 2017, 6.







Full Name in Arabic: Full name in English:(As you write it in Int. publications, underline family name): Del Prete Sonia

Date of Birth:

National ID AT 7551325


Faculty, University, Country: University of Florence

Graduation Date: February 2017

Title: Researcher Field of specialization: Protein Biochemistry

Affiliation: CNR-Institute of Bioscience and Bioresource (CNR-IBBR)

Current Position: Researcher

Contact Information: Mobile Phone: +39 3392140228 E-mail: [email protected]


h index 18

Citations 221



1

an, S.M.; AlOthman, Z.; Donald, W.A.; Supuran, C.T.; ; Vullo, D.; Di Fonzo, P.; OsmDel Prete, S.Capasso, C. Sulfonamide inhibition profile of the gamma-carbonic anhydrase identified in the genome of the pathogenic bacterium burkholderia pseudomallei the etiological agent responsible

of melioidosis. Bioorg Med Chem Lett 2017, 27, 490-495.

2 , S.; Vullo, D.; Osman, S.M.; AlOthman, Z.; Supuran, C.T.; Capasso, C. Sulfonamide Del Preteinhibition profiles of the beta-carbonic anhydrase from the pathogenic bacterium francisella

tularensis responsible of the febrile illness tularemia. Bioorg Med Chem 2017, 25, 3555-3561.

3 Del Prete, S.; Vullo, D.; di Fonzo, P.; Carginale, V.; Supuran, C.T.; Capasso, C. Comparison of the anion inhibition profiles of the beta- and gamma-carbonic anhydrases from the pathogenic bacterium burkholderia pseudomallei. Bioorg Med Chem 2017, 25, 2010-2015.








هاني سعيد إبراهيم عبدالحافظ

Full name in English:(As you write it in Int. publications, underline family name): Hany S. Ibrahim

Date of Birth: 13 / 3 / 1983

National ID 28303130100153


Faculty, University, Country: Faculty of Pharmacy, Cairo University, Egypt

Graduation Date: Mar.-2016

Title: Lecturer Field of specialization: Medicinal Chemistry

Affiliation: Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Egyptian Russian University, Egypt

Current Position: Lecturer

Contact Information: Mobile Phone: 01017566960 E-mail: [email protected] and [email protected]


h index 7

Citations 115



1

H. S. Ibrahim, S.M. Abo-Seri, H.A. Abdel-Aziz (2016) 3-Hydrazinoindolin-2-one derivatives: Chemical classification and investigation of their targets as anticancer agents. Eur. J. Med. Chem. 122, 366-381.

2 H. S. Ibrahim, S.M. Abo-Seri, N.S.Ismail, M. M. Elaasser, H.A. Abdel-Aziz (2016) Bis-isatin hydrazones with novel linkers: synthesis and biological evaluation as cytotoxic agents. Eur. J. Med. Chem. 108, 415-422.

3 H. S. Ibrahim, S.M. Abo-Seri, M. Tanc, M. M. Elaasser, H.A. Abdel-Aziz, C. T. Supuran (2016) Isatin-pyrazole benzenesulfonamide hybrids potently inhibit tumor-associated carbonic anhydrase isoforms IX and XII. Eur. J. Med. Chem. 103, 583-593.








Full Name in Arabic: تامر محمد إبراهيم

Full name in English: Tamer Mohamed Ibrahim

Date of Birth: 10/09/1938

National ID:

28309010206231

Last University Degree PhD. In Pharmaceutical Chemistry

Faculty, University, Country Tuebingen University, Germany

Graduation Date 2015

Title: Lecturer

Field of specialization: Molecular Design and Medicinal Chemistry

Affiliation: Department of Pharmaceutical Chemistry, Faculty of Pharmacy, KafrElsheikh University

Current Position: Lecturer in Pharmaceutical Chemistry, Faculty of Pharmacy, Kafrelsheikh University

Contact Information: Mobile Phone: 01153187856 Fax: E-mail: [email protected]


h index (SCOPUS only) 3

Citations (SCOPUS only) 77

Total no. of Int. publications in SCOPUS 6


1

Tamer M. Ibrahim, Matthias R. Bauer, Alexander Dörr and Frank M. Boeckler, 2105, “pROC-Chemotype Plots Enhance the Interpretability of Benchmarking Results in Structure-Based Virtual Screening”, Journal of Chemical Information and Modeling, Oct 5; 55 (11): 2297–2307.

2 Tamer M. Ibrahim, Matthias R. Bauer and Frank M. Boeckler. 2105, “Applying DEKOIS 2.0 in Structure-Based Virtual Screening to Probe the Impact of Preparation Procedures and Score Normalization”, Journal of Cheminformatics, May 20; 7(21).

3 Adnan A. Bekhit, Ahmed M. Farghaly, Ragab M Shafik, Mona M. A. Elsemary, Mai S. El-Shoukrofy, Alaa El-Din A. Bekhit and Tamer M Ibrahim. 2017, “Synthesis, evaluation and modeling of some triazolothienopyrimidinones as anti-inflammatory and antimicrobial agents”. Future Medicinal Chemistry, June 9(9), 881-897.

Part B: Proposal description

B.1. English Summary:

It is worth noting that breast cancer in Egypt is the most commonly diagnosed cancer and the leading cause of cancer death in women. This urgent necessity to fight the breast cancer motivated us to search for novel treatments targeting tumor associated carbonic anhydrase isoforms hCA IX and XII as a novel approach for breast cancer therapy. Whereas, several studies revealed the overexpression of CA IX and CA XII in breast cancer, therapeutic strategies based on the inhibition of CA IX and CA XII have stood out as an auspicious tactic for discovering therapies for human breast malignancy.

Recently, incorporation of urea functionality emerged as the most promising trend in the design of CAIs with good selectivity profile for inhibition of the transmembrane, tumor associated isoforms (CAs IX & XII) over the off-target cytosolic CAs I and II. SLC-0111, an ureido benzenesulfonamide derivative, is currently in clinical evaluations for treatment of breast cancer. In 2016, we developed novel series of ureidosubstituted benzenesulfonamides incorporating isatin moieties which displayed good selectivity profile for the tumor associated isoforms CAs IX (KIs: 5–240 nM) and XII (KIs: 0.47–2.83 nM).

In this proposal, we adopted two strategies to design and synthesize two novel series of carbonic anhydrase inhibitors; (1) Bioisosteric replacement phenyl ring of SLC-0111 with another heteroaryl rings such as pyridine, indole, etc.. (2) Ring expansion approach of the indole moiety, of our recently reported and promising ureido-benzenesulfonamides, to a quinolie moiety. The newly synthesized sulfonamides will be in vitro evaluated for their inhibitory activity against a panel of hCA I, II, and IX isoforms, using an applied Photophysics stopped-flow instrument. Then, the most active derivatives with good selectivity profile for inhibition of the tumor associated isoforms will be examined for their anti-proliferative activity and apoptotic induction potential against breast cancer cell line. Furthermore, a molecular docking of the tested compounds will be carried out in order to investigate their binding pattern with the carbonic anhydrase isoforms CAs IX. Also, the physicochemical properties and ADME profiling for all the synthesized compounds will be performed using Discovery Studio 2.5 (Accelrys, San Diego, CA, USA). In summary, the prime goal is to develop novel sulfonamides with potent and effective anti-proliferative activity towards breast cancer cells targeting tumor associated carbonic anhydrase isoforms CAs IX.

Figure 1. Design and general structures of the two proposed sulfonamides series.

B.2. Arabic Summary: is an understandable translation of the English summary.

هذه الضرورة الملحة .لوفاة لدى النساءلومن الجدير بالذكر أن سرطان الثدي في مصر هو األكثر شيوعا والسبب الرئيسي

التاسع والثاني عشر انزيمات كاربونك أنهيدريزلمحاربة سرطان الثدي دفعنا للبحث عن عالجات جديدة تستهدف أشكال

أن سرطان الثدي مرتبط باإلفراطفي حين كشفت العديد من الدراسات .ثديكنهج جديد لعالج سرطان الالمرتبطة باألورام

هذه االنزيمات طرقا تثبيطلعالجية الستراتيجيات اال باتت التاسع والثاني عشر ، النزيمات كاربونك أنهيدريزفي التعبير

.الثدي البشريسرطان ل الكتشاف عالجات

نزيمات كاربونك أنهيدريزمثبطات الاالتجاه الواعد في تصميم وظيفة اليوريا ال يعتبر دمج المجموعةنة األخيرة، ي اآلوف

انزيمات كاربونك تستهدف أشكال ) الغير مرتبطة باألورامجيدة لهذه االنزيمات عن غيرها انتقائية ب التاسع والثاني عشر

.واحد واثنان أنهيدريز

SLC-0111 في عام .يا في التقييمات السريرية لعالج سرطان الثدي، ويعمل حالأوريدو بنزينسولفوناميد مشتق من وهو

انتقائية جيدة إساتين التي عرضت مجموعاتدمج مع أوريدو بنزينسولفوناميد، قمنا بتطوير سلسلة جديدة من 6102

-5: التاسع كاربونك أنهيدريزلبثابت تثبيط باألورام المرتبطةالتاسع والثاني عشر انزيمات كاربونك أنهيدريزشكال أل

.موالرانون 2..6-1.40الثاني عشر كاربونك أنهيدريزل ابت تثبيطوبث وموالرنانو 641

حلقة (1):أنهيدريز انزيمات كاربونكشكال ألمن مثبطات سلسلتين في هذا االقتراح، اعتمدنا استراتيجيتين لتصميم وتوليف

نهج توسيع ( 6.. )ندول، الخ مع حلقات هيتيرواريل أخرى مثل البيريدين، اإل SLC-0111فينيل من مجموعة استبدال

.نكينوليحلقات حلقة اندول من مركباتنا المنشورة إلي

انزيمات كاربونكشكال ألسوف تكون السلفوناميدات المصنعة حديثا في المختبر تقييمها لنشاطها المثبطة ضد لوحة

انزيمات أشكال الجيدة لتثبيط النتقائية اثم، سيتم فحص المشتقات األكثر نشاطا مع .، باستخدام أداة فوتوفيسيكس أنهيدريز

وعالوة .خط خلية سرطان الثدي تثبيطالتكاثري المضادة وإمكانية تحريض الورم لنشاطهم بالمرتبطة كاربونك أنهيدريز

انزيمات شكال أللها مع االرتباطعلى ذلك، سيتم إجراء االلتحام الجزيئي للمركبات اختبار من أجل التحقيق في نمط

التنميط لجميع المركبات المصنعة باستخدام أيضا، سيتم تنفيذ الخصائص الفيزيائية والكيميائية .كاربونك أنهيدريز

وباختصار، فإن الهدف الرئيسي (. أسيلريس، سان دييغو، كاليفورنيا، الواليات المتحدة األمريكية) 6.5ديسكفري ستوديو

الة لمكافحة التكاثري النشاط نحو خاليا سرطان الثدي التي تستهدف الورم هو تطوير سولفوناميدس جديدة مع فعالية وفع

.التاسع والثاني عشر انزيمات كاربونك أنهيدريزشكال ألالمرتبطة

.سالسل المركبات المراد تصنيعهاتصميم و األشكال العامة ل. 1شكل

B.3. Objectives.

No. Objective

1. Design and Synthesis of the targeted heteroaryl- and quinazoline-based sulfonamides

(about 30 novel compounds) using convenient chemical reactions. As well as Purification

of the synthesized compounds.

2. Verification of the structures of the synthesized derivatives using spectral and elemental

methods of analysis.

3. Biological Evaluation of the inhibitory activity of the synthesized sulfonamides against the

cytosolic carbonic anhydrase isoforms (CAs I and II) and tumor associated (CAs IX and XII).

4. Investigation of the possible binding pattern of the tested compounds with carbonic

anhydrase isoforms CAs IX and XII, through carrying out a molecular docking study.

5. Prediction of several ADME descriptors for the synthesized compounds via a theoretical

computational kinetic study, using the Discovery Studio 2.5 software (Accelrys, San Diego,

CA, USA).

6. Evaluation of anti-proliferative activity of the most potent CAs IX and XII inhibitors against

breast cancer cell lines.

7. Apoptosis Study; determination of the relative levels of 35 human apoptosis-related

proteins.

8. Data analysis, their discussion, presentations in figures and tables, evaluation the

biological data of the synthesized compounds, suggesting the recommendations for the

applicability of these compounds, and finally documentation of the analyzed data.

B.4. Nature & Significance of collaboration:

We proposed a bilateral research proposal between ASRT of Egypt with CNR of Italy that uses a multidisciplinary team of medicinal, organic and computational chemists together

with molecular biologists to develop and evaluate novel sulfonamides derivatives that could fight the breast cancer.

According to the International Agency for Research on Cancer (IARC) estimates for 2012: there are 14.1 million new cancer cases in 2012 compared with 12.7 million in 2008, and 2.8 million cancer-related deaths occurred in 2012 compared with 7.6 million in 2008. Projections predict a substantive increase to 19.3 million new cancer cases by 2025. Regarding the breast cancer; the incidence has increased by more than 20%, while mortality has increased by 14%, since the 2008 estimates. It is worth noting that breast cancer in Egypt is the most commonly diagnosed cancer and the first leading cause of cancer death in women. This finding bring into sharp focus the need to pay much attention to modify and update drug leads from the point of view of pharmaceutical chemistry and drug design to fulfill more effective and affordable approaches for breast cancer treatment.

To this end, our interdisciplinary and co-operative research to discover new breast cancer therapy with novel modes of action is timely and extremely relevant to tackle this global health, wellbeing and socio-economic challenge. Each partner is complementary and essential to achieve the objectives of the project. The results of this interdisciplinary investigation will generate therapeutic leads against breast cancer thereby underpinning the later stages of drug discovery. Generally, the Egyptian side will be responsible for the design and synthesis of the proposed compounds, in addition to an apoptotic study, while the Italian side will be responsible for the biological evaluation of the developed molecules towards carbonic anhydrase isoforms. Efficient accomplishment of this proposal relies on the combined and complementary individual expertise of our team members.

On the other hand, this interdisciplinary collaboration will provide new training opportunities in a multidisciplinary environment and prospects for Egyptian academic researchers to interact with respected international colleagues. Also, it will set up strong ties with the CNR of Italy for future collaboration.

B.5. Action Plan:

Work package 1: Pharmaceutical Chemistry WP1a; Library design and synthesis: (in Egypt)

The Egyptian partners have relied upon medicinal chemistry and drug design to further their ongoing efforts to develop potent carbonic anhydrase inhibitors and anticancer therapy (8-11). Motivated by the promising obtained results from our previous studies, we propose here to design and synthesise novel series of small molecules based mainly on ureido benzenesulfonamide scaffold. Distinctive drug design strategies will be adopted to develop the proposed derivatives as illustrated in Schemes1-3. The proposed compounds passed the filters of Lipinski’s rule of five and the Veber rule, have good drug likeness scores. We are planning to prepare about 30 novel target compounds through this project.

Scheme 1. Synthesis of target sulfonamides 6a-o. (i) SOCl2 / catalytic amount of DMF / reflux 4h; (ii) NaN3 / acetone; (iii) Xylene / reflux 1h; (iv) Dioxane / reflux 4h.

Scheme 2. Synthesis of the key intermediates 10. Reagents and conditions: (i) THF / TEA / r.t. 5h; (ii) H2 / Pd/C / MeOH / r.t. 3h.

Scheme 3. Synthesis of target sulfonamides 17a-o. Reagents and conditions: (i) H2SO4 / Ac2O / 1hr; (ii) 90 0C / 1hr; (iii) 90 0C / 90 min; (iv) Diphenyl etther / 250 0C / 1hr (v) POCl3 / 100 0C / 5hr; (vi) 21a-f / THF / reflux 4h.

WP1b; Characterisation and structural elucidation of compounds: (in Egypt)

Verification of the structures of the synthesized compounds will be carried out mainly in Egypt using spectral (as; 1H NMR, 13C NMR, IR) and elemental methods of analysis.

WP1c; Molecular modelling studies: (Jointly in Egypt and Italy)

A. Molecular docking study:

The molecular docking of the prepared sulfonamides will be performed using the Discovery Studio 2.5/CDOCKER protocol (Accelrys, San Diego, CA, USA) or Molecular Operating Environment (MOE, 10.2010) software, to investigate their possible binding modes with carbonic anhydrase isoforms CAs IX and XII.

The protein crystallographic structure, hCA IX (PDB id: 3IAI) and hCA XII (PDB id: 1JD0) is downloaded from the Protein Data Bank (PDB). The protein was prepared for docking process according to the standard protein preparation procedures. The binding energy was calculated as a score to rank the docking poses. The top 10 docking poses would be finally saved. Docking poses were ranked according to their -CDOCKER interaction energy, and the top pose was chosen for analysis of interactions for each compound.

B. Physicochemical Properties and ADME Profiling:

Physicochemical properties and ADME profiling for all the synthesized compounds are performed using Discovery Studio 2.5 (Accelrys, San Diego, CA, USA). All the tested compounds are drawn as a small library and prepared using prepare ligand protocol to find

the suitable orientation in 3D. For studying physicochemical properties, the prepared library is filtered using the Lipinski and Veber rules protocols. ADME profiling is predicted for the designed library using ADME descriptors protocol.

Synthetic chemistry and Computational resources: The synthetic chemistry pathways employed to prepare the new targeted sulfonamides

will be conducted in the Kafrelsheikh University, where the basic facilities such as heaters, magnetic stirrers, rotary evaporator, oven, glassware, common organic solvents and UV-lamp are available. In addition, we will also need to use the analytical facilities (Mass and NMR spectrometers) in Kafrelsheikh and Cairo Universities. The rent costs of using these facilities have been involved within the budget under “analytical costs”. We already have a version of software needed for establishing the molecular docking and ADME Profiling, and we are well-trained to use it.

Work package 2: Biological Evaluation

WP2a; Library screening against different Carbonic Anhydrase isoforms: (in Italy)

The CA inhibitory ability of the synthesized sulfonamides will be assessed against four physiologically relevant hCA isoforms, hCA I, II (cytosolic) as well as hCA IX and XII (transmembrane, tumor associated isoforms).

An Applied Photophysics stopped-flow instrument has been used for assaying the CA catalysed CO2 hydration activity [42]. Phenol red (at a concentration of 0.2 mM) has been used as indicator, working at the absorbance maximum of 557 nm, with 20 mM Hepes (pH 7.5) as buffer, and 20 mM Na2SO4 (for maintaining constant the ionic strength), following the initial rates of the CA-catalyzed CO2 hydration reaction for a period of 10-100 s. The CO2 concentrations ranged from 1.7 to 17 mM for the determination of the kinetic parameters and inhibition constants. For each inhibitor at least six traces of the initial 5-10% of the reaction have been used for determining the initial velocity. The uncatalyzed rates were determined in the same manner and subtracted from the total observed rates. Stock solutions of inhibitor (0.1 mM) were prepared in distilled-deionized water and dilutions up to 0.01 nM were done thereafter with the assay buffer. Inhibitor and enzyme solutions were preincubated together for 15 min at room temperature prior to assay, in order to allow for the formation of the E-I complex. The inhibition constants were obtained by non-linear least-squares methods using PRISM 3 and the Cheng-Prusoff equation, as reported earlier [43-36], and represent the mean from at least three different determinations. All CA isofoms were recombinant ones obtained in-house as reported earlier [43-46].

The selectivity index (SI) of the tested compounds will be calculated to measure their differential inhibitory activity towards isoforms hCA I/XII and hCA II/XII.

WP2b; In vitro anti-proliferative activity against breast cancer cell lines: (in Egypt)

This work package involves screening of the most active derivatives with good selectivity profile for inhibition of the tumor associated isoforms (CAs IX and XII) over the off-target cytosolic (CAs I and II) for their anti-proliferative activity against one or two breast cancer cell lines.

Anti-proliferative activity of the newly prepared sulfonamides will be evaluated against breast cancer cell lines using sulforhodamine B (SRB) colorimetric assay as described by Skehan et al. [48]. Being a well-known broad spectrum anticancer drug, doxorubicin is chosen as a positive control. The anti-proliferative activity is expressed as median growth inhibitory concentration (IC50) values which represent the compounds concentrations required to produce a 50% inhibition of cell growth after 48 hours of incubation compared to untreated controls. This evaluation will be carried out in the National Research Center (NRC), Cairo, Egypt.

WP2c; Apoptosis study: (in Egypt)

We will investigate the potential pro-apoptotic effects of the active hits attempting to explore another underlying mechanism for their anti-proliferative activity. The levels of 35 apoptotic markers will be assessed using Proteome Profiler Human Apoptosis Array Kit (Rndsystems, UK) as per the manufacturer’s instructions. This evaluation will be carried out in the National Research Center (NRC), Cairo, Egypt.

B.6 Outcomes and Impact:

The proposed project has broad implications for many areas of science, including drug discovery, chemistry, and tackling breast cancer.

To the chemistry community, this research is expected to provide new materials and chemical methods, as well as a wealth of structure-property information on ureido benzenesulfonamides that can be used in drug development.

To the academic institution, it will provide new training opportunities in a multidisciplinary environment and prospects for Egyptian academic researchers to interact with respected international colleagues. Also, this collaboration positively contributes to capacity building. From the rigorous synergetic application of our team we expect by the end of the funding period:

Identifying novel compounds useful in controlling breast cancer with drug–like properties.

One master or one PhD theses is expected to be fully completed. At least 3 publications, in highly-reputable international journals and/or conferences are

expected. Active compounds will be patented. Set up strong ties with the CNR of Italy for future collaboration.

Part C: Gantt-chart and Logical Framework Matrix (LFM)

FIRST YEAR

Task No.

Task Title

Star

t D

ate

End

Dat

e

Du

rati

on

(M

on

ths)

M1

M2

M3

M4

M5

M6

M7

M8

M9

M1

0

M1

1

M1

2

Expected measurable outcomes of

successful implementation of

the task

1 Literature survey, ordering and delivery of chemicals, and supplies

1 Arrival of chemicals

and supplies

2 Preparing intermediates and target compounds in Scheme 1

4 Spectral Data

3 Verification of all the synthesized structures in schemes 1

1 Spectral Data

4 Preparing intermediates in Scheme 2 1 Spectral Data

5 Preparing intermediates and target compounds in Scheme 3

4 Spectral Data

6 Verification of all the synthesized

structures in schemes 2 and 3 1 Spectral Data

SECOND YEAR

Task No.

Task Title

Star

t D

ate

End

Dat

e

Du

rati

on

(M

on

ths)

M 1

3

M 1

4

M 1

5

M 1

6

M 1

7

M 1

8

M 1

9

M 2

0

M 2

1

Expected measurable

outcomes of successful

implementation of the task

1 Molecular docking study 2 2D and 3D diagrams

for the docked compounds

2 Physicochemical Properties and

ADME Profiling 1 The profiling data

3 Library screening against different

Carbonic Anhydrase isoforms 6 KI values for the

tested compounds

4 Evaluating the anti-proliferative

activity against breast cancer cell lines 1 IC50 values for the

tested compounds

5 Apoptosis study 1 Results of the study

6 Data collection, analysis and

preparation of drafts of papers 1

Submission of the manuscript

Part: D Budget

item 1st year 2

nd year percentage

Travel expenses 00 00 00

Small equipment 00 00 00

Chemicals and

Consumables 49,250 23,500 78.9 %

Training 00 00 00

Publications 00 00 00

Other expenses 19,500 (Spectral and Elemental

analysis and shipping to Italy)

21.1 %

Total 68,750 23,500

academy of scientifc research and technology · vii and molecular docking studies. eur. j. med....

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