Name: development of inhibitors of protein-protein interactions through replace: application to the design and development non-atp competitive cdk inhibitors
Uploaded: 2015-10-26T14:00:00.000+00:00
Duration: 10 min 33 s
Description: Watch this Scientific Journal Video about Development of Inhibitors of Protein-protein Interactions through REPLACE: Application to the Design and Development Non-ATP Competitive CDK Inhibitors at JoV

We thank Dr&#8217;s. Douglas Pittman and Michael Wyatt for their assistance with cell culture and Dr Wyatt and Ms. Erin Anderson for help in development of the binding assays. We acknowledge Mike Walla and Bill Cotham in the Department of Chemistry and Biochemistry at the University of South Carolina for assistance with Mass Spectrometry, Helga Cohen and Dr. Perry Pellechia for NMR spectrometry. This work was funded by the National Institutes of Health through the research project grant, 5R01CA131368.

<ol>
	<li>Andrews, M. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=REPLACE:+a+strategy+for+iterative+design+of+cyclin-binding+groove+inhibitors.">REPLACE: a strategy for iterative design of cyclin-binding groove inhibitors.</a> Chembiochem. 7, 1909-1915 (2006).</li><li>Liu, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimization+of+Non-ATP+Competitive+CDK/Cyclin+Groove+Inhibitors+through+REPLACE-Mediated+Fragment+Assembly.">Optimization of Non-ATP Competitive CDK/Cyclin Groove Inhibitors through REPLACE-Mediated Fragment Assembly.</a> J Med Chem. 56, 1573-1582 (2013).</li><li>McInnes, C., Bernstein, M., Desai, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chapter+29.">Chapter 29.</a> Annual Reports in Medicinal Chemistry. 47, 459-474 (2012).</li><li>Bower, J. F., Pannifer, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Using+fragment-based+technologies+to+target+protein-protein+interactions.">Using fragment-based technologies to target protein-protein interactions.</a> Curr Pharm Des. 18, 4685-4696 (2012).</li><li>Makley, L. N., Gestwicki, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expanding+the+number+of+'druggable'+targets:+non-enzymes+and+protein-protein+interactions.">Expanding the number of 'druggable' targets: non-enzymes and protein-protein interactions.</a> Chemical biology, and drug design. 81, 22-32 (2013).</li><li>Walensky, L. D., Bird, G. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hydrocarbon-Stapled+Peptides:+Principles,+Practice,+and+Progress.">Hydrocarbon-Stapled Peptides: Principles, Practice, and Progress.</a> J Med Chem. , (2014).</li><li>Shapiro, G. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cyclin-dependent+kinase+pathways+as+targets+for+cancer+treatment.">Cyclin-dependent kinase pathways as targets for cancer treatment.</a> J Clin Oncol. 24, 1770-1783 (2006).</li><li>Thais, M., Sielecki, J. F. B., Enfield, P. A., Trainor, G. l. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cyclin+dependant+kinase+inhibitors:+Useful+targets+in+cell+cycle+regulation.">Cyclin dependant kinase inhibitors: Useful targets in cell cycle regulation.</a> Journal of medicinal chemistry. 43, 1-18 (2000).</li><li>Morphy, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selectively+nonselective+kinase+inhibition:+striking+the+right+balance.">Selectively nonselective kinase inhibition: striking the right balance.</a> J Med Chem. 53, 1413-1437 (2010).</li><li>Chen, Y. N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selective+killing+of+transformed+cells+by+cyclin/cyclin-dependent+kinase+2+antagonists.">Selective killing of transformed cells by cyclin/cyclin-dependent kinase 2 antagonists.</a> Proc Natl Acad Sci USA. 96, 4325-4329 (1999).</li><li>Orzaez, M., Gortat, A., Mondragon, L., Bachs, O., Perez-Paya, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ATP-noncompetitive+inhibitors+of+CDK-cyclin+complexes.">ATP-noncompetitive inhibitors of CDK-cyclin complexes.</a> ChemMedChem. 4, 19-24 (2009).</li><li>Kontopidis, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Insights+into+cyclin+groove+recognition:+complex+crystal+structures+and+inhibitor+design+through+ligand+exchange.">Insights into cyclin groove recognition: complex crystal structures and inhibitor design through ligand exchange.</a> Structure. 11, 1537-1546 (2003).</li><li>McInnes, C., Andrews, M. J., Zheleva, D. I., Lane, D. P., Fischer, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Peptidomimetic+design+of+CDK+inhibitors+targeting+the+recruitment+site+of+the+cyclin+subunit.">Peptidomimetic design of CDK inhibitors targeting the recruitment site of the cyclin subunit.</a> Current medicinal chemistry. Anti-cancer agents. 3, 57-69 (2003).</li><li>Premnath, P. N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fragment+based+discovery+of+arginine+isosteres+through+REPLACE:+towards+non-ATP+competitive+CDK+inhibitors.">Fragment based discovery of arginine isosteres through REPLACE: towards non-ATP competitive CDK inhibitors.</a> Bioorganic, and medicinal. 22, 616-622 (2014).</li><li>Venkatachalam, C. M., Jiang, X., Oldfield, T., Waldman, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=LigandFit:+a+novel+method+for+the+shape-directed+rapid+docking+of+ligands+to+protein+active+sites.">LigandFit: a novel method for the shape-directed rapid docking of ligands to protein active sites.</a> J Mol Graph Model. 21, 289-307 (2003).</li><li>Mendoza, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selective+cyclin-dependent+kinase+2/cyclin+A+antagonists+that+differ+from+ATP+site+inhibitors+block+tumor+growth.">Selective cyclin-dependent kinase 2/cyclin A antagonists that differ from ATP site inhibitors block tumor growth.</a> Cancer Res. 63, 1020-1024 (2003).</li><li>Denizot, F., Lang, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+colorimetric+assay+for+cell+growth+and+survival.+Modifications+to+the+tetrazolium+dye+procedure+giving+improved+sensitivity+and+reliability.">Rapid colorimetric assay for cell growth and survival. Modifications to the tetrazolium dye procedure giving improved sensitivity and reliability.</a> J Immunol Methods. 89, 271-277 (1986).</li><li>Zheleva, D. I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Highly+potent+p21(WAF1)-derived+peptide+inhibitors+of+CDK-mediated+pRb+phosphorylation:+delineation+and+structural+insight+into+their+interactions+with+cyclin+A.">Highly potent p21(WAF1)-derived peptide inhibitors of CDK-mediated pRb phosphorylation: delineation and structural insight into their interactions with cyclin A.</a> The journal of peptide research : official journal of the American Peptide Society. 60, 257-270 (2002).</li><li>Ivanov, A. A., Khuri, K. F. R., Fu, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeting+protein-protein+interactions+as+an+anticancer+strategy.">Targeting protein-protein interactions as an anticancer strategy.</a> Trends in Pharmacological Sciences. 34, (2013).</li><li>Thayer, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improving+peptides.">Improving peptides.</a> Chemical and Engineering News. 89, 13-20 (2011).</li><li>Yin, H., Hamilton, A. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Strategies+for+targeting+protein-protein+interactions+with+synthetic+agents.">Strategies for targeting protein-protein interactions with synthetic agents.</a> Angew Chem Int Ed Engl. 44, 4130-4163 (2005).</li><li>Jubb, H., Higueruelo, A. P., Winter, A., Blundell, T. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+biology+and+drug+discovery+for+protein-protein+interactions.">Structural biology and drug discovery for protein-protein interactions.</a> Trends Pharmacol Sci. 33, 241-248 (2012).</li><li>Cannon, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pharmaceutics+and+drug+delivery+aspects+of+heme+and+porphyrin+therapy.">Pharmaceutics and drug delivery aspects of heme and porphyrin therapy.</a> J Pharm Sci. 82, 435-446 (1993).</li></ol>

The authors declare that they have no competing financial interests.

Targeting protein-protein interactions (PPI) in drug discovery is highly challenging as these typically involve a large shallow contact interface comprised of numerous and diffuse contacts19. Furthermore, peptidic compounds which inhibit PPI&#8217;s that are amenable to drug discovery are problematic due to their higher molecular mass, metabolic instability and poor bioavailability20. Current strategies that have been applied for the development of PPI inhibitors include design of proteomimetics and...

في هذه المقالة، وصفت دراسة حالة لتطبيق استبدال (استبدال ببدائل يجند جزئية باستخدام الحسابية التخصيب) استراتيجية لتحويل مثبطات ببتيدية من تفاعلات البروتين البروتين إلى مزيد من صيدليا الجزيئات ذات الصلة 1-3. بينما تمثل مثبطات مضخة البروتون مصدرا غنيا لكن مستغلة من الأهداف المحتملة المخدرات والمنهجيات الحالية غير كافية إلى حد كبير لجعل هذه يمكن الوصول إليها على نطاق واسع. الاستراتيجيات الحالية بما في ذلك جزء يستند تصميم 4، الإنتاجية العالية فحص 5 والببتيدات تدبيس وفرت 6 التقدم، ولكن هذه هي في كثير من الحالات غير فعالة. ونتيجة لذلك، يطلب المزيد من التقدم ونهج أكثر كفاءة. استبدال تم التحقق من صحة تماما في تطوير مثبطات كيناز التي أدت إلى تحسين الخصائص مثل المخدرات ولها إمكانات لمزيد من التطوير كما العلاجات المضادة للورم. ويتمثل هذه الاستراتيجية في تطوير مثبطات غير ATP من الخلاياCDKs دورة وينطوي على النحو التالي: 1) الحصول على المعلومات الهيكلية 3D على تفاعلات HAKRRLIF / RRLIF مع السيكلين الأخدود ملزمة. 2) تحديد محددات ملزمة هامة للتفاعل الببتيد. 3) الاقتطاع من الببتيد N-محطة تحتوي على واحد أو أكثر من محددات ملزمة؛ 4) تحديد الحسابية بدائل جزيء صغير المحتملة (بدائل يجند جزئية، بلاس) للجزء المقتطعة من الببتيد والتي تحتفظ التفاعلات الرئيسية للالببتيد الأم. 5) توقع توليف أو مصادر تجارية البلاس لربط بشوق مع موقع فرعي المحتلة من قبل بقايا الببتيد حذف (ق)؛ 6) توليف تقلب من خلال ربط من أفضل بلاس لالببتيد اقتطاع باستخدام تركيب الحالة الصلبة. 7) اختبار تقلب في في المختبر ملزمة أو فحص وظيفي (الاستقطاب مضان في سياق / السيكلين معلمين) تليها مزيد من التوصيف في مقايسة بقاء الخلية. تمثيل تخطيطي لاستبدال الاستراتيجيههو مبين في الشكل 1 ذ. في هذه المقالة، وتناقش تكرار لاستراتيجية استبدال وتطبيق لCDK2 / السيكلين A وصف بالتفصيل. ويعتقد CDKs أن حررت مباشرة أو غير مباشرة في معظم الأورام وبالتالي تعتبر أهدافا المخدرات سرطان المناسب 7. CDKs تتطلب بالتعاون مع الحلقيات لتفعيل كامل وبعد ذلك الفوسفات البروتينات الرئيسية المشاركة في تنظيم دورة الخلية 8. الفئات الرئيسية اثنين من CDKs هي isotypes التي تتحكم في نقاط التفتيش دورة الخلية [G1 / S (CDK4 / السيكلين D، CDK6 / السيكلين D وCDK4 / السيكلين E)، مرحلة S (CDK2 / السيكلين A) وG2 / M (CDK1 / السيكلين B)] والمنظمين من RNA البلمرة من خلال الفسفرة (CDK7 / السيكلين H، CDK8 / السيكلين C، CDK9 / السيكلين T). خطوة رئيسية في S مرحلة تطور يحدث عندما يشكل عامل E2F1 النسخ مجمع مع البروتين الذي يربط موانئ دبي ثم إلى DNA ويبدأ النسخ الجيني. مطلوب CDK2 / السيكلين A لتحييد E2F1 النسخيالنشاط خلال الفسفرة مما يؤدي إلى الإفراج عن مجمع E2F1-DP وتدهور لاحقا. ويعتقد تثبيط CDK2 / السيكلين A للحفاظ على E2F1 في DNA حالته ملزمة مما يؤدي إلى تنشيط دائم. فإن مستوى الناتجة من E2F-1 النشاط تتجاوز الحد الأدنى المطلوب للحث على البروتين p53 الخلايا المستقلة بالتالي يشير إلى استراتيجية علاجية. بسبب البروتين p53 حررت ومسارات PRB، ومستويات عالية من E2F-1 تحدث بشكل متكرر في الخلايا السرطانية وتثبيط CDK2 / السيكلين A ينبغي أن يؤدي إلى موت الخلايا المبرمج انتقائية في الأورام ويمكن اعتباره هدفا سرطان صادق 7. سريريا مثبطات معلمين التحقيق تستهدف موقع ATP الحفظ جدا ملزمة مما يؤدي إلى عبور التفاعل بين تحركات البروتينات أكبر من 500 في kinome الإنسان ويحتمل أن تكون مما أدى إلى آثار جانبية وسمية 9. نهج بديل غير قابلة للATP تثبيط المنافسة من خلال استهداف توظيف الركيزة خلال CBGموجودة على السيكلين فرعية التنظيمية الإيجابية والتي هي بالتالي متميزة وبعيدة عن موقع ATP 10،11 ملزمة. وCBG هو في المقام الأول الأخدود مسعور الحالي في السيكلين A، D السيكلين والسيكلين E ولقد ثبت للتعرف على تسلسل إجماع وجدت في ركائز والمكثفات الورم. كما الببتيد معزولة، والسيكلين ملزمة عزر (CBM) بربط CBG ولقد ثبت لكبح النشاط كيناز من CDKs دورة الخلية. وقد تم تحسين CBM إلى ثماني الببتيد (HAKRRLIF، CDK2 / السيكلين A IC 50 0.07 ± 0.02 ميكرومتر، CDK4 / السيكلين D، IC 50 0.88 ± 0.34 ميكرومتر) واقتطاع علاوة على ذلك لخماسي الببتيد تمثل حلا وسطا جيدا بين الوزن الجزيئي للبالمخدرات الشبه ورجولية (RRLIF، CDK2 / السيكلين A IC 50 1.01 ± 0.17 ميكرومتر، CDK4 / السيكلين D، IC 50 25.12 ± 2.97 ميكرومتر) 12،13. تتألف CBGs من الانتخابات التمهيدية كبير وجيب مسعور الثانوي أصغر التي يتم سدها من قبل الحمضةج المنطقة (تشمل Glu220، Glu224 وAsp283). وتشمل محددات ملزمة الرئيسية HAKRRLIF تفاعل Ala2 مع السندات جيب مسعور الثانوي، الاقتران أيون الهيدروجين وللLys3، الارجنتين 4 و Arg5 مع المنطقة الحمضية وعلى درجة عالية من التكامل بين Leu6 وPhe8 مع موقع دهون الأساسي. بالإضافة إلى ذلك، ساهم السندات الهيدروجين العديد من العمود الفقري الببتيد في حين يعمل Ile7 كبقايا هل يسمح اتصال الأمثل مع جيب الأساسي. يظهر وضع ملزمة وتفاعلات HAKRRLIF مع CBG في الشكل 2. سوف تستهدف CBM / CBG تفاعل البروتين البروتين تمنع النشاط كيناز من CDK2 / السيكلين A، CDK2 / السيكلين E &amp; CDK4 / السيكلين D وهذا ينبغي أن يؤدي E2F1 الخلايا بوساطة الخلايا السرطانية في حين لا تؤثر الخلايا الطبيعية 7. على الرغم من أن CBM المستمدة الببتيدات هي مثبطات فعالة من CDKs دورة الخلية، فمن غير المحتمل أن يكون مفيدا كدواء بسبب الايضعدم الاستقرار ونقص عام في نفاذية الخلية. تحقيقا لهذه الغاية، قمنا بتطبيق استراتيجية استبدالها من أجل تحويل هذه المثبطات ببتيدية واسعا إلى مزيد من مركبات شبيهة المخدرات للحصول على مزيد من العلاجات المضادة للورم استغلال E2F1 حررت خلال CDK2 / السيكلين A تثبيط التنمية. بروتوكول التالية يلخص العمل الذي تم الانتهاء منه في تطبيق استبدال لالأخدود السيكلين. في المقام الأول، تم تحديد بدائل فريق السد مثل المخدرات للرباعي البيبتيد N-محطة من HAKRRLIF. وعلاوة على ذلك تم التحقيق التحسينات في هذه المجموعات في دراسة التحقق من صحة إضافية لاستبدال. وتعرض أيضا ممثل النتائج من هذه الدراسات.

<table><tbody><tr><td colspan="4">&lt;strong&gt;Computational Chemistry&lt;/strong&gt;</td></tr><tr><td>Accelyrs Discovery studio 3.0</td><td></td><td></td><td></td></tr><tr><td>Dell Optiplex Workstations</td><td></td><td></td><td></td></tr><tr><td colspan="4">&lt;strong&gt;Synthetic Organic Chemistry&lt;/strong&gt;</td></tr><tr><td colspan="4">Silica gel (GF-254 plates) for TLC, Biotage (Uppsala, Sweden) for flash chromatography, Waters Alliance 2695 HPLC with a 2996 diode-array detector and equipped with a C18 (2) 100 A, 250 x 4.6&amp;nbsp;mm, 5&amp;nbsp;&amp;mu;m column (Phenomenox Luna) for purity determination, &lt;sup&gt;1&lt;/sup&gt;H NMR and &lt;sup&gt;13&lt;/sup&gt;C NMR spectra were recorded with a Varian Mercury 300 and 400 Spectrometer, respectively. Mass spectra were measured with a Micromass QTOF (Tandem quadruple-1 time of flight mass spectrometer), electrospray ionization (ESI) and VG 70S (Double-focusing magnetic sector mass spectrometer, EI).</td></tr><tr><td colspan="4">&lt;strong&gt;Flourescence Polarization Assay&lt;/strong&gt;</td></tr><tr><td>384 micro well plates, Micro pipets</td><td>Grenier Bio-one</td><td>110256602</td><td></td></tr><tr><td>CDK4D1 and CDK2CA (well purified recombinant human kinase complex)</td><td>BPS Bio Sciences</td><td>40094(CDK4/Cyclin D), 41101(CDK2/Cyclin A)</td><td></td></tr><tr><td colspan="4">assay buffer (25 nM HEPES pH 7, 10 mM NaCl, 0.01% Nonidet P-40, 1&amp;nbsp;mM dithiothretiol (DTT))</td></tr><tr><td>25 nM HEPES</td><td>CALBIOCHEM</td><td>375368</td><td></td></tr><tr><td>NaCl</td><td>Fisher</td><td>127838</td><td></td></tr><tr><td>Nonidet P-40</td><td>US Biological</td><td>N3500</td><td></td></tr><tr><td>DTT</td><td>Aldrich</td><td></td><td></td></tr><tr><td>-70 &amp;#176;C freezer</td><td>Revco (Ultima II)</td><td></td><td></td></tr><tr><td>DTX880 multimode detector fitted with 485&amp;nbsp;nm/535&amp;nbsp;nm excitation/emission filters and a dichroic mirror suitable for fluorescein</td><td>Beckman Coulter, Brea, CA</td><td></td><td></td></tr><tr><td colspan="4">&lt;strong&gt;Cell Culture&lt;/strong&gt;</td></tr><tr><td>96 well plates</td><td>Fisher</td><td></td><td></td></tr><tr><td>Frozen stocks of U2OS (osteosarcoma) and DU145 (prostate cancer) cell lines</td><td>ATCC</td><td></td><td></td></tr><tr><td>NU serum, DMEM media, trypsin, PEN/STRIP, MTT reagent</td><td>Fisher, Life technology, Alfa Aesar</td><td></td><td></td></tr><tr><td>Heamocytometer</td><td>VWR</td><td></td><td></td></tr><tr><td>-70 &amp;#176;C freezer</td><td>Revco (Ultima II)</td><td></td><td></td></tr><tr><td>Incubator</td><td>Thermo electron corporation</td><td></td><td></td></tr><tr><td>Centrifuge</td><td>Eppendorf</td><td>5804 R</td><td></td></tr><tr><td>Refrigerator 4-8 &amp;#176;C</td><td>Isotemp Fisher</td><td></td><td></td></tr><tr><td>DTX880 multimode detector fitted with 595&amp;nbsp;nm filter</td><td>Beckman Coulter, Brea, CA</td><td></td><td></td></tr></tbody></table>

development of inhibitors of protein-protein interactions through replace: application to the design and development non-atp competitive cdk inhibitors

استبدال هو استراتيجية فريدة من نوعها وضعت لاستهداف أكثر فعالية البروتين البروتين التفاعلات (مثبطات مضخة البروتون). انها تهدف الى توسيع المساحة المتوفرة على الهدف المخدرات من خلال توفير منهجية محسنة لتحديد مثبطات لمثل هذه المواقع الملزمة والتي تمثل غالبية الأهداف المحتملة المخدرات. والهدف الرئيسي من هذه الورقة هو تقديم لمحة المنهجية لاستخدام وتطبيق استراتيجية استبدال الذي ينطوي على النهج الكيمياء الحسابية والاصطناعية. استبدل يتجلى من خلال تطبيقها لتطوير تحركات غير ATP السيكلين تنافسية تعتمد (للمعلمين) مثبطات كما العلاجات المضادة للورم. وكثيرا ما حررت CDKs في السرطان، وبالتالي تعتبر أهدافا هامة للتنمية المخدرات. تم الإبلاغ عن تثبيط CDK2 / السيكلين A في المرحلة S لتعزيز موت الخلايا المبرمج انتقائية من الخلايا السرطانية بطريقة مستقلة البروتين p53 من خلال مسار E2F1. استهداف تفاعل البروتين البروتين في المربوطة السيكلينز الأخدود (CBG) هو النهج الذي سيسمح للتثبيط محددة من دورة الخلية على CDKs النسخي. يتم التعرف على CBG عن طريق سلسلة إجماع المستمدة من ركائز معلمين وورم البروتينات الكابتة يسمى السيكلين عزر (CBM) ملزمة. وقد سبق الأمثل CBM إلى ثماني الببتيد من p21Waf (HAKRRIF) ثم اقتطاع المزيد لخماسي الببتيد الاحتفاظ النشاط الكافي (RRLIF). الببتيدات بصفة عامة ليست قابلة للاختراق الخلية، غير مستقرة عملية الأيض وبالتالي تم تطبيق استبدال (مع استبدال الجزئي يجند البدائل من خلال الحاسوبية التخصيب) استراتيجية من أجل توليد المزيد من مثبطات مثل المخدرات. تبدأ استراتيجية مع تصميم جزء ligated الببتيد المثبطة (تقلب) التي تحول دون انتقائية دورة الخلية مجمعات معلمين / السيكلين. تم إنشاء تقلب عن طريق استبدال تكرارا مخلفات HAKRRLIF / RRLIF مع شظية مثل الجزيئات الصغيرة (متوجا مجموعات)، بدءا من N-محطة (Ncaps)، تليها بديل عنمحطة سي. هذه المركبات هي نقطة الانطلاق لتوليد مثبطات معلمين تنافسية غير ATP كما العلاجات المضادة للورم.

وتظهر تفاعلات HAKRRLIF مع الأخدود السيكلين في الشكل 2، وبقايا الببتيد التي تمثل محددات ملزمة رئيسية تشمل Ala2، Arg4، Leu6 وPhe8 مع المخلفات الأخرى التي تقدم مساهمات أصغر 12،13،18. في دراسة الحالة هذه قد استخدمت استراتيجية استبدالها من أجل إيجاد بدائل شظية م...

Watch this Scientific Journal Video about Development of Inhibitors of Protein-protein Interactions through REPLACE: Application to the Design and Development Non-ATP Competitive CDK Inhibitors at JoV

Development of Inhibitors of Protein-protein Interactions through REPLACE: Application to the Design and Development Non-ATP Competitive CDK Inhibitors

We describe implementation of the REPLACE strategy for targeting protein-protein interactions. REPLACE is an iterative strategy involving synthetic and computational approaches for the conversion of optimized peptidic inhibitors into drug like molecules.

تطوير مثبطات البروتين البروتين التفاعلات استبدال من خلال: تطبيق لتصميم وتطوير غير ATP مثبطات معلمين تنافسي

REPLACE is a unique strategy developed to more effectively target protein-protein interactions (PPIs). It aims to expand available drug target space by ...

development-inhibitors-protein-protein-interactions-through-replace

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

11.2K Views.  University of South Carolina. We describe implementation of the REPLACE strategy for targeting protein-protein interactions. REPLACE is an iterative strategy involving synthetic and computational approaches for the conversion of optimized peptidic inhibitors into drug like molecules.

Video: Development of Inhibitors of Protein-protein Interactions through REPLACE: Application to the Design and Development Non-ATP Competitive CDK Inhibitors

Identification of Cyclin-dependent Kinase 1 Specific Phosphorylation Sites by an In Vitro Kinase Assay

Cyclin-dependent kinase 1 (Cdk1) is a master controller for the cell cycle in all eukaryotes and phosphorylates an estimated 8 - 13% of the proteome; however, the number of identified targets for Cdk1, particularly in human cells is still low. The identification of Cdk1-specific phosphorylation sites is important, as they provide mechanistic insights into how Cdk1 controls the cell cycle. Cell cycle regulation is critical for faithful chromosome segregation, and defects in this complicated process lead to chromosomal aberrations and cancer.
Here, we describe an in vitro kinase assay that is used to identify Cdk1-specific phosphorylation sites. In this assay, a purified protein is phosphorylated in vitro by commercially available human Cdk1/cyclin B. Successful phosphorylation is confirmed by SDS-PAGE, and phosphorylation sites are subsequently identified by mass spectrometry. We also describe purification protocols that yield highly pure and homogeneous protein preparations suitable for the kinase assay, and a binding assay for the functional verification of the identified phosphorylation sites, which probes the interaction between a classical nuclear localization signal (cNLS) and its nuclear transport receptor karyopherin &#945;. To aid with experimental design, we review approaches for the prediction of Cdk1-specific phosphorylation sites from protein sequences. Together these protocols present a very powerful approach that yields Cdk1-specific phosphorylation sites and enables mechanistic studies into how Cdk1 controls the cell cycle. Since this method relies on purified proteins, it can be applied to any model organism and yields reliable results, especially when combined with cell functional studies.

Identification of Cyclin-dependent Kinase 1 Specific Phosphorylation Sites by an In Vitro Kinase Assay

Cyclin-dependent kinase 1 (Cdk1) is activated in the G2 phase of the cell cycle and regulates many cellular pathways. Here, we present a protocol for an in vitro kinase assay with Cdk1, which allows the identification of Cdk1-specific phosphorylation sites for establishing cellular targets of this important kinase.

تحديد كيناز Cyclin تعتمد على 1 مواقع الفسفرة محددة من قبل كيناز في المختبر الاعتداء

Cyclin-dependent kinase 1 (Cdk1) is a master controller for the cell cycle in all eukaryotes and phosphorylates an estimated 8 - 13% of the proteome; ...

Biochemistry

Identification of Novel CK2 Kinase Substrates Using a Versatile Biochemical Approach

The study of kinase-substrate relationships is essential to gain a complete understanding of the functions of these enzymes and their downstream targets in both physiological and pathological states. CK2 is an evolutionarily conserved serine/threonine kinase with a growing list of hundreds of substrates involved in multiple cellular processes. Due to its pleiotropic properties, identifying and characterizing a comprehensive set of CK2 substrates has been particularly challenging and remains a hurdle in the study of this important enzyme. To address this challenge, we have devised a versatile experimental strategy that enables the targeted enrichment and identification of putative CK2 substrates. This protocol takes advantage of the unique dual co-substrate specificity of CK2 allowing for specific thiophosphorylation of its substrates in a cell or tissue lysate. These substrate proteins are subsequently alkylated, immunoprecipitated, and identified by liquid chromatography/tandem mass spectrometry (LC-MS/MS). We have previously used this approach to successfully identify CK2 substrates from Drosophila ovaries and here we extend the application of this protocol to human glioblastoma cells, illustrating the adaptability of this method to investigate the biological roles of this kinase in various model organisms and experimental systems.

The objective of this protocol is to label, enrich, and identify substrates of protein kinase CK2 from a complex biological sample such as a cell lysate or tissue homogenate. This method leverages unique aspects of CK2 biology for this purpose.

تحديد ركائز كيناز CK2 الرواية باستخدام نهج بيوكيميائية تنوعاً

The study of kinase-substrate relationships is essential to gain a complete understanding of the functions of these enzymes and their downstream targets ...

Kinase Inhibitor Screening In Self-assembled Human Protein Microarrays

The screening of kinase inhibitors is crucial for better understanding properties of a drug and for the identification of potentially new targets with clinical implications. Several methodologies have been reported to accomplish such screening. However, each has its own limitations (e.g., the screening of only ATP analogues, restriction to using purified kinase domains, significant costs associated with testing more than a few kinases at a time, and lack of flexibility in screening protein kinases with novel mutations). Here, a new protocol that overcomes some of these limitations and can be used for the unbiased screening of kinase inhibitors is presented. A strength of this method is its ability to compare the activity of kinase inhibitors across multiple proteins, either between different kinases or different variants of the same kinase. Self-assembled protein microarrays generated through the expression of protein kinases by a human-based in vitro transcription and translation system (IVTT) are employed. The proteins displayed on the microarray are active, allowing for measurement of the effects of kinase inhibitors. The following procedure describes the protocol steps in detail, from the microarray generation and screening to the data analysis.

A detailed protocol for the generation of self-assembled human protein microarrays for the screening of kinase inhibitors is presented.

فحص مثبطات كيناز في ميكروصفائف البروتين البشري المجمعة ذاتيا

The screening of kinase inhibitors is crucial for better understanding properties of a drug and for the identification of potentially new targets with ...

High-Throughput Cellular Profiling of Targeted Protein Degradation Compounds Using HiBiT CRISPR Cell Lines

Targeted protein degradation compounds, including molecular glues or proteolysis targeting chimeras, are an exciting new therapeutic modality in small molecule drug discovery. This class of compounds induces protein degradation by bringing into proximity the target protein and the E3 ligase machinery proteins required to ubiquitinate and ultimately degrade the target protein through the ubiquitin-proteasomal pathway (UPP). Profiling of target protein degradation in a high-throughput fashion, however, remains highly challenging given the complexity of cellular pathways required to achieve degradation. Here we present a protocol and screening strategy based on the use of CRISPR/Cas9 endogenous tagging of target proteins with the 11 amino acid HiBiT tag which complements with high affinity to the LgBiT protein, to produce a luminescent protein. These CRISPR targeted cell lines with endogenous tags can be used to measure compound induced degradation in either real-time, kinetic live cell or endpoint lytic modes by monitoring luminescent signal using a luminescent plate-based reader. Here we outline the recommended screening protocols for the different formats, and&#160;also describe the calculation of key degradation parameters of rate, Dmax, DC50, Dmax50, as well as multiplexing with cell viability assays. These approaches enable rapid discovery and triaging of early stage compounds while maintaining endogenous expression and regulation of target proteins in relevant cellular backgrounds, allowing for efficient optimization of lead therapeutic compounds.

This protocol describes the quantitative luminescent detection of protein degradation kinetics in living cells that have been engineered using CRISPR/Cas9 to express antibody free endogenous protein detection tag fused to a target protein. Detailed instructions for calculating and obtaining quantitative degradation parameters, rate, Dmax, DC50, and Dmax50 are included.

التنميط الخلوي عالي الإنتاجية لمركبات تحلل البروتين المستهدفة باستخدام خطوط خلايا HiBiT CRISPR

Targeted protein degradation compounds, including molecular glues or proteolysis targeting chimeras, are an exciting new therapeutic modality in small ...

Cancer Research

Identification of Mediators of T-cell Receptor Signaling via the Screening of Chemical Inhibitor Libraries

The T-cell receptor (TCR) signaling pathway comprises a multitude of mediators that transmit signals upon the activation of the TCR. Different strategies have been proposed and implemented for the identification of new mediators of TCR signaling, which would improve the understanding of T-cell processes, including activation and thymic selection. We describe a screening assay that enables the identification of molecules that influence TCR signaling based on the activation of developing thymocytes. Strong TCR signals cause developing thymocytes to activate apoptotic machinery in a process known as negative selection. Through the application of kinase inhibitors, those with targets that affect TCR signaling are able to override the process of negative selection. The method detailed in this paper can be used to identify inhibitors of canonical kinases with established roles in the TCR signaling pathways and also inhibitors of new kinases yet to be established in the TCR signaling pathways. The screening strategy here can be applied to screens of higher throughput for the identification of novel druggable targets in TCR signaling.

Identification of Mediators of T-cell Receptor Signaling via the Screening of Chemical Inhibitor Libraries

This paper uses a flow-cytometry-based assay to screen libraries of chemical inhibitors for the identification of inhibitors and their targets that influence T-cell receptor signaling. The methods described here can also be expanded for high-throughput screenings.

تحديد وسطاء من خلايا تي مستقبلات الإشارات عن طريق فحص مكتبات المانع الكيميائية

The T-cell receptor (TCR) signaling pathway comprises a multitude of mediators that transmit signals upon the activation of the TCR. Different strategies ...

Immunology and Infection

Protein WISDOM: A Workbench for In silico De novo Design of BioMolecules

The aim of de novo protein design is to find the amino acid sequences that will fold into a desired 3-dimensional structure with improvements in specific properties, such as binding affinity, agonist or antagonist behavior, or stability, relative to the native sequence. Protein design lies at the center of current advances drug design and discovery. Not only does protein design provide predictions for potentially useful drug targets, but it also enhances our understanding of the protein folding process and protein-protein interactions. Experimental methods such as directed evolution have shown success in protein design. However, such methods are restricted by the limited sequence space that can be searched tractably. In contrast, computational design strategies allow for the screening of a much larger set of sequences covering a wide variety of properties and functionality. We have developed a range of computational de novo protein design methods capable of tackling several important areas of protein design. These include the design of monomeric proteins for increased stability and complexes for increased binding affinity.
To disseminate these methods for broader use we present Protein WISDOM (<a href="http://www.proteinwisdom.org" target="_blank">http://www.proteinwisdom.org</a>), a tool that provides automated methods for a variety of protein design problems. Structural templates are submitted to initialize the design process. The first stage of design is an optimization sequence selection stage that aims at improving stability through minimization of potential energy in the sequence space. Selected sequences are then run through a fold specificity stage and a binding affinity stage. A rank-ordered list of the sequences for each step of the process, along with relevant designed structures, provides the user with a comprehensive quantitative assessment of the design. Here we provide the details of each design method, as well as several notable experimental successes attained through the use of the methods.

Protein WISDOM: A Workbench for In silico De novo Design of BioMolecules

We developed computational de novo protein design methods capable of tackling several important areas of protein design. To disseminate these methods we present Protein WISDOM, an online tool for protein design (<a href="http://www.proteinwisdom.org" target="_blank">http://www.proteinwisdom.org</a>). Starting from a structural template, design of monomeric proteins for increased stability and complexes for increased binding affinity can be performed.

الحكمة البروتين: A منضدة لل<em&gt; في السيليكون</em&gt;<em&gt; دي نوفو</em&gt; تصميم الجزيئات الحيوية

The aim of de novo protein design is to find the amino acid sequences that will fold into a desired 3-dimensional structure with improvements in specific ...

A Method for Screening and Validation of Resistant Mutations Against Kinase Inhibitors

The discovery of BCR/ABL as a driver oncogene in chronic myeloid leukemia (CML) resulted in the development of Imatinib, which, in fact, demonstrated the potential of targeting the kinase in cancers by effectively treating the CML patients. This observation revolutionized drug development to target the oncogenic kinases implicated in various other malignancies, such as, EGFR, B-RAF, KIT and PDGFRs. However, one major drawback of anti-kinase therapies is the emergence of drug resistance mutations rendering the target to have reduced or lost affinity for the drug. Understanding the mechanisms employed by resistant variants not only helps in developing the next generation inhibitors but also gives impetus to clinical management using personalized medicine. We reported a retroviral vector based screening strategy to identify the spectrum of resistance conferring mutations in BCR/ABL, which has helped in developing the next generation BCR/ABL inhibitors. Using Ruxolitinib and JAK2 as a drug target pair, here we describe in vitro screening methods that utilizes the mouse BAF3 cells expressing the random mutation library of JAK2 kinase.

Emergence of genetic resistance against kinase inhibitor therapy poses significant challenge for effective cancer therapy. Identification and characterization of resistant mutations against a newly developed drug helps in better clinical management and next generation drug design. Here, we describe our protocol for in vitro screening and validation of resistant mutations.

وهناك طريقة لفحص والتحقق من الطفرات المقاومة ضد كيناز مثبطات

The discovery of BCR/ABL as a driver oncogene in chronic myeloid leukemia (CML) resulted in the development of Imatinib, which, in fact, demonstrated the ...

Identification of Kinase-substrate Pairs Using High Throughput Screening

We have developed a screening platform to identify dedicated human protein kinases for phosphorylated substrates which can be used to elucidate novel signal transduction pathways. Our approach features the use of a library of purified GST-tagged human protein kinases and a recombinant protein substrate of interest. We have used this technology to identify MAP/microtubule affinity-regulating kinase 2 (MARK2) as the kinase for a glucose-regulated site on CREB-Regulated Transcriptional Coactivator 2 (CRTC2), a protein required for beta cell proliferation, as well as the Axl family of tyrosine kinases as regulators of cell metastasis by phosphorylation of the adaptor protein ELMO. We describe this technology and discuss how it can help to establish a comprehensive map of how cells respond to environmental stimuli.

Protein phosphorylation is a central feature of how cells interpret and respond to information in their extracellular milieu. Here, we present a high throughput screening protocol using kinases purified from mammalian cells to rapidly identify kinases that phosphorylate a substrate(s) of interest.

تحديد أزواج كيناز الركيزة عن طريق فحص إنتاجية عالية


We have developed a screening platform to identify dedicated human protein kinases for phosphorylated substrates which can be used to elucidate novel ...

Developing and Testing Methylated Nano-Structured Dipeptides that Inhibit Src Kinase Activity In Vitro for Anti-Cancer Applications

Here, with the aim of developing a novel anti-cancer treatment, seven dipeptides were designed that contained methylated tryptophan and/or methylated arginine and were produced using Fmoc solid-phase peptide synthesis. Overexpression of the Src tyrosine kinase enzyme has been implicated in the development of different cancers. Dipeptides containing unnatural amino acids such as methylated arginine (RCH3), dimethylated arginine (R(CH3)2), and/or methylated tryptophan (WCH3) residues have earlier been shown to inhibit Src kinase. In this study, three such dipeptides, W-RCH3, WCH3-RCH3, and W-R(CH3)2, were tested using acellular assays and were found to have IC50 values (the concentration at which 50% inhibition occurs) of 510 nM, 916 nM, and 1 &#181;M, respectively. These values were comparable to those obtained for cyclic penta- to nano-W-R peptides ([W-R]5-[W-R]9) synthesized in previous studies. However, the unmethylated versions of the dipeptides did not show any inhibitory activity against Src kinase. All of these dipeptides (50 &#181;M) did not show any cytotoxicity after incubation up to 72 h with three different cancer cell lines, including leukemia (CCRF-CEM), breast adenocarcinoma (MDA-MB-231), and ovarian adenocarcinoma (SK-OV-3) cell lines, indicating the limited permeability of the peptides through the cell membrane. Therefore, further study is needed to improve the permeability of these peptides for anticancer applications, such as by using a peptide carrier or additional peptide functionalization. In summary, this study provides a protocol to synthesize and test peptides that inhibit Src kinase activity, and thus possess promising anticancer ability, as demonstrated using acellular and cellular assays.

Developing and Testing Methylated Nano-Structured Dipeptides that Inhibit Src Kinase Activity In Vitro for Anti-Cancer Applications

Presented here is a protocol for designing and testing dipeptides that can inhibit Src kinase enzyme activity using acellular and cellular assays for anti-cancer applications. The peptides formulated here (W-RCH3, WCH3-RCH3, and W-R(CH3)2)&#160;inhibited Src kinase with IC50 values of 510 nM, 916 nM, and 1 &#181;M, respectively.

Here, with the aim of developing a novel anti-cancer treatment, seven dipeptides were designed that contained methylated tryptophan and/or methylated ...

تطوير مثبطات البروتين البروتين التفاعلات استبدال من خلال: تطبيق لتصميم وتطوير غير ATP مثبطات معلمين تنافسي

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تحديد كيناز Cyclin تعتمد على 1 مواقع الفسفرة محددة من قبل كيناز في المختبر الاعتداء

تحديد ركائز كيناز CK2 الرواية باستخدام نهج بيوكيميائية تنوعاً

فحص مثبطات كيناز في ميكروصفائف البروتين البشري المجمعة ذاتيا

التنميط الخلوي عالي الإنتاجية لمركبات تحلل البروتين المستهدفة باستخدام خطوط خلايا HiBiT CRISPR

تحديد وسطاء من خلايا تي مستقبلات الإشارات عن طريق فحص مكتبات المانع الكيميائية

الحكمة البروتين: A منضدة لل

وهناك طريقة لفحص والتحقق من الطفرات المقاومة ضد كيناز مثبطات

تحديد أزواج كيناز الركيزة عن طريق فحص إنتاجية عالية

Developing and Testing Methylated Nano-Structured Dipeptides that Inhibit Src Kinase Activity In Vitro for Anti-Cancer Applications

تثبيط نشاط CDK