STRUCTURE FUNCTION RELATIOSHIP IN PROTEASES AND ITS RELEVANCE

STRUCTURE FUNCTION RELATIOSHIP IN PROTEASES AND ITS RELEVANCE IN PATHOLOGY REVIEW PAPER PRESENTED BY DR. SUBRATA GANGULI AT INDUSTRIAL PHARMACY CONFERENCE, DUBAI, HELD ON APRIL 28 -29, 2016

HEALTH CARE AND INDUSTRIAL PHARMACY HEALTH EXPENDITURE in 2014 in US$ (WORLD BANK STATISTICS) BIODIVERSITY BIOTECHNOLOGY MATRIX (Taken from APJ Abdul Kalam “India 2020”) Country Per capita expenditure RICH-RICH --- NIL ---- Bangladesh 31 POOR-RICH (USA, Japan, France, Germany, UK) Cuba 817 Greece 1743 India 75 POOR-POOR (some desertic countries) RICH-POOR (Brazil, China, India, Mexico) Israel 2910 USA 9403 Technology flow from NORTH to SOUTH and Diversity from SOUTH to NORTH is one model of investment (after Prof. Kalam) Dr S. ganguli at the Industrial Pharmacy Conference, Dubai on April 28 -29, 2016.

LIFESTYLE AND HEALTH CAUSES OF DEATH (WORLD BANK STATISTICS) TRADITIONAL HEALTH WORKERS (per 1000 population from WHO STATISTICS) Country dentist pharmacist Comments (traditional worker) Bangladesh 0. 019 . 064 0. 334 in 2011 India 0. 095 0. 529 Not listed China 0. 039 0. 266 1. 029 in 2000 USA 1. 627 0. 876 Not listed Communica Nonble (% of communica total) ble (% of total) Bangladesh 32 59 Cuba 6 86 Greece 6 91 India 28 60 Israel 9 86 Dr S. ganguli at the Industrial Pharmacy Conference, Dubai on April 28 -29, 2016.

PROTEIN DEGRADATION PHYSIOLOGICAL CONDITION • Normal lifecycle and recycling • Activation by cleavage of precursors • Removal of signal peptides • Processing of polypeptides • Destroy potentially toxic proteins PATHOLOGICAL CORRELATION • Invasion of host machinery Interfering with normal lifecycle • Implicated in diseases like rheumatoid arthritis and Alzheimer’s disease, and in tumor growth. Small molecule inhibitors can work as therapeutics. Dr S. ganguli at the Industrial Pharmacy Conference, Dubai on April 28 -29, 2016.

PROTEIN BIOINFORMATICS DATABASE ANALYSIS Dr S. ganguli at the Industrial Pharmacy Conference, Dubai on April 28 -29, 2016.

6. 50% 3. 50% 4. 00% taxonomic classification INFORMATION EXPLOSION IN PROTEIN BIOINFORMATICS NCBI site (www. ncbi. nlm. nih. gov) lists 207018196067 bases and 190250235 sequences in its Feb. 2016 statistics on Gen. Bank PDB database(www. pdb. org) contains 117882 macromolecular structures MEROPS (merops. sanger. ac. uk) includes 17729 organisms containing 900806 proteases Top. FIND database (clipserve. clip. ubc. ca/topfind) lists 290000 termini and 33000 cleavage sites 50. 50% 35. 40% Eukaryota Archaea Bacteria Rest A plot (taken from NCBI statistics) on a log-log scale illustrating the phenomenal growth of availability of protein structures after the advent of the web-based approaches and development of fast computation technologies. The blue vertical line indicates availability of the world wide web in the year 1990. Dr S. ganguli at the Industrial Pharmacy Conference, Dubai on April 28 -29, 2016. Virus

VIEWING THE GROWTH IN PROTEIN BIOINFORMATICS Pyramid of knowledge of high resolution protein structures (<2 Å) PROTEIN DATABASE EXPANDING ALONG WITH THE DEVELOPMENTS IN INFORMATION TECHOLOGY 18747 (2011— 2015) 43489 during 2010 -2015 15336 (2006— 2010) 8748(2001— 2005) 33119 during 2005 -2010 17754 During 2000 --2005 3842 (1996 — 2000) 10964 Upto 2000 1711 (upto 1995) Dr S. ganguli at the Industrial Pharmacy Conference, Dubai on April 28 -29, 2016.

Summary of Structures at the PDB Database ENZYME (EC) CLASSIFICATION 8% 4% 4% 37% 17% 29% Hydrolases Oxidoreductases Isomerases Transferases Lyases Ligases SCOP CLASSIFICATION Type of Fold % of Total α-β 25. 5 α+β 23. 5 All β 22. 7 All α 16. 2 Dr S. ganguli at the Industrial Pharmacy Conference, Dubai on April 28 -29, 2016.

Summary of Structures at the PDB Database Methods employed to determine the structure Taxonomic groups 3% 4% 7% 35% 51% 96. 2% 1 0. 6% 0. 4% X-ray (96. 2%) eukaryotes Bacteria Solution nmr (2. 9%) virus archaea Electron microscopy (0. 6%) rest Rest (<0. 4%) Dr S. ganguli at the Industrial Pharmacy Conference, Dubai on April 28 -29, 2016.

ANALYSIS OF THE MEROPS DATABASE PDB ID (768) 296062 260 Dr S. ganguli at the Industrial Pharmacy Conference, Dubai on April 28 -29, 2016. Unclassifiable Unknown Threonine Serine Mixed Asparagine 396 3879 34426 7704 4 6894 Metallo- Cysteine Unknown Threonine Serine 32 5 Aspartic 7 Mixed Asparagine 18 Metallo Cysteine Glutamic 3 43927 Glutamic 221 54 332627 174887 168 Aspartic 300 250 200 150 100 50 0 Sequences (900806)

SEQUENCES IN MEROPS DATABASE SEQUENCES 4 ut am M ic et As allo pa ra gin e M ixe d Se r Th ine re on i Un ne Un kno cla wn ss ifi ab le e in Gl st e Cy As p ar tic 350000 300000 250000 200000 332627 150000 296062 100000 174887 50000 43927 34426 7704 396 3879 6894 0 Dr S. ganguli at the Industrial Pharmacy Conference, Dubai on April 28 -29, 2016. SEQUENCES

STRUCTURE FUNCTION PARADIGM SEQUENCE TO STRUCTURE TO FUNCTION Dr S. ganguli at the Industrial Pharmacy Conference, Dubai on April 28 -29, 2016.

SEQUENCE TO STRUCTURE TO FUNCTION STRUCTURE FUNCTION PARADIGM: • The primary sequence of the protein encoded by the gene contains enough information to form a functional protein capable performing cellular work • The structural features are reproducible and universal CONVERSELY • ONE CAN MAKE CHANGES IN THE PRIMARY SEQUENCE IN VITRO AND EXPRESS DESIRABLE FUNCTION IN VIVO THE CONCEPT IS REQUIRED FOR MAKING NEW THERAPEUTIC MOLECULES Dr S. ganguli at the Industrial Pharmacy Conference, Dubai on April 28 -29, 2016.

UNLIMITED SEQUENCES WITH LIMITED FOLDS Growth of Unique SCOP Folds per Year in PDB DATABASE YEAR SCOP FOLD TOTAL STRUCT URE TO URE FOLD RATIO 2016 1393 117882 84. 6 2015 1393 114660 82. 2 2010 1393 69605 50 2005 1240 34065 27. 5 2000 723 13591 18. 8 Yearly growth of Total Structures in PDB DATABASE Dr S. ganguli at the Industrial Pharmacy Conference, Dubai on April 28 -29, 2016.

THE STRUCTURE FUNCTION PARADIGM SEQUENCE TO STRUCTURE: • • • Caltech and Cambridge groups established importance of hydrogen bond and the sequence to determine the geometry of a three dimensional objects. SEQUENCE AND FUNCTION: • • Ramachandran plot established the stereochemical constraints Privalov’s consideration of protein stability described role of hydrophobic cores • Stability criteria explained limited number of folds despite unlimited number of sequences. Accordingly these folds were associated with, and invoked for explaining, molecular basis of biological functions • • Sequence homology predicts comparable function and thus became basis for many search algorithms for recognition sites. Emphasis on stability necessitated consideration for degradation. The sequences described are called degrons. Conditional expression of degrons became important in biotechnology and medicine. The entire degradation criterion in vivo is discussed in N-end rule by Varshavsky With the enormous understanding of the structural features one may conclude function from sequence, but structural data is important for designing novel molecules important in biotechnology and medicine. We shall briefly comment on structure and sequence identity at the end of this talk. Dr S. ganguli at the Industrial Pharmacy Conference, Dubai on April 28 -29, 2016.

MMP in ANGIOGENESIS EARLY EVIDENCES Dr S. ganguli at the Industrial Pharmacy Conference, Dubai on April 28 -29, 2016.

MMP in ANGIOGENESIS • The following slides represent the early experiments demonstrating the role of matrix metallo-proteases in angiogenesis • The slides were downloaded from the internet for review purposes • The authors in this paper are: T. L. Haas et al. Am J Physiol Heart Circ Physiol 2000; 279: H 1540 -H 1547 (PMID 11009439) Dr S. ganguli at the Industrial Pharmacy Conference, Dubai on April 28 -29, 2016.

Basement membrane disturbances in chronically stimulated muscle. T. L. Haas et al. Am J Physiol Heart Circ Physiol 2000; 279: H 1540 H 1547 © 2000 by American Physiological Society Dr S. ganguli at the Industrial Pharmacy Conference, Dubai on April 28 -29, 2016.

Matrix metalloproteinase (MMP)-2 and membrane type 1 (MT 1)-MMP m. RNA and protein increase with chronic stimulation. T. L. Haas et al. Am J Physiol Heart Circ Physiol 2000; 279: H 1540 H 1547 © 2000 by American Physiological Society Dr S. ganguli at the Industrial Pharmacy Conference, Dubai on April 28 -29, 2016.

SEQUENCES IN MEROPS DATABASE SEQUENCES 4 ut am M ic et As allo pa ra gin e M ixe d Se r Th ine re on i Un ne Un kno cla wn ss ifi ab le e in Gl st e Cy As p ar tic 350000 300000 250000 200000 332627 150000 296062 100000 174887 50000 43927 34426 7704 396 3879 6894 0 Dr S. ganguli at the Industrial Pharmacy Conference, Dubai on April 28 -29, 2016. SEQUENCES

Table 1. A summary of sequence and structure data of MMP proteins (updated April 15, 2016) Note: The nomenclature of MMP’s in this table is consistent with www. wikipedia. org. The data are collected from the NCBI and PDB database. X and N in column 4 refer to X-ray Diffraction and NMR spectroscopy respectively. Symbol Other Name Gene ID (Entrez) PDB ID MMP 1 Interstitial collagenase, fibroblast collagenase 4312 1 ayk (N) MMP 2 72 k. Da Type 4 Collagenase, gelatinase A 4313 1 ck 7 (X) MMP 3 Stromelysin 1 4314 1 b 3 d (X) MMP 7 Matrilysin 4316 1 mmp(X) MMP 8 Neutrophil collagnase 4317 1 a 85 (X) MMP 9 92 k. Da Type 4 collagenase, gelatinase B 4318 1 itv(X) MMP 10 Stromelysin-2, Transin-2 4319 1 q 3 a (X) MMP 11 Stromelysin- 3 4320 1 hv 5 (X) MMP 12 Macrophage metalloelastase 4321 1 jiz(X) MMP 13 Collagenase 3 4322 1 cxv (X) MMP 14 4323 1 bqq (X) MMP 15 4324 1 rm 8 (X) MMP 16 4325 MMP 17 4326 MMP 19 Matrix metalloprotase RASI 4327 MMP 20 enamelysin 9313 MMP 21 118856 MMP 24 10893 MMP 25 MMP 26 64386 Matrilysin-2 MMP 27 MMP 28 epilysin 56547 Dr S. ganguli at the Industrial Pharmacy Conference, Dubai on April 28 -29, 2016. 64066 79148

EXAMPLE OF MULTIPLE ALIGNMENT USING blastp at the NCBI server The blastp analysis software on the NCBI sever was used to make a pair wise multiple alignment of MMP sequences in FASTA format. The MMP 1 sequence was used as a query against 7 other MMP sequences (2, 3, 7, 8, 9, 10, 11). The sequence identities ranged between 40% to 58%. One sample output is shown in the right panel. Dr S. ganguli at the Industrial Pharmacy Conference, Dubai on April 28 -29, 2016.

3 -D STRUCTURES 5 MMP MOLECULES Dr S. ganguli at the Industrial Pharmacy Conference, Dubai on April 28 -29, 2016.

3 -D Structure of MMP 1 NMR 30 structures High-resolution structure of the inhibitor-free catalytic fragment of human fibroblast collagenase determined by multidimensional NMR. , Moy FJ, Chanda PK, Cosmi S, Pisano MR, Urbano C, Wilhelm J, Powers R, Biochemistry 1998 Feb 10; 37(6): 1495 -504. PMID: 9484219 Dr S. ganguli at the Industrial Pharmacy Conference, Dubai on April 28 -29, 2016.

3 -D Structure of MMP 2 X-Ray at 2. 80 Å Structure of human pro-matrix metalloproteinase-2: activation mechanism revealed. , Morgunova E, Tuuttila A, Bergmann U, Isupov M, Lindqvist Y, Schneider G, Tryggvason K, Science 1999 Jun 4; 284(5420): 1667 -70. PMID: 10356396 Dr S. ganguli at the Industrial Pharmacy Conference, Dubai on April 28 -29, 2016.

3 -D Structure of Stromelysin 1 (MMP 3) I complex with inhibitor at 2. 30 Å Crystal structure of the stromelysin catalytic domain at 2. 0 A resolution: inhibitor-induced conformational changes. , Chen L, Rydel TJ, Gu F, Dunaway CM, Pikul S, Dunham KM, Barnett BL, J Mol Biol 1999 Oct 29; 293(3): 545 -57. PMID: 10543949 Dr S. ganguli at the Industrial Pharmacy Conference, Dubai on April 28 -29, 2016.

3 -D Structure of Matrilysin X-Ray at 2. 30 Å Matrilysin-inhibitor complexes: common themes among metalloproteases. , Browner MF, Smith WW, Castelhano AL, Biochemistry 1995 May 23; 34(20): 6602 -10. PMID: 7756291 Dr S. ganguli at the Industrial Pharmacy Conference, Dubai on April 28 -29, 2016.

3 -D Structure of Stromelysin-3 A, B, C, D, E, F chains at 2. 60 Å Crystal structure of the stromelysin-3 (MMP-11) catalytic domain complexed with a phosphinic inhibitor mimicking the transition-state. , Gall AL, Ruff M, Kannan R, Cuniasse P, Yiotakis A, Dive V, Rio MC, Basset P, Moras D, J Mol Biol 2001 Mar 23; 307(2): 577 -86. PMID: 11254383 Dr S. ganguli at the Industrial Pharmacy Conference, Dubai on April 28 -29, 2016.

3 -D Structure of Catalytic Domain of MMP 16 X-Ray at 1. 80 Å Crystal structure of the catalytic domain of MMP-16/MT 3 -MMP: characterization of MT-MMP specific features. , Lang R, Braun M, Sounni NE, Noel A, Frankenne F, Foidart JM, Bode W, Maskos K, J Mol Biol 2004 Feb 6; 336(1): 213 -25. PMID: 14741217 Dr S. ganguli at the Industrial Pharmacy Conference, Dubai on April 28 -29, 2016.

STRUCTURE OF 5 MMP PROTEINS 5 MMP proteins are displayed (clockwise MMP 1, MMP 2, MMP 3, MMP 7 and MMP 16). The images are cropped to focus on active site and metal binding Dr S. ganguli at the Industrial Pharmacy Conference, Dubai on April 28 -29, 2016.

STRUCTURE SUPERIMPOSTION METHOD USED: TMALIGN server through the PDB site QUERY: MMP 16(1 rm 8) 169 aa Color blue SUBJECT: MMP 7 (1 mmp) 166 aa color red TM SCORE: 0. 89082 RMSD: 1. 43 Å OVERALL IDENTITY IN BLAST: 45% Dr S. ganguli at the Industrial Pharmacy Conference, Dubai on April 28 -29, 2016.

SEQUENCE AND STUCTURE: COMPARISON AND INTROSPECTION VAST SEARCH NCBI DATABASE 18/04/2016 The VAST search in the NCBI database is good for comparing related structures. Using 1 rm 8 molecule as a query the database search yielded * 1601 entries * A Cutoff of 1Å rms deviation included 133 structures with sequence identities between 52 -55% * The 1601 entries included a range of rmsd of 0. 73 Å to 4. 04 Å • Manual inspection of the output from the search included several counter-intuitive examples. EXAMPLE OF FEW ODDITIES: PDB ID Rmsd (Å) % Identity 3 KRY 0. 73 54 4 ILW 1 52 3 HDA 1. 17 31 3 HB 2 1. 18 30 3 MA 2 1. 05 70 3 KRY 0. 73 72 1 SRP 1. 38 2 4 KXL 1. 39 2 1. 45 2 4 FYI Pharmacy Dr S. ganguli at the Industrial Conference, Dubai on April 28 -29, 2016.

THANK YOU PLEASE FEEL FREE TO CONTACT Dr. Subrata Ganguli vvbusy@yahoo. com

Dr S. ganguli at the Industrial Pharmacy Conference, Dubai on April 28 -29, 2016.

MODERATOR’S SPEECH SUBRATA GANGULI, Ph. D. Jitendra Apt, 59 D. P. Road, Baguiati, KOLKATA 700028 E-mail: ganguli_subrata@yahoo. com R&D, CTRD, Salt Lake, Kolkata 700064 Formerly Professor, CIPT &AHS, Howrah 711316 Cell: +91 9007744387