Cross-Linked bis-tetramers (Octamers) of Human Hemoglobin: Routes to State-ofthe-art

Transcription

1 Cross-Linked bis-tetramers (ctamers) of Human Hemoglobin: Routes to State-ofthe-art HBCs Ying Yang, Jonathan Foot, Francine Lui, Erika Siren and Ronald Kluger Department of Chemistry University of Toronto 1

2 Conflict of interest disclosure Do you have any relevant financial relationship(s) to disclose? No Will your presentation and/or paper include information or discussion of off-label drug use? No 2

3 For circulation of Hb: Cross-link subunits to prevent dimer release Selective reagents - homogeneous and defined product β β bis trimesyl lysyl -amide at -82 β β β bis fumaryl lysyl -amide at -99 (DCLHB) β VAL 1 + LYS LYS 82 + ' VAL 1 verall: Efficiency in production (cost) and storage, safety, efficacy 3

4 Cooperative oxygen binding for efficient delivery xygen Binding and Release Lungs Maximize oxygen delivery f = Hb/(Hb+ Hb) tissues p2 X-linked Hb RBCs f = (p 2 )n / ( p50 n + p 2n ) 4

5 xygen affinity can be controlled xygen affinity inversely proportional to length Short leash prevents full relaxation

6 Additional concern: Dealing with nitric oxide N activates guanyl cyclase to produce cyclic GMP (Phosphodiesterases cleave cgmp) Acellular Hb can permeate endothelium Scavenging of N by Hb tetramers prevents relaxation of blood vessels N and 2 are very similar The Nobel Prize in Physiology or Medicine 1998 Robert F. Furchgott, Louis J. Ignarro, Ferid Murad "for their discoveries concerning nitric oxide as a signalling molecule in the cardiovascular system" 6

7 Enlarged Hb to avoid N scavengingadding PEG Attach via thiol-michael addition to α99-fumaryl-α99-cross-linked Hb Traut s reagent (iminothiolane) converts NH 2 to NH---SH (Acharya) to provide additional sites for PEGylation MP4 does not cause blood pressure increase Not designed to deliver oxygen Vandegriff KD, Malavalli A, Wooldridge J, Lohman J, Winslow RM. MP4, a new nonvasoactive PEG-Hb conjugate. Transfusion, 43: ,

8 Bis-Tetramer size increase Cross-link and connect tetramers Extra weight from functional material No reactions known to cross-link and couple Lys-82 NH 2 H 2 N NH 2 'Lys-82 connector H 2 N

9 Modular construction of linkers 3-amino-isophthalic acid as coupling site Any dicarboxylic acid chloride R= Br H Br 9

10 Cross-linked bis-tetramers - properties Hb P 50 torr n 50 Native NH X HN Bis-tetramer X = Bis-tetramer X = N N Bis-tetramer X = Hu and Kluger Biochemistry 2008, 47, Problem: Reduced Hill coefficient 10

11 Create closer interactions From reagents with sp 3 in bridge NH X HN 1 2 S CF 3 C CF

12 Single-point torsional flexibility Changes alignment and interactions of tetramers 12

13 Bis-tetramers with torsional flexibility 13

14 Inter-tetramer interactions 14

15 Cooperativity and 2 affinity Tetramers interact p 50 n 50 Native hemoglobin Bis-tetramer bent Bis-tetramer bent Bis-tetramer bent Bis-tetramer linear X = are highly cooperative S CF 3 C CF 3 NH X HN 4 = bis-naphthyl 15

16 Molecular size distributions R Cross-linked bis-tetramer (128 kda) Cross-linked tetramer (64 kda) Non-cross-linked S 2 42% 55% < 5% C(CF 3 ) 27% 70% < 5% 37% 60% < 5% Competition with hydrolysis lowers yield of bis-tetramers: Separate protein modification and connections Hu & Kluger Biochemistry 2008, 47,

17 Click reactions can we use them? Make complex molecules by forming C X rather than C-C bonds (Sharpless) Mimics nature, expands to materials science, bioconjugation Example: Huisgen 1,3-dipolar addition: azide and diene (but - very slow) 17

18 Cu (I)-catalyzed Azide-Alkyne Cycloaddition (CuAAC) Fast formation of anti 1,4-triazoles in water, ut-competes all other reagents Tornøe, Christensen, Meldal "Peptidotriazoles on Solid Phase: [1,2,3]-Triazoles by Regiospecific Copper(I)-Catalyzed 1,3-Dipolar Cycloadditions of Terminal Alkynes to Azides". J rg Chem V. V. Rostovtsev, L. G. Green, V. V. Fokin, K. B. Sharpless, Angew. Chem. Int. Ed. 2002, 41,

19 Bio-orthogonal click coupling Between functional groups not in present in biomolecules (Bertozzi) Works in a cell or in presence of proteins, nucleic acids Can this apply to Hb coupling? Hb Hb Hb Hb 19

20 CuAAC and Hb coupling Bio-orthogonal No hydrolysis Hb Hb N N N N N N asymmetric symmetric 20

21 Directed reaction needed Unavoidable competition? series parallel 21

22 Solubility-controlled serial addition through autocatalysis Hb Hb Hb Soluble Insoluble Slow Soluble Fast, cooperative Avoids single modification Hb Hb J. S. Foot, F. E. Lui and R. Kluger, Chem. Commun., 2009,

23 Introduction of bio-orthogonal groups Full cross-linked Mixed cross-linked products C4 HPLC analysis of HbN 3. J. S. Foot, F. E. Lui and R. Kluger, Chem. Commun., 2009,

24 Bis-tetramer from sequential click reactions (a) G200 HPLC analysis, bis-tetramer at 32 min, (b) authentic bis-tetramer nly β-β azide reacts in CuAAC implies α-α azide is internal 24

25 DPG site α-α cross-link R Chatterjee et al J. Biol. Chem

26 DPG site β-β cross-link N 3 NH Lys NH NH Lys Schumacher et al Nature , 84 26

27 DPG-site β-β cross-link isomers Fernandez et al J. Mol. Biol

28 Convert Hb to a acylation reagent Br β β NH 2 C NH 2 C C Br Br β β Br Br C Br Bulky anionic electrophile selects Lys-β-82 exclusively R. Kluger and Y. Song J. rg. Chem ,

29 Attaching azide to hemoglobin after β-β specific cross-linking Br NH M MPS, ph TTDS N 2,37 o C, 3 h NH 2 Br Br Br CH CH HC Br H 2 N N 3 DBST-Hb H N H N H N Br HC 0.1 M MPS, ph 8.0, rt, 24 h Br N 3 Specific modification. Accompanied by hydrolysis Br TTDS N H HN Y. Yang and R. Kluger, Chem. Commun., 2010, Review: R. Kluger, J. S. Foot and A. A. Vandersteen Chem. Commun. 2010,

30 Single bis-tetramer 30

31 Reaction with other bis-alkynes HPLC analysis Hb-Hb Hb-CH Y. Yang 31

32 Ligand affects efficiency Bathophenanthroline TBTA 32

33 Reduction of Cu(II) to Cu(I) necessary Ascorbic acid is most effective reductant TCEP 33

34 CuAAC coupling with PEG-alkyne Protected protein 34

35 Animal models Zapol lab Conventional clinical tests of HBCs Patients have existing health disorders Many have endothelial dysfunction (impaired N response) Animal toxicity models do not mimic in-hospital tests Improved animal toxicity models compared: Healthy wild-type (WT) mice Diabetic db/db mice N sensistive A db/db mouse (right) next to a control white mouse. NCBI: Information about Energy Balance Silverman, T. A., Weiskopf, R. B., Transfusion, 2009, 47,

36 Blood pressure: WT mice HBC design target: No increase in BP yet can deliver oxygen Positive Control (tetrameric Hb): increase in Systolic BP (SBP) BT: No increase in SBP BT-PEG: No increase in SBP Non-invasive BP measurement PBS = Phosphate Buffer Saline mtet = Murine Tetrameric Hb 36

37 Blood pressure: db/db mice Model for pre-existing endothelial dysfunction Extremely sensitive to N scavenging BT: No increase in SBP BT-PEG: No increase in SBP Positive Control (tetrameric Hb): 27 mm Hg increase in SBP Francine Lui in Zapol lab 37

38 Systemic vascular resistance Blood pressure alone does not predict constriction Systemic vascular resistance (SVR) is an indicator of vasoconstriction Invasive hemodynamic measurements BT: Small increase in SVR BT-PEG: No increase in SVR ΔSVR (mmhg.min.ml 1) Baseline mtet BT BT PEG Dr. David M. Baron, Department of Anesthesia, Massachusetts General Hospital 38

39 BT or BT-PEG? S n S n BT-Hb n BT-PEG n No increase in blood pressure in healthy or diabetic mice No significant increase in systemic vascular resistance (vasoconstriction) BT Pure, homogenous Increased size P 50 = 9.3 n 50 = 2.7 Moderate NiR BT-PEG Pure, homogenous Further increased size P 50 = 4.1 n 50 = 2.4 High NiR PEG not necessary 39

40 Conclusions Coupled tetramers can be formed as amides or triazoles CuAAC provides efficient bioorthogonal coupling Solubility-directed reactions assure bis-tetramer formation Cross-link stereoisomerism controls reaction access In vivo results for bis-tetramers are promising Rational design and known structures permit correlative analysis 40

41 Thanks Toronto Ying Yang Jon Foot (Pharmaxis. Sydney, Australia) Francine Lui Erika Siren MGH Warren Zapol David Baron Chong Lei Binglan Yu Francine Lui 41