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Recognition And Selectivity Of Binding: Molecular Correlates

Professor T.R.C.Boyde, University of Hong Kong Hong Kong Society for Biochemistry and Molecular Biology, CUHK, February 22nd 2008

Birthplace of the Hong Kong Biochemical Association

Words

• antibody, AB unit, binding unit
• specificity
• discrimination, selectivity
• homobivalent, heterobivalent
• homopolyvalent, heteropolyvalent
• target, ligand, epitope
• hybrid, adduct

Recognition And Selectivity Of Binding: Molecular Correlates Specific recognition depends upon complementarity in all respects First example well studied at atomic scale is enzyme specificity

• now defined as the

ratio of rates for a principal as compared with a comparator substrate

Recognition And Selectivity Of Binding: Molecular Correlates Specificity

• qualitative, the identity of the target

Selectivity or ‘Discriminatory Power’

• quantitative, a ligand binds better to one

target than another, and by how much

• expressed in terms of affinity, avidity or

potency (including downstream effects)

• again, ratio to a comparator

Recognition And Selectivity Of Binding: Molecular Correlates

Discrimination Constant, D, ‘The ratio of the affinity constants of a ligand for two targets’

XDUV = KXU/ KXV

• r, “The ratio of the affinity constants of a

ligand for a defined target and a defined cross- reactant (comparator).”

Recognition And Selectivity Of Binding: Molecular Correlates

Discrimination, d, ‘The equilibrium ratio of the concentrations of the bound forms of a ligand, bound to two targets’

d = CXU/CXV = D.CU/CV

• r, ‘The equilibrium ratio of the concentrations
• f the bound forms of a ligand, bound to a defined

target and a defined comparator’

Recognition And Selectivity Of Binding: Molecular Correlates

Why add complications? Why bother with ‘selectivity’? Why not stick with affinity constants?

High affinity alone is unhelpful: it is the relationship of affinities that matters for discrimination. Why not say so? Plenty of room for confusion: ‘affinity’ is often identified incorrectly with the monovalent or intrinsic affinity of the single binding site. Why not clarify? Precision is needed to deal with the new ideas surrounding polyvalent antibodies. Let us recognise the principal and comparator targets explicitly.

Recognition And Selectivity Of Binding: Molecular Correlates

Molecular correlates for high affinity and selectivity

1] Number, 2] strength, 3] and nature of bonds formed, through the 4] matching of complementary features, which may allow 5] formation of strongly directional bonds. 6] Total area of bonding surface (relates to 1). 7] Molecular geometry, for all these, and to yield a 8] neat fit 9] without strain

Recognition And Selectivity Of Binding: Molecular Correlates

Drug discovery

Uses all these approaches, not forgetting the participation of bridging molecules, and taking into account also druggability (low molecular weight etc.), downstream effector functions (through conformational changes in the target), ease of synthesis, solubility and potential for safety in use The means of search vary enormously

Recognition And Selectivity Of Binding: Molecular Correlates Can these ideas be useful in studying specific binding by proteins?

Yes, but a lot of what goes on is not under our control, the protein and its target have co-evolved so that many of these features may be observed but hardly modified Also, we have to look with more care at conformational change in ligand as well as target We treat a single binding site as a unitary phenomenon, capable of being combined but not modified. Its intrinsic affinity is NOT the same as the measured affinity.

Bivalency

• bound to its homobivalent target

Recognition And Selectivity Of Binding: Molecular Correlates More than one binding site: affinity

1] enhanced contact area – greater affinity 2] offset by molecular strain 3] also by loss of freedom of movement [entropy] 4] participation of other parts of the molecule in bonding may go either way So energies NOT expected to be additive, though an increase over monovalent is the general case

Distortion

epitopes too far apart

Distortion

both target and antibody must bend,

if binding is to occur

Flexible linker

homo- or hetero-polyvalent

Recognition And Selectivity Of Binding: Molecular Correlates

A digression on adding together binding energies ΔGº = -RTlnK

Adding energies is equivalent to multiplying equilibrium constants, but since the first is invalid so is the latter. There is no general way to go from the K of a monovalent reaction to that of the corresponding bivalent reaction (Jencks 1981) Entropic losses may be high if the linker is too floppy. Note too that K is dimensionless. A parallel description

• f the standard state conveys that concentrations are in

molar terms. Thermodynamics is not deduced from kinetics.

Recognition And Selectivity Of Binding: Molecular Correlates More than one binding site: selectivity

1] A bivalent antibody not only binds with higher affinity than its Fab, it also shows greater selectivity; selects the polyvalent target from interfering monovalent epitope molecules 2] Evolution of polyvalency in antibodies 3] IgM decavalent, high affinity and selectivity (for a polyvalent target) despite low intrinsic Fab affinity

Bivalency

• bound to its homobivalent target

Distortion

both target and antibody must bend,

if binding is to occur

Recognition And Selectivity Of Binding: Molecular Correlates Heteropolyvalency:

Always artificial? Examples to the contrary. New light on specificity: Parallel to the homopolyvalent case, a heteropolyvalent ligand selects for its corresponding target, and this constitutes a new specificity, not observed in nature (Crosslinking two distinct epitope-bearing molecules, not present on the same target, is a quite different objective and not instructive for us)

Flexible linker

homo- or hetero-polyvalent

Recognition And Selectivity Of Binding: Molecular Correlates Heteropolyvalency:

Always artificial? Examples to the contrary. New light on specificity: Parallel to the homopolyvalent case, a heteropolyvalent ligand selects for its corresponding target, and this constitutes a new specificity, not observed in nature (Crosslinking two distinct epitope-bearing molecules, not present on the same target, is a quite different objective and not instructive for us)

Heteropolyvalency

Heteropolyvalency

‘diabody’ connecting two distinct targets

Flexible linker

homo- or hetero-polyvalent

Nucleic acid linkers

‘adducts’ with single-strand DNA tails

Nucleic acid linker

duplex forms between complementary segments

Recognition And Selectivity Of Binding: Molecular Correlates

• A remarkably simple model is two complementary nucleic acid sequences

joined by a non-hybridising sequence, e.g ACCCCC(A)nGGGGGA.

• n ΔG

ΔH ΔS Tm

• 1
• 3.5
• 32.4
• 93.2

74.4

• 2
• 4.5
• 39.4
• 112.6

76.9

• 3
• 5.2
• 40.4
• 113.5

82.7

• 4
• 5.3
• 38.2
• 106.1

86.8

• 5
• 6.4
• 43.2
• 118.7

90.8

• 6
• 5.7
• 43.2
• 120.9

84.0

• 7
• 5.4
• 43.2
• 121.9

81.2

• 8
• 5.4
• 43.2
• 121.9

81.2

• 9
• 5.4
• 43.2
• 121.9

81.2

• 10
• 5.4
• 43.2
• 121.9

81.2

• 15
• 5.0
• 43.2
• 123.2

77.5

• 20
• 4.6
• 43.2
• 124.5

73.9

• Conditions: NaCl 0.150mol/l,

MgCl2 0.002 mol/l, 37degCelsius

• Free energy and enthalpy kcal/mol; entropy (cal/mol/K); Tm deg Celsius.

Nucleic acid linkers

three or more AB units, no problem add functional components, no problem

Recognition And Selectivity Of Binding: Molecular Correlates

Nucleic acid linkers are neat and advantageous 1] Adducts with an oligonucleotide tail are easily made, 2] may be of low molecular weight, e.g. about 20kDa, 3] readily self-assemble forming a stable link 4] having also a flexible segment as long as necessary 5] Hybrids can be made with >2 Fab’s 6] plus additional effector or marker components 7] in a few moments (given stock of the adducts) 8] tailored for the individual case 9] even within the body, on site, in vivo 10] thus avoiding problems of tissue penetration 11] and may even provide for intra-cellular access.

Recognition And Selectivity Of Binding: Molecular Correlates. Conclusions.

Between macromolecules, provided ligand sites binding to epitopes on the target avoid undue strain and floppiness:- Polyvalent binding gives enhanced affinity and selectivity, plus 3] a distinctive specificity, namely for the polyvalent target. 4] Artificial heteropolyvalent ligands exhibit novel specificities. 5] Some flexibility in ligand or target is essential for binding. 6] Oligonucleotide or aminoacid linkers – less entropic loss? 7] Oligonucleotides – many other advantages. 8] Hetereopolyvalent ligands based on natural proteins, and with oligonucleotide linkers may prove very useful.

Applications in cancer treatment?

Next steps

1] Database of effective target epitopes 2] Database of available antibodies and AB units – suppliers, contractors, laboratories 3] Competitive contracts for testing methods 4] Prepare battery of adducts, evaluate safety 5] Enrol patients, test and treat

Applications in cancer treatment?

Barriers Individualised treatment is not open to formal evaluation as presently understood Therefore pharmaceutical companies find difficulty if taking this on board A new kind of financial partner is needed, and a far-sighted clinical organisation

Structure of cooperation?

• Regional or country by country basis
• Free hand within the region agreed
• Full two-way scientific cooperation - to

secure maximum benefit, quickly, for patients world-wide

• Modest capital outlay as compared with

the potential business returns

Applications in cancer treatment?

Relevant characteristics of cancer:

*Microscopy may mislead as to the biochemical signature *Very few cancer ‘stem cells’ serve to renew the growth *Many genetic changes as the growth develops *Loss or change of proteins, including surface epitopes *Personal cancer signature[s]: personal treatment *Exceedingly high discriminatory power is required both in the testing system and in the treatment, *The treatment must be assembled very quickly, because

• therwise the patient may be dead

Nucleic acid advantages

*rapid choice and assembly of adducts *choose according to the particular target *easily prepare and purify adducts *use artificial or natural binding units *smaller binding units to penetrate tissues *assembly even in situ, within the body *potentially good immune tolerance *enzyme resistant forms possible