Module 5- Interactions/ Analysis/ Modifications

Question 1

Protein abundance in a yeast cell

Answer 1

~42 million proteins per yeast cell
Abundance varies from 3/5 copies to 7.5E5 copies per cell
Median abundance of 2622

Question 2

Protein abundance in a human cell roughly

Answer 2

Cell is ~1/2 protein
~300mg/mL of protein in a cell
Human has ~20,000 protein encoding genes

Question 3

Generation of protein diversity in humans

Answer 3

Comes from isoforms and PTMs
1 gene can lead to a range of different proteins which are different due to genetic (splicing creating isoforms) and PTM

Question 4

Different protein interactions leading to biological functions

Answer 4

Genetic pathways- signalling pathways/ sequential interactions
Pathway scaffolding
Enzymatic reactions
Molecular machines- form stable complexes, not much changes

Question 5

Things important in protein-protein interactions

Answer 5

Domains are cornerstone
Intrinsically disordered regions important

Question 6

Features of intrinsically disordered regions

Answer 6

Not well defined structures
Highly modified and therefore have a lot of diversity- more tolerant to PTMs, insertions and deletions
Domains bind to disordered regions

Question 7

What is a yeast-two-hybrid assay

Answer 7

Where a DNA-binding domain and activation domain which normally bind together are separated and have tags put on them (bait and prey)
Can use two methods to validate this: either causes transcription of essential gene and get growth or leads to transcription of reporter gene such as causing fluorescence
Pair-wise interactions

Question 8

Affinity purification mass spectrometry

Answer 8

Extract proteins and complexes from cells, use antibodies to do mass spec on these complexes and get data on them. Doesn’t just give pair-wise interactions

Question 9

First example of experiment done- what does it mean that they had the largest data set compared to literature curated data but there was still heaps missing?

Answer 9

Luck et al. 2020
They compared their dataset obtained from their experiment with data curated from literature and had the biggest and best dataset
Isnt complete because it was a yeast two-hybrid test so therefore, interactions in mammalian cells wont all be in yeast cells such as ones needing PTM, if a complex is required for interaction to occur, or membrane proteins being mis-localised and wont be with their partner

Question 10

First example of experiment done- what two methods were used to validate interactions found in yeast two-hybrid, making hypothesis to be tested

Answer 10

Protein x onto jak protein, protein y onto stat
If x and y interact then jak P stat and then the P stat causes transcription of the reporter gene
X and y both attached to proteins that when they react cause a fluorescent result

Question 11

Different types of protein-protein interaction interfaces

Answer 11

Pre-formed interface
Conformational change leading to an adaptation of shape to cause interaction
Folded/ ordered domains binding disordered structure
Disordered structure folding upon binding with domain
Two disordered structures coming together and folding

Question 12

General properties of interfaces/ how are they characterised

Answer 12

Overall amount of surface area buried
Chemical composition of the buried surfaces (enriched for aromatic resides)
Shape and charge complementarity of occluded surfaces (close packing)
Specific interactions such as hydrogen bonds and decreased flexibility

Question 13

Features of size of the interface/ buried surface area (BSA)

Answer 13

Difference in surface area of two proteins alone compared to when they are in a complex
Can cause underestimation as it assumes that proteins come together without conformational change which isnt always the case
Average ~18,000 which is overestimate as mainly only large stable proteins have been sized
Less weaker transient complexes have been solved which tend to be smaller

Question 14

Foglizzo and how interface was found

Answer 14

Size exclusion of protein showed that it was a dimer and each subunit had three functional units. Interface area was small and there was three possible ways it could have come together with this small interface
Did mutations in each possible interface and size exclusion on each, showed that one interface mutation led to a monomer which must be the interface as mutation meant they could not come together as dimer
More experiments could then be done to fond out how it interacts

Question 15

Composition of interface residues

Answer 15

Ratio of <1= residue less likely to be in interface eg acidic or polar
>1= residue more likely to be in interface eg aromatic and arg

Question 16

Core and rim of interface

Answer 16

Core of interface is buried and has no contact with solvent, likely to have aromatic and hydrophobic residues, look like protein interior
Rim of interface has parts buried and exposed to solvent, likely to have more polar residues here, looks like rest of non-binding surface

Question 17

Water molecules in interface interactions

Answer 17

Often make key connections, often sitting around edge of rim near polar residues
May be found in unexpected areas, not likely to be there though

Question 18

Obligate and non-obligate

Answer 18

Obligate= always in a complex
Non-obligate= regulated interactions, sometimes monomers, sometimes dimers, sometimes in complexes

Question 19

Shape and charge complementarity- scoring

Answer 19

Different scores used to measure packing of proteins in complex
How close residues are, how many holes there are, how they come together- complementarity
When interfaces are small this is hard to determine
Close packed= 0.7, likely real interface
Crystal packing, not close= 0.3/0.4

Question 20

Conformational change and complementarity and size

Answer 20

Smaller interface has less conformational change- less entropic cost= more pre-organised and fit together
Larger interface has more conformational change- more entropic cost

Question 21

Conformation and anchor residues

Answer 21

Small interfaces tend to have anchor residues which come together and help binding interactions
Other residues then move around these anchors to optimise contacts

Question 22

Three types of ways proteins may come together and interact and which is more likely

Answer 22

Compact and interact together- conformational change
No conformational change, just come together
Extended interaction- residues reach and contact each other- optimise contact= most likely

Question 23

What is alphafold

Answer 23

Take knowledge of sequence and structure and builds a protein model
Early days, can possibly be applied to protein complexes

Question 24

Alphafold in yeast-two-hybrid being used to predict complexes example

Answer 24

Humphreys et al. 2021
Many structures were predicted
Not good for transient interactions
Better prediction for large stable complexes
Some binary complexes that were found actually normally fit into a complex but needs more info
Again, reiterates early days

Question 25

Things predicted to be at the interfaces in human protein interaction network

Answer 25

Predict disease causing mutations are at interface and phosphorylation sites which lead to regulation of protein interactions

Question 26

What is a pDockQ based on

Answer 26

Proximity- number of residues in close proximity, beta carbons within 10 angstroms, greater number in close proximity, more likely to be a contact PLDDT- alpha fold prediction score, how good the prediction is

Question 27

Orthogonal data eg cross-links in protein interaction determination

Answer 27

Cross linking reagents can be used, bifunctional with cross-linker and functional ends which bind to different proteins Mass spec can be used to identify cross-linked residues Can sort cross-link data based on pDockQ, scores with higher confidence observed more significant cross-links (spacer less than a certain number of angstroms)

Question 28

Location of proteins and affinity of interactions

Answer 28

Obligate oligomers- high affinity Non-obligate permanent PPIs dont need to interact but when they do have high affinity Non-obligate triggered transient PPIs- high affinity, regulatory Non-obligate co-localised PPIs- moderate affinity, regulatory Non-obligate weak transient PPIs- dependent entirely on concentration, low affinity

Question 29

Ways and approaches to validate data out of high throughput

Answer 29

Qualitative and quantitative GST pulldown (qualitative) Isothermal calorimetry (quantitative) Surface plasmon resonance SPR (quantitative)

Question 30

Features of protein-protein interaction domains

Answer 30

Independently folded, 35-150 aas, can still bind target if expressed independently Binding properties of isolated domain reflect those of intact proteins N- and C-termini close in space with ligand binding site on opposite face Folding allows domains to be connected without disrupting function

Question 31

What are SH2 (~115) and SH3 (~300) domains in human genome

Answer 31

Src homology First discovered cancer causing protein, showed cancer caused by mutations Regulate kinase activity (SH1), has Tyr527 on end of gene commonly mutated in cancer

Question 32

Features of proline binding motifs making them play a key role as a docking site for signalling proteins

Answer 32

Unusual shape of pyrrolidine ring Constrained dihedral angles Substituted amide nitrogen Relative stability of cis isomer

Question 33

How does isothermal calorimetry work

Answer 33

Reaction cell filled with protein solution and injected syringe filled with ligand solution Small volumes of ligand injected into cell triggering binding reaction Exothermic- samples becomes warmer and causes downward peak sequence When binding saturation reached, remaining heat effects (if present) due to mechanical and dilution Area under peak plotted versus molar ratio, gives Ka

Question 34

How does surface plasmon resonance (SPR) work

Answer 34

Measures based on kinetics, measures kon and koff rates One substrate immobilised on a surface, the other run over top Reflected light tells how much is bound, can measure the association and dissociation Compare sensorgrams for different interactions

Question 35

PPI paper 1 and how they validated novel interactions found at hugh confidence

Answer 35

Burke et al. 2023 Cross-linking data between proteins Disease causing mutations prevalent at interfaces Phosphorylation sites at interfaces

Question 36

Recognition of prolines for proline binding motifs

Answer 36

Often stretches of proline (polyproline type II helices) are favourable for binding 3 residues per turn, ring and carboxyl regions regularly positioned- backbone restricted Carbonyls are free so make no intra-molecular H bonds and are free to make interactions which binding proteins take advantage of for interaction

Question 37

SH3 domain and proline rich regions

Answer 37

SH3 has two antiparallel B-sheets and two variable loops (RT and n-Src) Binds ligand in polyproline II helix Recognition relies on N-substitutionm proline Variability in loops confers some selectivity

Question 38

More on how SH3 binds proline-rich regions

Answer 38

One or two residues at end of PPII helix recognised by loops eg Arg- loops required for specificity Two xP grooves for proline binding Three aromatic residues make areas for proline to bind (xP grooves)

Question 39

How to break SH3 binding and how was it found that three aromatic residues are important and loops are important

Answer 39

Lim and Richards 1994 Break by determining if residue is in interface and mutating or by unfolding the protein Can mutate residues to see if they are in interface and if no binding occurs, shown that mutating 3 aromatics the binding is broken= evidence for being in interface Mutating regions in RT-loop can make SH3 better or worse at binding (if was bad before, mutations can make better and vice versa)

Question 40

Features of the WW domain

Answer 40

Triple stranded B-sheet with 2 conserved Trps with conserved length of the loop, where the Trps are tells where binding interface is Binds ligand in PPII helix and recognition relies on N-substitution of proline Usually has one xP binding pocket

Question 41

WW domain binding to proline rich region and compared to SH3

Answer 41

One xP groove where proline sits Specificity loops recognise a specific residue which confers binding Both little domains which bind proline rich regions, aromatics in the exact same place- have evolved to have residues in the same space showing that having aromatics making xP grooves is best for binding

Question 42

What does an SH2 domain do

Answer 42

Docking site for signalling proteins as tyrosine phosphorylation allows recognition to be tightly regulated by kinases Various evolution has occurred, used in many places and in many proteins

Question 43

Features of SH2 domains

Answer 43

4 stranded anti-parallel B-sheets, 2 helices conserved and a positively charged pocket with Arg binds pTyr (neg charged) specifically provided by C-terminal- it always binds in the same place

Question 44

SH2 conserved regions binding to pTyr

Answer 44

BetaB5 Arg and alphaA2 arg/lys BetaB7 Ser and betaD His All important for the interaction and shows that SH2 recognise pTyr in the same way, gives specificity for the pTyr

Question 45

Importance of loop conformation in SH2- how it was found

Answer 45

Kaneko et al. 2011 Took 70 SH2 and evaluated binding at semi-high throughput, screened against peptide libraries containing pTyr residues, led to 2 models of binding Loops block different pockets (+2 +3 or +4) allowing one pocket to be free for recognition and binding of proline rich protein

Question 46

Surrounding residues in SH2

Answer 46

Permissive and non-permissive interactions combine to influence binding Surrounding residues have an impact and influence binding and can cause unfavourable interactions and block binding Additional sequences in each protein can give specific interactions

Question 47

SH domains and kinase interactions

Answer 47

The Tyr527 on the end is phosphorylated and goes into SH2 which inactivates it. The SH3-SH2 linker is proline-rich and binds to SH3 which keeps it inactivated and overall, kinase is inactive For kinase to be active, interactions need to be turned off by a phosphatase removing the P or a competing proline rich sequence binding instead of the linker

Question 48

How cancer affects kinase of SH domains

Answer 48

Elevated phosphatases shifting the equilibrium allowing the phosphate to be removed and kinase to be turned on Mutated Tyr so cant be phosphorylated Suppression of the kinase that adds the phosphate onto Tyr527

Question 49

Ways to help specific binding for SH2 and SH3 domains

Answer 49

RT loops Conformation of loops, determines if +2 +3 pr +4 is important for binding (not much specificity as most SH2 belong to +3) Domains have additional regions for interaction eg flanking or extended specificity surface, multiple modules, multiple recognition surfaces

Question 50

Approaches to achieve high-fidelity signal transmission

Answer 50

Co-localisation- organise on cell surface Compartmentalization (organelle) Scaffold-mediated complex assembly

Question 51

GRB2 structure and domains

Answer 51

Adapter protein with no enzymatic function Has 2 SH3 domains and 1 SH2 domain SH3 domains on outside binding polyproline sequences at the ends with slightly different specificity SH2 domain in the middl binding phosphotyrosine

Question 52

SOS and GRB2

Answer 52

SOS most commonly binds to SH3 domains in GRB2 as it has polyproline sequences SOS is a GTP exchange factor causing MAPK signalling Weak interactions with GRB2 for controlling signalling

Question 53

Transduction of TCR in experiment results

Answer 53

Su et al 2016 LAT is embedded in membrane on a microscope slide due to His-tags in the N-terminus= immobilised LAT is also phosphorylated and when GRB2 and SOS are added they mediate the formation of clusters Adding phosphatase removes the clusters High concentration of SH3 binding sites on GRB2 for SOS so if one interaction is knocked off, another can be made and is the bridge for cross-link formation

Question 54

Clustering in TCR with valency of GRB2 binding sites

Answer 54

When Tyr is mutated to phenylalanine clusters struggle to form By increasing the concentration of GRB2 and SOS, even with lower [tyrosine] still get clusters No tyrosine= no clustering Multivalent binding- need to bring molecules together to get interactions/ results

Question 55

Clustering in TCR and phosphatases

Answer 55

As clusters form phosphatases are excluded- builds a wall to keep out phosphatases allowing the signal to occur

Question 56

GRB2 and EGFR (example of RTK) signalling

Answer 56

Kinase receptor tyrosine is phosphorylated, which GRB2 SH2 domain binds to SOS binds to GRB2 SH3 domains, and nucleotide exchange occurs on RAS which causes signalling and MAPK activation

Question 57

GRB2 structure- is dimerisation important?

Answer 57

Forms a dimer in crystal structure Tyr160 is phosphorylated normally, but this is buried in the dimer so cant be phosphorylated- evidence to think that dimerisation is not important or always done

Question 58

Ways it is shown GRB2 is a dimer in solution- gel

Answer 58

Ahmed et al. 2015 Have the wildtype, one with a mimic of phospho-tyrosine, one with two asparagines mutated to glutamic acid for force interactions apart When run on a gel, evidence shows they are dimers

Question 59

Ways it is shown GRB2 is a dimer in solution- fluorescence

Answer 59

Ahmed et al. 2015 FRET used to measure interactions Co-expression with mutant shows no decrease in fluorescence lifetime Wildtypes show decrease in lifetime= have dimers Monomer:dimer equilibrium in solution

Question 60

Monomeric GRB2 in tumours

Answer 60

Ahmed et al. 2015 Shown with antibodies specific for phospho-tyrosine When monomer dominates= have high proliferative cells Phosphorylated Tyr160 more prevalent in high grade tumours= monomer

Question 61

Clustering of EGFR and GRB2- experiment done

Answer 61

Lin et al. 2022 EGFR sumo molecule attached to his tag= embedded in membrane on microscope slide With ATP and Kinase, when wt-GRB2 added clusters form When GRB2 with P-Tyr160 (=monomer) no clusters When mutated GRB2 mimicking P-Tyr160 with glutamic acid, no clusters Shows GRB2 dimers are required for cross linking ang cluster formation

Question 62

GRB2 and EGFR clusters and RAS

Answer 62

Linked to RAS activation: when there are clusters there is more RAS activation

Question 63

SOS and RAS activation with EGFR

Answer 63

SOS is not needed for cluster formation and can get clusters without it However, SOS presence causes better cross-linking, enhancing clustering and RAS activation Clusters recruit SOS and activate RAS signalling

Question 64

Characteristics of protein interfaces

Answer 64

Generally flat, large, highly variable, featureless and flexible Involve many intermolecular contacts

Question 65

Drugs and protein binding

Answer 65

Small (<500 kDa) and bind to pockets= hydrophobic

Question 66

Current FDA approved protein interaction drugs

Answer 66

Cell surface receptors and enzymes represent ~80% Adv of enzymes is can do assays and have binding pockets

Question 67

Important drug targets

Answer 67

Nuclear receptors, ion channels and transporters

Question 68

How many human proteins thought of as ‘druggable’

Answer 68

~10-15%

Question 69

Features of GH interaction with GHR

Answer 69

Binds in asymmetric way 1:1 interaction 3nm= tight Buried SA of 1300 angstroms with 33 residues DeltaG= -12.3kCa/mol

Question 70

Mutating 33 residues in GHR binding pocket- two important conclusions made from this

Answer 70

Clackson and Wells 1995 All residues mutated to Ala except 2 cys residues, measured binding Importance of residue for binding doesnt correlate to BSA (extent to which residue correlates with interface doesnt correspond to binding) A couple of residues are important for the binding and interaction

Question 71

Hot spots in GH:GHR interaction

Answer 71

When mutated residues mapped onto binding SA, it is seen that certain areas or ‘hot spots’ are important for binding affinity- sit close together Tend to not have water Hot spots in both GH and GHR interact with each other containing key residues for the interaction

Question 72

GH:GHR hydrophobic residues and importance

Answer 72

Hydrophobic residues are important but important residues dont correlate with hydrophobicity

Question 73

Abundance of SOS compared to GRB2

Answer 73

SOS is in much lower abundance than GRB, might not act in a 1:1 stoichiometric ratio

Question 74

Overall: multivalent binding interactions, triggers and recruitment of proteins

Answer 74

Multivalency critical to the way interactions form Triggers shift equilibrium- causes molecules to bind and increases interactions while allowing repulsion of other interactions Favourable and unfavourable interactions cause phase separation Clusters can allow the recruitment of other client proteins eg SOS to further enhance interactions Clustering causes high concentrations of molecules, meaning weak interactions become more powerful

Question 75

Intrinsic cell death pathway

Answer 75

Regulated by BCL-2 protein abundance Many inhibitors of inhibitors In healthy cells, high BCL-2, inhibits BAX, BH3 only is low and free or absent, mitochondria intact Apoptotic cell, BH3 only increases and binds to BCL-2 leaving BAX free to put pores in mitochondria BCL-2 elevated in cancer= promoting

Question 76

Structure and homology of BCL-2 (B-cell lymphoma) proteins

Answer 76

BCL-2 pro-survival and pro-apoptotic all share homology domains (BH domains) Survival include Bcl-2, Bcl-xL, A1, Mcl-1 and Bcl-w Apoptotic bax-like include Bax, Bak and Bok Apoptotic BH3-only include Bim, Puma, Bmf, Bid, Noxa, Bik, Bad and Blk

Question 77

Bcl-2 protein interactions

Answer 77

For survival, Bax-like bind into Bcl-2 and are inhibited For apoptosis, BH3-only bind in the same place as Bax-like in Bcl-2

Question 78

Features of BH3 motif

Answer 78

4 hydrophobic residues (h1-h4) all on one side Conserved aspartic acid and leucine Gly/Ala conservation in other position

Question 79

Binding of Bax to Bcl-2 (pro-surivival complex)

Answer 79

Bax is disordered and folds as it binds Four hydrophobic pockets in Bcl-2 BH3 create areas for hydrophobic residues to interact/ go to for interaction

Question 80

BH3 and selective binding

Answer 80

Chen et al. 2005 BIM and PUMA both bind to every Bcl-2 protein Others dont bind to all Bcl-2 All BH3-only have core elements but differ in sequence which confers selective binding Interactions were different

Question 81

BH3 selective binding and drug development

Answer 81

Selective binding by BH3 id important for drug development due to underlying key selectivity Could get selective elements that only bind to key Bcl-2 elements= selective binding and selective inhibition

Question 82

Bcl-2 flexibility

Answer 82

Bcl-2 proteins undergo slight conformational change and arrange differently to accomodate different peptides Flexible grooves, some differences in core elements End state differs depending on the ligand= plasticity

Question 83

Killing correlation with Bcl selectivity

Answer 83

Different cells have different profiles of pro-survival molecules and rely on different pro-survival molecules Selective binding correlates to selective killing

Question 84

Development of ABT-737- screening for binders

Answer 84

Petros et al. 2006 NMR used as could use high concentrations to find weak binders and can look for on target binding- ones that bind in the grooves Firstly screened 10,000 compounds with Bcl-xL, added 10 at a time, 210Da average mass of fragments If got shifts in NMR then deconvoluted (one at a time) to find the binder Second screen where they took compound 1 from first screen and screened another 3500 with 125Da average mass and see if better binding occurs

Question 85

Development of ABT-737- compound optimisation

Answer 85

Took compounds from first and second NMR screens and added them together which gave a compound with good binding (took two weak binders and made them strong binders) Made many libraries of this and looked for even tighter binders- got higher gain in affinity

Question 86

Development of ABT-737- issue with albumin and overcoming

Answer 86

Oltersdorf 2005 Compound also bound to albimun so was mopped up and couldnt do its thing Overlayed structure from optimisation with where it is known to bind in structural visualisation and overlayed albumin with its site, looked for sites to target so that they would be solvent exposed when bound to Bcl-xL but were required in albumin binding Anti-design to overcome binding to albumin, led to ABT-737

Question 87

Binding of ABT-737 in BH3 binding groove

Answer 87

Lessene et al. 2008 Bound to Bcl-xL Selects for a deeper binding pocket in h2 and h4- makes Bcl-xL adopt a conformation allowing for deeper binding- takes advantage of plasticity

Question 88

ABT-737 to Abt-263 and clinical trial outcomes

Answer 88

Navitoclax Caused tumour lysis syndrome- destroyed tumours too quick, body could not deal with all of the debris and led to death Showed it was effective and that the therapeutic window needed to be tweaked Also caused rapid and constant loss of platelets

Question 89

Why was Abt-263 causing a loss of platelets

Answer 89

The interaction being targeted was Bad-Bcl-xL However, Bad is not completely specific and binds to Bcl-xL, Bcl-2 and Bcl-w Cells have different pro-survival molecules that they rely on, platelets only have Bcl-xL to rely on so it led to a decrease in tumours and also a decrease in platelets as their survival molecules were also lost

Question 90

Development of Abt-199 (venetoclax)

Answer 90

Souers et al. 2013 Goal was to make Bcl-2 selective Altered Abt-263, removed a group leading to a molecule with increased selectivity but decreased affinity Looked at crystal packing of new molecule, saw there was a Trp from somewhere else that fit in well, so added indol ring which went into the same place the Trp was in crystal packing Abt-199 more Bcl-2 selective with Ki of 0.01nm (Ki of Bcl-xL 48nm and of Bcl-w 245nm)

Question 91

Outcomes of venetoclax

Answer 91

Much less decrease in platelets Many lymphoid tumours rely on Bcl-2 so led to decrease in lymphoid malignancies First drug in this class/ first drug to target this new pathway

Question 92

Why do cancer cells have increased sensitivity to venetoclax

Answer 92

Cancer cells are in an equilibrium of wanting to die due to damage but cant because cancer causes increased pro-survival molecules (Bcl-2) Adding venetoclax causes disruption of this equilibrium so killing occurs rapidly Not as much collateral damage to other cells Good for any tumour over expressing Bcl-2

Question 93

Venetoclax and resistance

Answer 93

A mutation in the p4 pocket develops in patients who have taken this for a number of years: G101V BH3-only proteins still bind the same in this p4 pocket The mutation causes a change in position of E152, which shifts Y18 leading to a small rearrangement of the bottom of the p4 binding pocket leading to a decrease in affinity for venetoclax

Question 94

S55746 compared to venetoclax

Answer 94

Birkinshaw et al. 2019 Binds to Bcl-2 in a different way- binds in the p1-p3 pockets, not in p4 Therefore, the mutation in p4 of G101V would not affect binding of this molecule as it does for venetoclax

Question 95

Optimising protein interactions and biochemistry for Abt-263- antibody

Answer 95

By adding an antibody which is specific for tumour cells to Abt-263 in conjugate, could cause specific delivery of the molecule to tumour cells causing death of only those cells as only targets Bcl-xL in tumour cells- under trials

Question 96

Optimising protein interactions and biochemistry for Abt-263- PROTAC

Answer 96

Khan et al. 2019 Abt-263 is linked to another molecule which binds to an E3 ligase This would cause the ubiquitination of the target protein (Bcl-xL) for degradation Advantage is that a low dose is needed to destroy many targets, but with other approaches higher doses are needed as they are single use molecules for inhibition of Bcl-xL Trials have shown loss of target, no off-target loss eg platelets and a reduction in tumour= promising