SAR BY NMR

Lubna Mohammad

What is SAR by NMR?

To drug or not to drug that’s the question !

• an HTS of a relatively large (~106) compound collection typically serves as the entry point to lead identification and optimization, however, this approach is failing to produce high-quality clinical candidates at a rate commensurate with the resources that have been dedicated to these efforts.

• There are several very specific reasons for these trends:1. the quality of the leads that have resulted from these large-scale

screens tend to lack the appropriate physicochemical properties for clinical success.

2. only about 50% of all screens produce quality leads that are worthy of further evaluation so we need to screen a larger compounds libraries to enhance the screening.

Conventional HTS vs. fragment-based

screening• One of the greatest advantages of fragment-based

screening as compared to conventional HTS is the substantially greater coverage of chemical diversity space

• even small fragment libraries represent a substantially larger fraction of diversity space than conventional compound repositories.

• Another factor contributing to this increased diversity is that the ultimate goal is to link or tether the individual fragments found at the multiple sub sites, as described below. This means that the effective number of ligands that is virtually assessed in a fragment screen is a power function of the number of subsites evaluated and the number of different ways to link the fragments together

Conventional HTS vs. fragment-based

screening• Figure 3B gives one estimate for these values for a

binding site that contains two independent subsites and 10 linking possibilities. In this example, a fragment library of 104 compounds virtually represents 109 compounds far larger than all corporate HTS repositories combined These dramatic increases in the coverage of chemical diversity space allow a fragment screen to be a robust indicator of protein druggability In the scenario described above, obtaining no hits from conventional HTS suggests either an undruggable target or the lack of appropriate chemical matter.

Conventional HTS vs. fragment-based

screening• In contrast, obtaining no hits from a fragment screen

is an excellent predictor that the protein simply cannot be targeted with small molecules. This ability of a fragment screen to predict protein druggabililty has been borne out experimentally (Figure 3C) (24, 25). Obtaining high fragment hit-rates (>0.3%) is an excellent predictor that high-affinity, small molecule ligands can be identified, whereas low hit rates (<0.1%) strongly suggest an undruggable pocket. Thus, positive results from a fragment screen can be used to reliably redirect discovery resources for those targets that might initially fail

• B. A plot of the virtual number of ligands assessed as a function of the actual number of compounds tested for fragment screening (red line) and conventional high-throughput screening (black line) For HTS, as no linking of the individual hits is proposed, the numberof ligands assessed is exactly equal to the number tested However in a linked fragment approach, the number of virtual ligands is M*N , where

• N is the number of compounds tested, P is the number of independent but proximal subsites on the protein, and M is the number of linking possibilities

• (23). The example shown here is for (P=2, M=10) as a function of N. C. The observed frequency of being able to inhibit protein targets with potent, nonpeptde, non-covalent

• small molecules as a function of the experimental hit

The 1H-15N HSQC spectrum is avery powerful tool for rapidmonitoring of binding processes. Ifthe protein is 15N labeled then wemonitor chemical shift changescaused by protein-protein interactions,protein DNA interactions, protein-ligandinteractions.

Examples right. Top, a 1H-15N HSQC ofan acyl carrier protein in the apo-form(no fatty acid bound). In the lowerpanel the effect of increasing fattyacid chain length is monitored.

The NMR Bandshift and binding site mapping

A case study - Leukocyte function associated protein-1 (LFA-1)

This protein is involved in tethering a leukocyte to a endothelium, allowing migration through the tissue to a site of inflammation.

One domain of LFA-1, the I-domain is 181 amino acids and undergoes a conformational change where helix 7 slides down the protein, switching it into an active open form. This open form is competent for cell surface binding.

If we can stop this switch, we may have an anti-inflammatory mechanism

Inflammation (chronic) is responsible for asthma and arthritis.

LFA-1LFA-1

S

N

N

N

O

N

O-O

A B

C

D

O

Developed small molecule inhibitors and test binding

Weak bindingmM to mMsee a migration of the peaks

Unbound1H 7.4ppm

100% bound1H 8.0ppm

Total LFA-1 = 80mM = [P]+[PL]

Total ligand 20 50 100 150 200 400m

NH of 7.487 7.595 7.720 7.796 7.843 7.921L132 0.087 0.195 0.320 0.396 0.443 0.521

0.145 0.325 0.534 0.660 0.738 0.869

Bound Ligand 11.6 26.0 42.7 52.8 59.0 69.5

Free Ligand 8.4 24.0 57.3 97.2 141.0 330.5

L132 1H shift

= [PL][P] + [PL]

[PL] /80

i.e 0.145* 80 11.6 [PL]

A more successful inhibitor- nM ‘tight’ binding.

See unbound and boundpopulations

Solve NMR structure of complex…

Helix 7 isprevented fromshifting

NMR is a diverse tool with which we can study protein structure.

It gives us information in solution under ‘physiological’ conditions

2D and 3D techniques combined with modern assignment methodshave allowed proteins up to 40 kDa to be solved.

The power of NMR lies not just with its ability to solve structuresbut also its ability to probe binding of ligands and partner proteinsin ‘real’ time.

Many aspects we have not had time to deal with. NMR reveals howproteins move in solution - can see domains flexing with differenttimescale motions. These often correlate with binding patcheson the protein.

References • SAR by NMR butting pieces together • Author : Philip J. Hajduk ,Pharmaceutical

Discovery Division, Abbott Laboratories, Abbott Park, IL 60064