12 February 2014

Fragments vs NAMPT, maximum ligand efficiencies, and off-target activities

The enzyme nicotinamide phosphoribosyltransferase (NAMPT) is essential for the synthesis of the important cofactor NAD and thus an intriguing target for blocking cancer cell metabolism. In two recent papers, researchers from Genentech, Forma Therapeutics, Pharmaron Beijing, and Crown Biosciences describe how they used fragment-based approaches to discover new inhibitors of this enzyme.

In the first (J. Med. Chem.) paper, Peter Dragovich (Genentech) and collaborators start with a screen of 5000 fragments using surface plasmon resonance (SPR) at the relatively low concentration of 100 µM. This yielded 283 hits which were retested at 150 µM and also competed with a known high-affinity inhibitor; noncompetitive fragments, which presumably bind outside the active site, were discarded. This winnowed interesting hits to 118 fragments, each of which was characterized in full dose-response curves. Only 6 were extremely weak (KD> 2 mM) or nonspecific, while 35 were quite potent (KD< 100 µM).

As an interesting aside, the substrate for NAMPT is nicotinamide, and this was characterized by SPR as having a remarkably high ligand efficiency (LE) approaching the “soft limit” Teddy recently discussed. The researchers suggest:

The LE exhibited by nicotinamide for NAMPT is the highest we have observed for a fragment lead and, given that NAMPT is highly optimized to efficiently bind this substrate, may approach an upper limit of this parameter for such molecules.

Keep in mind that Genentech has done lots of screens, so this is a significant statement. Indeed, I can think of only a few fragments (here and here) with comparable LE values.

But back to NAMPT. More than 30 co-crystal structures of fragments bound to the enzyme were solved, and several of these fragments were advanced. In doing so a variety of information was used, including data from molecules previously discovered in-house and elsewhere. Lots of nice SAR are presented, and if you’re into structure-based drug design I’d strongly encourage a close reading of the paper. Just to give you a flavor, compounds 12 and 13 (blue), despite their structural similarity, bound in very different orientations. A bit of engineering led to compound 15, and crystallography revealed that only a single enantiomer of a racemic mixture binds to the enzyme. Borrowing information from other NAMPT inhibitors led to the potent single enantiomer compound 17; the other enantiomer is 250-fold less active. Further modification yielded an orally active molecule with activity in a mouse xenograft model.

In the Bioorg. Med. Chem. Lett. paper, members of the same team describe two other series of molecules derived from fragments – and provide some important warnings about interpreting data.

One series (not shown here) was optimized to nanomolar potency in biochemical assays and antiproliferative cell assays. However, the team did a series of careful follow-up studies to show that these molecules are probably acting through off-target mechanisms. For example, the molecules do not reduce NAD levels as they should, and addition of the product of NAMPT did not rescue the cells, as it would were NAMPT the primary target.

For the other series, compound 7 (red above) was characterized crystallographically bound to NAMPT. Initial attempts to improve affinity were unsuccessful, but the co-crystal structure of another fragment suggested that replacing the pyrazole moiety with a simple phenyl group would be tolerated, leading to compound 25. Subsequent fragment growing ultimately led to Compound 51, with low nanomolar potency in both biochemical and cell-based assays. Importantly, this molecule did reduce NAD levels in cells, and the antiproliferative effects could be rescued by adding the product of NAMPT. Taken together, these data show that compound 51 is a nanomolar inhibitor of NAMPT both biochemically and in cells.

The importance of such rigorous characterization is driven home by a footnote, in which the researchers reveal that compound 51 was previously alleged to be an inhibitor of glucose transporter 1 (GLUT1). This was published in a high-profile journal, and several chemical suppliers now sell this compound (called STF-31). Although the current paper does not explicitly say so, it is possible the results in the earlier paper could be attributed to NAMPT inhibition rather than GLUT1 inhibition.

In the hope that views on STF-31 will evolve, I’ll close this Darwin Day post with a quote from The Descent of Man:

False facts are highly injurious to the progress of science, for they often long endure; but false views, if supported by some evidence, do little harm, as every one takes a salutary pleasure in proving their falseness; and when this is done, one path towards error is closed and the road to truth is often at the same time opened.


Anonymous said...

We found a fragment that bound to the zymogen form of caspase-6 with a LE of 0.75, which I will call a "tie" with 0.76...

Anonymous said...

I'm surprised that Compound 51 had low nanomolar potency in both biochemical and cell-based assays.

I get the impression that the cellular concnetraton of NAD isn't as well worked out as ATP, but I'd still expect some drop-off due to increased competition with NAD on going into cells.

Dan Erlanson said...

I agree that the low nanomolar potency is remarkable; the researchers discuss this in a footnote:

We currently believe that many cell-potent NAMPT inhibitors form PRPPderived phosphoribosylated adducts in the protein’s active site which block the function of the enzyme. This belief is consistent with the repeated observation of these adducts by mass spectrometry in biochemical and/or crystallographic experiments (e.g., compounds 5 and 6; see Ref. 10a,c,e). Once formed, the PRPP-adducts may accumulate intracellularly and thereby enhance cell culture antiproliferation effects (see Ref. 6a for additional information and discussion). However, there are many other factors that also likely influence NAMPT inhibitor cell potency including: biochemical inhibition activity, the ability of a given inhibitor and/or its corresponding PRPP-derived ribose adduct to effectively compete with the NAM substrate, cell membrane permeability, and/or protein binding.