Do You Need Structure To Prosecute Fragments?

As pointed out here, the majority of people think you need to have structure to successfully move fragments forward.  I, as has been noted previously, disagree and vehemently so.  However, if you fall in the category where you need that crutch you may be familiar with INPHARMA.  X-ray crystallography is the workhorse of structures, but in many cases (far more as we get into more and more complex targets), X-ray fails.  Old-fashioned solution NMR (with 15N, 13C, etc.) is just not fast enough to be a viable tool.  A few years back, INPHARMA was introduced to try to bridge the gap between the two methods.

  

INPHARMA (INter-ligand mapping for PHARmacophore MApping) is based upon robust ligand-based screening data.  Two weakly binding, and competitive, fragments are put into solution with the target.  If the compounds are weak enough (>10uM) it is possible to detect a NOE between protons on the two ligands.  This NOE is mediated by the active site, so the compounds must be binding in the active together (see below).  It is then possible to run very intense computational methods to determine the orientation of the ligands in the binding site (if the structure of the target is known).  

In this paper, Isabelle Krimm looks at INPHARMA's ability to discriminate different binding sites on a single target.  For this she uses, glycogen phosphorylase (a type 2 diabetes target) that has four distinct binding sites: active site, inhibitor site, allosteric site, and new allosteric site. 

Cpd 1 and 2 bind the inhibitor site, where Cpd 3 binds the new allosteric site.  Cpds 4 and 5 are proposed analogs of 2, Cpds 6 and 7 are analogs of 3, and Cpds 8 and 9 are "frequent-hitters".  NOESY experiments were recorded for the six fragments in the presence of 2 and 3.  All compounds exhibited intramolecular NOEs upon binding to the target.  Additionally, Cpds 2 and 4/5 showed intermolecular NOEs, as did Cpd 3 with 6, 7, 8, and 9.  Cpds 4 and 5 did not shows NOEs with 3, nor did 6, 7, 8, and 9 show NOEs to Cpd 2.  For fragments 4-7, competition data support that these are inter-ligand NOEs and that Cpds 8 and 9 are non-specific binders.  No intermolecular NOEs were seen to Cpd 1.  Cpd 1 has a IC50 of 1 uM, while Cpd 2 is 100uM.  This supports theoretical calculations that the two competitive binders must have binding constants no more than 8x different.  

In this paper, Lee et al. demonstrate the use of hyperpolarization to increase the sensitivity of INPHARMA. Dynamic Nuclear Polarization (DNP) uses electrons to transfer magnetization to nuclei leading to orders of magnitude increases in signal.  Lee et al. use DNP to increase the signal in INPHARMA significantly by hyperpolarizing one of the two competitive compounds, in a method they call HYPER-BIPO-NOE (Hyperpolarized binding pocket NOE)[which may be one of the worst not-even-acronyms ever]. 

1D HYPER-BIPO-NOE spectra, a) in full scale and
b) expanded to show transferred signals. Stacked spectra are, from top
to bottom: hyperpolarized ligand 1 with ligand 2 and protein (HYPERBIPO-
NOE); hyperpolarized ligand 1 with ligand 2, but without protein
(Control 1); hyperpolarized [D6]DMSO/D2O, with only ligand 2 and
protein (Control 2); Thermal spectrum of the HYPER-BIPO-NOE
sample (Thermal). The resonance from DMSO, which was suppressed
using presaturation, is designated by *.

This tremendous increase in signal can be extraordinarily useful for INPHARMA applications because they are able to obtain the inter-ligand NOE in a single scan.  The authors then go on to demonstrate that HYPER-BIPO-NOE-INPHARMA data is similar to STD-INPHARMA and can be used to generate binding poses. 

These two papers show that INPHARMA can be a useful tool to orient fragments similarly in a binding pocket.  INPHARMA requires that you have a weak binder that binds in a known site on the target.  For the current generation of targets this may exclude INPHARMA from being used.  It is also important to note that the choice of mixing time (which can range from 70-800ms) is a critical parameter.  The incorrect choice of the mixing time can lead to poor signal intensity and false negative results.  DNP, while turnkey if you have enough money, is not common in industry at all.  I would be surprised if HYPER-BIPO-NOE-INPHARMA actually gets any traction there at all.  There is also a poll with this post, please read and answer.