MTSA--a Matlab program to fit thermal shift data.

Thermal shift analysis is becoming widely used as a method to identify initial hit ligands for inhibitor discovery or to identify ligands that may aid crystallization. The data analysis software provided by the equipment manufacturers or in the public domain is cumbersome to use. We have assessed a number of different approaches to generate a value for the melting temperature (T(m)) and implemented these methods in the program MTSA within the commercial software Matlab to provide an easy-to-use and rapid way to process experimental thermal shift data. The program outputs the T(m), the quality of the fit, and the deviation from a standard value, the thermal shift ΔT(m). Our analysis of these results includes a discussion of some issues with previous publications in this area. We conclude that the most suitable value for T(m) should be taken from the midpoint determined for a curve fitted to the experimental data with a five-parameter equation. In addition, we found that different ranking of ligand binding can be obtained using the different techniques when screening for binding of weak ligands such as fragments. Therefore, the technique should be used with caution for such screening.

A crystallographic fragment screen identifies cinnamic acid derivatives as starting points for potent Pim-1 inhibitors.

A crystallographic fragment screen was carried out to identify starting points for the development of inhibitors of protein kinase Pim-1, a potential target for tumour therapy. All fragment hits identified via soaking in this study turned out to bind to the unusually hydrophobic pocket at the hinge region. The most potent fragments, two cinnamic acid derivatives (with a best IC(50) of 130 µM), additionally form a well defined hydrogen bond. The balance between hydrophobic and polar interactions makes these molecules good starting points for further optimization. Pim-2 inhibitors from a recently reported high-throughput screening campaign also feature a cinnamic acid moiety. Two of these Pim-2 inhibitors were synthesized, their potencies against Pim-1 were determined and their cocrystal structures were elucidated in order to determine to what degree the binding modes identified by fragment screening are conserved in optimized inhibitors. The structures show that the cinnamic acid moieties indeed adopt the same binding mode. Fragment screening thus correctly identified binding modes which are maintained when fragments are grown into larger and higher affinity inhibitors. The high-throughput screening-derived compound (E)-3-{3-[6-(4-aminocyclohexylamino)-pyrazin-2-yl]phenyl}acrylic acid (compound 1) is the most potent inhibitor of the cinnamic acid series for which the three-dimensional binding mode is known (IC(50) = 17 nM, K(d) = 28 nM). The structure reveals the molecular basis for the large gain in potency between the initial fragment hit and this optimized inhibitor.

Recent progress in fragment-based lead discovery.

Fragment-based methods have emerged as a new strategy for drug discovery. The main advantages are that useful starting points for lead identification for most targets can be identified from a relatively small (typically 1000-member) library of low molecular weight compounds. The main constraints are the need for a method that can reliably detect weak binding and strategies for evolving the fragments into larger lead compounds. The approach has been validated recently as series of compounds from various programs have entered clinical trials. Current new developments are focussing on application of the methods to targets where conventional HTS fails and to integration of fragments alongside HTS for more druggable targets. Here, we provide a brief summary of the key elements of fragment-based lead discovery (FBLD), review recent progress and provide a perspective on the challenges that remain for the field.