Graduation Year


Document Type




Degree Granting Department


Major Professor

Roman Manetsch


Fluorescence, Fragment-based lead discovery, Protein-protein interactions



Fluorescent molecules used as detection probes and sensors provide vital information about the chemical events in living cells. Despite the large variety of available fluorescent dyes, new improved fluorogenic systems are of continued interest. The Diaryl-substituted Maleimides (DMs) exhibit excellent photophysical properties but have remained unexplored in bioscience applications. Herein we present the identification and full spectroscopic characterization of 3,4-bis(2,4-difluorophenyl)-maleimide and its first reported use as a donor component in Forster resonance energy transfer (FRET) systems. The FRET technique is often used to visualize proteins and to investigate protein-protein interactions in vitro as well as in vivo. The analysis of the photophysical properties of 3,4-bis(2,4-difluorophenyl)-maleimide revealed a large Stokes shift of 140 nm in MeOH, a very good fluorescence quantum yield in DCM (Ffl 0.61), and a high extinction coefficient ε(340) 48,400 M-1cm-1, thus ranking this molecule as superior over other reported moieties from this class. In addition, 3,4-bis(2,4-difluorophenyl)-maleimide was utilized as a donor component in two FRET systems wherein different molecules were chosen as suitable acceptor components - a fluorescent quencher (DABCYL) and another compatible fluorophore, tetraphenylporphyrin (TPP). It has been demonstrated that by designing a FRET peptide which contains the DM donor moiety and the acceptor (quencher) motif, a depopulation of the donor excited state occurred via intermolecular FRET mechanism, provided that the pairs were in close proximity. The Forster-Radius (R0) calculated for this FRET system was 36 % and a Forster-Radius (R0) of 26 % was determined for the second FRET system which contained TPP as an acceptor. The excellent photophysical properties of this fluorophore reveal a great potential for further bioscience applications. The 3,4-bis(2,4-difluorophenyl)-maleimide fluorescent moiety was also implemented in an alternative application targeting the enzyme carbonic anhydrase (CAs) are metalloenzymes that regulate essential physiologic and physio-pathological processes in different tissues and cells, and modulation of their activities is an efficient path to treating a wide range of human diseases. Developing more selective CA fluorescent probes as imaging tools is of significant importance for the diagnosis and treatment of cancer related disorders. The kinetic TGS approach is an efficient and reliable lead discovery strategy in which the biological target of interest is directly involved in the selection and assembly of the fragments together to generate its own inhibitors. Herein, we investigated whether the in situ click chemistry approach can be implemented in the design of novel CA inhibitors from a library of non-sulfonamide containing scaffolds, which has not been reported in the literature. In addition, we exploit the incorporation of the (recently reported by us) fluorescent moiety 3,4-bis(2,4-difluorophenyl)-maleimide) as a potential biomarker with affinity to CA, as well as two coumaine derivatives representing a newly discovered class of inhibitors. The screening of a set of library with eight structurally diverse azides AZ1-AZ8 and fifteen functionalized alkynes AK1-AK12 led to the identification of 8 hit combinations among which the most prominent ones were those containing the coumarine and fluorescent maleimide scaffolds. The syn- and anti-tirazole hit combinations, AK1AZ2, AK1AZ3, AK4AZ2, and AK4AZ3 were synthesized, and in a regioisomer-assignment co-injection test it was determined that the enzyme favored the formation of the anti-triazoles for all identified combinations. The mechanism of inhibition of these triazoles was validated by incubating the alkyne/azide scaffolds in the presence of Apo-CA (non-Zn containing) enzyme. It was demonstrated that the Zn-bound water/hydroxide was needed in order to hydrolyze the coumarins which generated the actual inhibitor, the corresponding hydroxycinnamic acid. The time dependent nature of the inhibition activity typical for all coumarine-based inhibitors was also observed for the triazole compounds whose inhibition constants (Ki) were determined in two independent experiments with pre-incubation times of 3 and 25 minutes, respectively. It was observed that the lower Ki values were determined, the longer the pre-incubations lasted. Thus, a novel type of coumarin-containing triazoles were presented as in situ generated hits which have the potential to be used as fluorescent bio-markers or other drug discovery applications.

The proteins from the Bcl-2 family proteins play a central role in the regualtion of normal cellular homeostasis and have been validated as a target for the development of anticancer agents. Herein, in a proof-of-concept study based on a previous kinetic TGS study targeting Bcl-XL, it was demonstrated that a multi-fragment kinetic TGS approach coupled with TQMS technology was successfully implemented in the identification of known protein-protein modulators. Optimized screening conditions utilizing a triple quadruple mass spectrometer in the Multiple Reaction Monitoring (MRM) mode was demonstrated to be very efficient in kinetic TGS hit identification increasing both the throughput and sensitivity of this approach. The multi-fragment incubation approach was studied in detail and it was concluded that 200 fragment combinations in one well is an optimal and practical number permitting good acylsulfonamide detectability. Subsequently, a structurally diverse liberty of forty five thio acids and thirty eight sulfonyl azides was screened in parallel against Mcl-1 and Bcl-XL, and several potential hit combinations were identified. A control testing was carried out by substituting Bcl-XL with a mutant R139ABcl-XL, used to confirm that the potential kinetic TGS hit combinations were actually forming at the protein's hot spot and not elsewhere on the protein surface. Although, the synthesis of all these kinetic TGS hit compounds is currently ongoing, preliminary testing of several acylsulfonamides indicate that they disrupt the Bcl-XL/Bim or Mcl-1/Bim interaction.