SHAPE-DEPENDENT MOLECULAR RECOGNITION OF SPECIFIC SEQUENCES OF DNA BY HETEROCYCLIC CATIONS by YI MIAO Under the Direction of Dr. W. David Wilson ABSTRACT DB921 and DB911 are biphenyl-benzimidazole-diamidine isomers with a central para- and meta-substituted phenyl group, respectively. Unexpectedly, linear DB921 has much stronger binding affinity with DNA than its curved isomer, DB911. This is quite surprising and intriguing since DB911 has the classical curved shape generally required for strong minor groove binding while DB921 clearly does not match the groove shape. Several biophysical techniques including thermal melting (Tm), circular dichroism (CD), biosensor-surface plasmon resonance (SPR), and isothermal titration calorimetry (ITC) have been utilized to investigate the interactions between these compounds and DNA. The structure of the DB921-DNA complex reveals that DB921 binds to DNA with a reduced twist of the biphenyl for better fit of DB921 into the minor groove. A bound water molecule complements the curvature of DB921 and contributes for tight binding by forming H-bonds with both DNA and DB921. Structure-affinity relationship studies of a series of DB921 analogs show that the benzimidazole group is one of the key groups of DB921 for its strong binding to the minor groove. Thermodynamic studies show that the stronger binding of DB921 is due to a more favorable binding enthalpy compared to DB911 even though the complex formation with DNA for these compounds are all predominantly entropically driven. DB921 also has more negative heat capacity change than DB911. The initial studies of inhibition of the interaction between an AT hook peptide of HMGA proteins and its target DNA by a set of diamidine AT-minor groove binders using biosensor-SPR technique show that the inhibitory ranking order is consistent with that of binding affinity and linear-shaped DB921 still has excellent inhibitory effects. These heterocyclic cations rapidly inhibit the binding of DBD2 peptide to the DNA and may only block the specific AT binding of the peptide without hindering the non-specific binding interaction. The results of this project have shown that DB921 represents a new novel effective minor groove binder that does not fit the traditional model and is a potential inhibitor for DNA/protein complexes. INDEX WORDS: Molecular recognition, DNA binding, Minor groove binding, Linear shape, Compound curvature, Binding affinity, Binding kinetics, Thermodynamics, Surface plasmon resonance, Isothermal titration calorimetry, Inhibition

Made available in DSpace on 2015-04-14T13:35:46Z (GMT). No. of bitstreams: 1 450558.pdf: 2693787 bytes, checksum: e4ffe9234a98b2ddfc9bc350c708c01c (MD5) Previous issue date: 2013-06-28 / Tuberculosis (TB), one of the oldest recorded human afflictions, is still one of the biggest killers among the infectious diseases. The HIV co-infection and the emergence of multidrug resistant TB have provided a very alarming challenge to global health and led us to focus on the research for new and more effective therapeutics against the disease. The modern approach to the development of new chemical compounds against complex diseases, especially the neglected endemic ones, such as TB, is based on the use of defined molecular targets. Enzymes from the pyrimidine biosynthesis pathway have been considered potential targets for identification or development of novel anti-mycobacterial agents since in bacteria, pyrimidine nucleotide interconvertion pathways are important in a number of essential processes, including DNA, RNA, and phospholipid biosynthesis. Cytidine 5 -monophosphate kinase from Mycobacterium tuberculosis (MtCMK) catalyzes the ATP-dependent phosphoryl group transfer preferentially to CMP and dCMP. Here, initial velocity studies and Isothermal Titration Calorimetry (ITC) measurements showed that MtCMK follows a random-order kinetic mechanism of substrate binding, and an ordered mechanism for product release. The thermodynamic signatures of CMP and CDP binding to MtCMK showed favorable enthalpy and unfavorable entropy, and ATP binding was characterized by favorable changes in enthalpy and entropy. The contribution of linked protonation events to the energetics of MtCMK:phosphoryl group acceptor binary complex formation suggested a net gain of protons. Values for the pKa of a likely chemical group involved in proton exchange and for the intrinsic binding enthalpy were calculated. The Asp187 side chain of MtCMK is suggested as the likely candidate for the protonation event. Data on thermodynamics of binary complex formation were collected to evaluate the contribution of 2 -OH group to intermolecular interactions. The data are discussed in light of functional and structural comparisons among CMP/dCMP kinases and UMP/CMP ones. / A tuberculose (TB), uma das doen?as mais antigas da humanidade, ainda ? uma das principais causas de morte entre as doen?as infecciosas. A coinfec??o com o HIV e a emerg?ncia de TB resistente a m?ltiplas drogas representam um desafio ? sa?de p?blica e tem estimulado a pesquisa por novos e mais efetivos agentes terap?uticos contra a doen?a. Novas abordagens para o desenvolvimento de compostos contra doen?as complexas, especialmente doen?as end?micas negligenciadas, s?o baseadas no uso de alvos moleculares definidos. Enzimas envolvidas no metabolismo de pirimidinas tornam-se alvos moleculares interessantes para compostos inibidores, uma vez que em bact?rias, as rotas de interconvers?o de nucleot?deos pirimid?nicos s?o importantes em in?meros processos essenciais, incluindo a bioss?ntese de DNA, RNA e fosfolip?dios. A citidina 5 -monofosfato quinase de Mycobacterium tuberculosis (MtCMK) em estudo, catalisa a transfer?ncia revers?vel de um grupamento fosforil a partir de ATP, preferencialmente para CMP e dCMP. Neste trabalho, os estudos de velocidade inicial e experimentos de Calorimetria de Titula??o Isot?rmica (ITC) demonstraram que a adi??o dos substratos (CMP e ATP) ? MtCMK segue um mecanismo cin?tico sequencial aleat?rio, e que a libera??o dos produtos ocorre de forma ordenada, onde o ADP ? o primeiro produto a ser liberado. As assinaturas termodin?micas da liga??o do CMP e do CDP ? MtCMK mostraram varia??es favor?veis da entalpia e desfavor?veis da entropia, e, a liga??o do ATP foi caracterizada por mudan?as favor?veis da entalpia e da entropia. As contribui??es energ?ticas oriundas dos eventos de protona??o, associados ? forma??o do complexo bin?rio MtCMK:receptor do grupamento fosforil, sugerem um ganho l?quido de pr?tons. Al?m disso, foram calculados os valores de pKa de um prov?vel grupo envolvido na troca de pr?tons, e da entalpia de liga??o intr?nseca. A cadeia lateral do Asp187 da MtCMK ? sugerido como prov?vel candidato para o evento de protona??o. As medidas termodin?micas da forma??o do complexo bin?rio foram coletados a fim de avaliar a contribui??o do grupo 2 -OH da pentose nas intera??es intermoleculares. Os dados obtidos foram discutidos comparando-se as caracter?sticas estrutural e funcional entre as CMKs j? estudadas e a UMP/CMP quinase humana.