The ability to synthesize pyrimidine nucleotides is essential for most organisms. Pyrimidines are required for RNA and DNA synthesis, as well as cell wall synthesis and the metabolism of certain carbohydrates. Recent findings, however, indicate that the pyrimidine biosynthetic pathway and its intermediates maybe more important for bacterial metabolism than originally thought. Maksimova et al., 1994, reported that a P. putida M, pyrimidine auxotroph in the third step of the pathway, dihydroorotase (DHOase), failed to produce the siderophore pyoverdin. We created a PAO1 DHOase pyrimidine auxotroph to determine if this was also true for P. aeruginosa. Creation of this mutant was a two-step process, as P. aeruginosa has two pyrC genes (pyrC and pyrC2), both of which encode active DHOase enzymes. The pyrC gene was inactivated by gene replacement with a truncated form of the gene. Next, the pyrC2 gene was insertionally inactivated with the aacC1 gentamicin resistance gene, isolated from pCGMW. The resulting pyrimidine auxotroph produced significantly less pyoverdin than did the wild type. In addition, the mutant produced 40% less of the phenazine antibiotic, pyocyanin, than did the wild type. As both of these compounds have been reported to be vital to the virulence response of P. aeruginosa, we decided to test the ability of the DHOase mutant strain to produce other virulence factors as well. Here we report that a block in the conversion of carbamoyl aspartate (CAA) to dihydroorotate significantly impairs the ability of P. aeruginosa to affect virulence. We believe that the accumulation of CAA in the cell is the root cause of this observed defect. This research demonstrates a potential role for pyrimidine intermediates in the virulence response of P. aeruginosa and may lead to novel targets for chemotherapy against P. aeruginosa infections.

Aspartate transcarbamoylase (ATCase) catalyzes the first committed step in pyrimidine biosynthesis. Bacterial ATCases are divided into three classes, A, B and C. Class A ATCases are largest at 450-500, are. dodecamers and represented by Pseudomonas ATCase. The overlapping pyrBC' genes encode the Pseudomonases ATCase, which is active only as a 480 kDa dodecamer and requires an inactive pyrC'-encoded DHOase for ATCase activity. ATCase has been studied in two non-pathogenic members of Mycobacterium, M. smegmatis and M. phlei. Their ATCases are dodecamers of molecular weight 480 kDa, composed of six PyrB and six PyrC polypeptides. Unlike the Pseudomonas ATCase, the PyrC polypeptide in these mycobacteria encodes an active DHOase. Moreover, the ATCase: DHOase complex in M. smegmatis is active both as the native 480 kDa and as a 390 kDa complex. The latter lacks two PyrC polypeptides yet retains ATCase activity. The ATCase from M. phlei is similar, except that it is active as the native 480 kDa form but also as 450,410 and 380 kDa forms. These complexes lack one, two, and three PyrC polypeptides, respectively. By contrast,.ATCases from pathogenic mycobacteria are active only at 480 kDa. Mycobacterial ATCases contain active DHOases and accordingly. are placed in class A1 . The class A1 ATCases contain active DHOases while class A2 ATCases contain inactive DHOases. ATCase has also been purified from Burkholderia cepacia and from an E. coli strain in which the cloned pyrB of B. cepacia was expressed. The B. cepacia ATCase has a molecular mass of 550 kDa, with two different polypeptides, PyrB (52 kDa) and PyrC of (39 kDa). The enzyme is active both as the native enzyme at 550 kDa and as smaller molecular forms including 240 kDa and 165 kDa. The ATCase synthesized by the cloned pyrB gene has a molecular weight of 165 kDa composed of three identical PyrB and no PyrC polypeptides. Nucleotide effectors ATP, CTP, and UTP inhibited all forms of enzymes. Because of its size and its activity as a trimer and smaller than native forms, the B. cepacia enzyme is placed in a new class.

In this research, the necessity of understanding and using bioinformatics is demonstrated using the enzyme aspartate transcarbamoylase (ATCase) as the model enzyme. The first portion of this research focuses on the use of bioinformatics. A partial sequence of the pyrB gene found in Enterococcus faecalis was submitted to GenBank and was analyzed against the contiguous sequence from its own genome project. A BLAST (Basic Local Alignment Search Tool; Atschul, et al., 1990) was performed in order to hypothesize the remaining portion of the gene from the contiguous sequence. This allowed a global comparison to other known aspartate transcarbamoylases (ATCases) and once deduced, a translation of the sequence gave the stop codon and thus the complete sequence of the open reading frame. When this was complete, upstream and downstream primers were designed in order to amplify the gene from genomic DNA. The amplified product was then sequenced and used later in phylogenetic analyses concerning the evolution of ATCase. The second portion of this research involves taking multiple ATCase nucleotide sequences and performing phenetic and phylogenetic analyses of the archaea and eubacter families. From these analyses, ancestral relationships which dictate both structure and function were extrapolated from the data and discussed.

Previous research in our laboratory established that pyrB, pyrC or pyrD knock-out mutants in Pseudomonas aeruginosa required pyrimidines for growth. Each mutant was also discovered to be defective in the production of virulence factors. Moreover, the addition of exogenous uracil did not restore the mutant to wild type virulence levels. In an earlier study using non-pathogenic P. putida, mutants blocked in one of the first three enzymes of the pyrimidine pathway produced no pyoverdine pigment while mutants blocked in the fourth, fifth or sixth steps produced copious quantities of pigment, just like wild type P. putida. The present study explored the correlation between pyrimidine auxotrophy and pigment production in P. aeruginosa. Since the pigment pyoverdine is a siderophore it may also be considered a virulence factor. Other virulence factors tested included casein protease, elastase, hemolysin, swimming, swarming and twitching motilities, and iron binding capacity. In all cases, these virulence factors were significantly decreased in the pyrB, pyrC or pyrD mutants and even in the presence of uracil did not attain wild type levels. In order to complete this comprehensive study, pyrimidine mutants blocked in the fifth (pyrE) and sixth (pyrF) steps of the biosynthetic pathway were examined in P. aeruginosa. A third mutant, crc, was also studied because of its location within 80 base pairs of the pyrE gene on the P. aeruginosa chromosome and because of its importance for carbon source utilization. Production of the virulence factors listed above showed a significant decrease in the three mutant strains used in this study when compared with the wild type. This finding may be exploited for novel chemotherapy strategies for ameliorating P. aeruginosa infections in cystic fibrosis patients.

Alzheimer’s disease (AD) is a highly complex and rapidly progressive neurodegenerative disorder characterized by the systemic collapse of cognitive function and formation of dense amyloid-β (Aβ) plaques and neurofibrillary tangles (NFTs). AD pathology is derived from the cholinergic, amyloid and tau hypotheses, respectively. Current pharmacotherapy with known anti-cholinesterases, such as Aricept ® and Exelon ®, only offer symptomatic relief without any disease-modifying effects (DMEs). It is now clear that in order to prevent the rapid progression of AD, new therapeutic treatments should target multiple AD pathways as opposed to the traditional “one drug, one target” approach. This research project employed medicinal chemistry tools to develop multifunctional small organic molecules against three key targets of AD pathology – the cholinesterases (AChE and BuChE), AChE-induced and self-induced Aβ1-40 aggregation and generation (β-secretase). A chemical library composed of 112 derivatives was generated to gather structure-activity relationship (SAR) data. The derivatives were based on a novel, non-fused, 2,4-disubstituted pyrimidine ring (2,4-DPR) template with substituents at the C-2 and C-4 position varying in size, steric and electronic properties. Molecular modeling was utilized to investigate their binding modes within the target enzymes and along with the acquired SAR, the chemical library was screened to identify lead multifunctional candidates.

The problem of forming a glycosidic bond between ribose and the free nucleoside bases to produce beta-nucleosides under plausible prebiotic conditions is commonly referred to in origin of life research as The Nucleoside Problem. The lack of a general solution to this problem currently represents one of the largest stumbling blocks to the RNA world hypothesis and many other theories regarding the origin of life. Over thirty years ago the purine nucleosides were successfully synthesized by drying the fully-formed bases and ribose together in the presence of divalent metal ion salts. However, glycosidic bond formation by the pyrimidine bases has never been achieved under similar reaction conditions. This thesis describes the first plausible prebiotic synthesis of a pyrimidine nucleoside, demonstrated with the pyrimidine base analogue 2-pyrimidinone. Information provided by nucleoside-formation reaction involving 2-pyrimidinone and related pyrimidine bases should provide valuable insights into the possible mechanism by which glycosidic bond formation was accomplished on the prebiotic Earth.

Drug discovery is reliant on new developments in natural product chemistry as well as advances in chemical synthesis. The interconnectivity and interdependence of natural and synthetic investigation in drug discovery is evident. The chemical exploration reported herein elaborates the relationship between natural product chemistry and chemical synthesis. Of particular interest are chemicals from organisms residing in less accessible environments, particularly Antarctica and endophytic microbial communities. Degradation via reductive ozonolysis of palmerolide A, a macrocyclic polyketide isolated from the Antarctic tunicate Synoicum adareanum, and subsequent synthetic preparation of the resulting polyols (1,2,6-hexanetriol and 1,2,3,6-hexanetetraol) led to a revision in the absolute configuration of the bioactive natural product (7R, 10R, 11R to 7S, 10S, 11S). A partial synthesis of palmerolide A (C3-14) was completed using Grubb’s 2nd generation catalyst to couple fragments formed using the previously developed methodology from the degradation study. Isolation of indole-pyrimidine containing alkaloids meridianins A, B, C, and E from the Antarctic tunicate Synoicum sp. prompted a synthetic investigation of psammopemmin A, a related alkaloid from the Antarctic sponge Psammopemma sp. resulting in reassignment of the structure of psammopemmin A to that of meridianin A. Both meridianin A and psammopemmin A were synthesized through a Suzuki coupling of the same 4-indolol nucleophile to the apposite pyrimidine electrophile. Several synthetic 3-pyrimidylindole analogs were also prepared and investigated for central nervous system, antimalarial, and cytotoxic activity. Chemical investigation of extracts from mangrove fungal endophytes that displayed antimalarial properties in vitro resulted in the isolation of several potent but cytotoxic and cytostatic compounds: cytochalasin D, roridin E, and 12,13-deoxyroridin E.

Malaria is a preventable and treatable disease caused by infection with Plasmodium parasites. Complex socioeconomic and political factors limit access to vector control and antimalarial drugs, and an estimated 600,000 people die from malaria every year. Rising drug resistance threatens to make malaria untreatable. As for all new traits, resistance is limited by fitness, and a small number of pathways are heavily favored by evolution. These pathways are targets for drug discovery. Pairing compounds active against the wild-type and the small emerging resistant population, a strategy we termed "targeting resistance," could block the rise of competitively viable resistance.

Šio darbo tikslas – pirimidino junginių – potencialių karboanhidrazių (CA) slopiklių –kūrimas. Teoriniai 4-[N-(pirimidin-4-il)]aminobenzensulfonamidų, turinčių pakaitus 2-, 5- ir 6-oje pirimidino žiedo padėtyje, sąveikos su aktyviuoju hCA centru tyrimai parodė, kad šie junginiai gali įsiterpti į aktyvųjį baltymo centrą ir su hCA turėtų sąveikauti kaip tipiški klasikiniai CA slopikliai. Tiksliniai 4-[N-(2,5,6-pakeisti pirimidin-4-il)amino]benzensulfonamidai sintetinti 4,6-dichlorpirimidinuose, 5-oje padėtyje turinčiuose cian-, formil- arba nitrogrupes, chloro atomą keičiant 4-aminobenzensulfonamidu. Bendradarbiaujant su Biotechnologijos instituto mokslininkais, kurie atliko hCA slopinimo susintetintais junginiais tyrimus, tobulintos šių junginių hCA slopinimo savybės. Slopiklių struktūros modifikuotos keičiant jungtuko tarp benzensulfonamido ir pirimidino fragmentų ilgį ir įvedant naujus pakaitus pirimidino žiede, kai kuriais atvejais taip sudarant naujas heterociklines sistemas. Šiuo tikslu ištirta pirimidin-5-karbaldehidų kondensacija su indolin-2-tionais. Nustatyta, kad 4-[N-(pirimidin-4-il)amino](metil-,etil-)benzensulfonamidai, 5-oje pirimidino žiedo padėtyje turintys cian-, formil- arba nitrogrupes, o 6-oje pirimidino žiedo padėtyje turintys benzilamino-, chlor-, metoksi- arba oksogrupes, yra nano- – mikromolinės eilės hCA slopikliai, galintys atrankiai slopinti hCAI, II ar XIII. Jų hCA slopinimo aktyvumą lemia sulfonamido grupės sąveika su katalitiniu cinko jonu ir... [toliau žr. visą tekstą] / The aim of the work was design of pyrimidine derivatives – potent carbonic anhydrase (CA) inhibitors. Theoretical investigation of interaction of 4-[N-(pyrimidin-4-yl)]aminobenzenesulfonamides containing substituents at 2-, 5- and 6- positions of pyrimidine ring with an active site of hCAs suggested that these compounds can fit into an active site of the enzymes and should interact with them as typical hCA inhibitors. Synthesis of target compounds was carried out by substitution of chloro group at 4,6-dichloropyrimidines containing cyano-, formyl- or nitro groups at position 5 of the pyrimidine ring with 4-aminobenzenesulfonamide. Regarding inhibition of hCAs with the synthesized compounds data acquired by the scientists of Institute of Biotechnology, structures of inhibitors were modified in order to improve their binding properties to hCA. It was performed by variation of a linker length between benzenesulfonamide and pyrimidine fragments and by introduction of new substituents at position 6 of the pyrimidine ring. In some cases these modifications led to the formation of new heterocyclic systems. For this purpose condensation of indoline-2-thiones with pyrimidine-5-carbaldehydes was investigated. It was determined that 4-[N-(pyrimidin-4-yl)amino](methyl-,ethyl-)benzenesulfonamides containing cyano-, formyl- or nitro groups at position 5 of the pyrimidine ring and benzylamino-, chloro-, methoxy- or oxo groups at position 6 of the pyrimidine ring inhibit hCA in... [to full text]

The aim of the work was design of pyrimidine derivatives – potent carbonic anhydrase (CA) inhibitors. Theoretical investigation of interaction of 4-[N-(pyrimidin-4-yl)]aminobenzenesulfonamides containing substituents at 2-, 5- and 6- positions of pyrimidine ring with an active site of hCAs suggested that these compounds can fit into an active site of the enzymes and should interact with them as typical hCA inhibitors. Synthesis of target compounds was carried out by substitution of chloro group at 4,6-dichloropyrimidines containing cyano-, formyl- or nitro groups at position 5 of the pyrimidine ring with 4-aminobenzenesulfonamide. Regarding inhibition of hCAs with the synthesized compounds data acquired by the scientists of Institute of Biotechnology, structures of inhibitors were modified in order to improve their binding properties to hCA. It was performed by variation of a linker length between benzenesulfonamide and pyrimidine fragments and by introduction of new substituents at position 6 of the pyrimidine ring. In some cases these modifications led to the formation of new heterocyclic systems. For this purpose condensation of indoline-2-thiones with pyrimidine-5-carbaldehydes was investigated. It was determined that 4-[N-(pyrimidin-4-yl)amino](methyl-,ethyl-)benzenesulfonamides containing cyano-, formyl- or nitro groups at position 5 of the pyrimidine ring and benzylamino-, chloro-, methoxy- or oxo groups at position 6 of the pyrimidine ring inhibit hCA in... [to full text] / Šio darbo tikslas – pirimidino junginių – potencialių karboanhidrazių (CA) slopiklių –kūrimas. Teoriniai 4-[N-(pirimidin-4-il)]aminobenzensulfonamidų, turinčių pakaitus 2-, 5- ir 6-oje pirimidino žiedo padėtyje, sąveikos su aktyviuoju hCA centru tyrimai parodė, kad šie junginiai gali įsiterpti į aktyvųjį baltymo centrą ir su hCA turėtų sąveikauti kaip tipiški klasikiniai CA slopikliai. Tiksliniai 4-[N-(2,5,6-pakeisti pirimidin-4-il)amino]benzensulfonamidai sintetinti 4,6-dichlorpirimidinuose, 5-oje padėtyje turinčiuose cian-, formil- arba nitrogrupes, chloro atomą keičiant 4-aminobenzensulfonamidu. Bendradarbiaujant su Biotechnologijos instituto mokslininkais, kurie atliko hCA slopinimo susintetintais junginiais tyrimus, tobulintos šių junginių hCA slopinimo savybės. Slopiklių struktūros modifikuotos keičiant jungtuko tarp benzensulfonamido ir pirimidino fragmentų ilgį ir įvedant naujus pakaitus pirimidino žiede, kai kuriais atvejais taip sudarant naujas heterociklines sistemas. Šiuo tikslu ištirta pirimidin-5-karbaldehidų kondensacija su indolin-2-tionais. Nustatyta, kad 4-[N-(pirimidin-4-il)amino](metil-,etil-)benzensulfonamidai, 5-oje pirimidino žiedo padėtyje turintys cian-, formil- arba nitrogrupes, o 6-oje pirimidino žiedo padėtyje turintys benzilamino-, chlor-, metoksi- arba oksogrupes, yra nano- – mikromolinės eilės hCA slopikliai, galintys atrankiai slopinti hCAI, II ar XIII. Jų hCA slopinimo aktyvumą lemia sulfonamido grupės sąveika su katalitiniu cinko jonu ir... [toliau žr. visą tekstą]