Mycobacterial Cell Wall Biosynthesis Research Articles

Integration of biophysical and biological approaches to validate fragment-like compounds targeting l,d-transpeptidases from Mycobacterium tuberculosis

Tuberculosis is one of the major causes of death worldwide; more than a million people die every year because of this infection. The constant emergency of Mycobacterium tuberculosis resistant strains against the most used treatments also contributes to the burden caused by this disease. Consequently, the development of new alternative therapies against this disease is constantly required. In recent years, only a few molecules have reached the market as new antituberculosis agents. The mycobacterial cell wall biosynthesis is for a longstanding considered an important target for drug development. Particularly, in M. tuberculosis, the peptidoglycan cross-links are predominantly formed by nonclassical bridges between the third residues of adjacent tetrapeptides. The responsible enzymes for these reactions are ld-transpeptidases (Ldts), for which M. tuberculosis has five paralogues. Although these enzymes are distinct from the penicillin-binding proteins (PBPs), they can also be inactivated by β-lactam antibiotics, but since M. tuberculosis has a chromosomal β-lactamase, most of the antibiotics of these classes can be degraded. Thus, to identify alternative scaffolds for the development of new antimicrobials against tuberculosis, we have integrated several fragment-based drug discovery techniques. Based on that, we identified and validated a number of small molecules that could be the starting point in the synthesis of more potent inhibitors against at least two Ldts from M. tuberculosis, LdtMt2 and LdtMt3. Eight identified molecules inhibited the Ldts activity in at least 20%, and three of them have antimycobacterial activity. The cell ultrastructural analysis suggested that one of the best compounds induced severe effects on the septum and cell wall morphologies, which corroborates our target-based approach to identifying new Ldts hits.

In silico virtual screening for the identification of novel inhibitors against dihydrodipicolinate reductase (DapB) of Mycobacterium tuberculosis, a key enzyme of diaminopimelate pathway.

Tuberculosis (TB) caused by Mycobacterium tuberculosis (M. tb) is one of the most devastating infectious diseases afflicting a large number of human lives. In spite of the availability of several drugs for treating TB, emergence of drug-resistant strains of the pathogen has made treatment and eradication of TB a challenging task. Hence, there is an imperative need for new intervention strategies to target M. tb. The enzymes of the diaminopimelate pathway, involved in lysine biosynthesis as well as in cell wall biosynthesis, have been considered as important anti-TB drug targets. One such enzyme is dihydrodipicolinate reductase (DapB), catalyzing the reduction of 2,3-dihydrodipicolinic acid to 2,3,4,5-tetrahydro-dipicolinic acid by utilizing NADH/NADPH as its cofactor. Here, we have generated an antisense knockdown mutant strain of dapB and demonstrated a crucial role of dapB in the in vitro and intracellular growth of M. tb. Further, in silico virtual screening was performed by employing a library of ~95,000 compounds for the identification of inhibitory molecules against DapB. The molecules with high docking scores were screened against the enzymatic activity of DapB to determine their IC50 values. Further, hit molecules that inhibited DapB were screened against the M. tb growth in broth culture and inside macrophages. The molecules exhibiting M. tb inhibition were also evaluated for their cytotoxicity against various mammalian cell lines. In summary, we have identified a lead molecule, B59, with an IC50 value of 11 µg/mL and MIC99 value of 20 µg/mL, which can be further optimized to develop potent inhibitory compounds against M. tb DapB. IMPORTANCE Non-compliance to lengthy antituberculosis (TB) treatment regimen, associated side effects, and emergence of drug-resistant strains of Mycobacterium tuberculosis (M. tb) emphasize the need to develop more effective anti-TB drugs. Here, we have evaluated the role of M. tb dihydrodipicolinate reductase (DapB), a component of the diaminopimelate pathway, which is involved in the biosynthesis of both lysine and mycobacterial cell wall. We showed that DapB is essential for the in vitro as well as intracellular growth of M. tb. We further utilized M. tb DapB, as a target for identification of inhibitors by employing in silico virtual screening, and conducted various in vitro screening assays to identify inhibitors with potential to inhibit DapB activity and in vitro and intracellular growth of M. tb with no significant cytotoxicity against various mammalian cell lines. Altogether, M. tb DapB serves as an important drug target and a hit molecule, namely, 4-(3-Phenylazoquinoxalin-2-yl) butanoic acid methyl ester has been identified as an antimycobacterial molecule in our study.

Structural insight into mycobacterial cell wall biosynthesis by AftB, a potential anti-TB drug target.

Tuberculosis (TB) is a severe infectious disease caused by Mycobacterium tuberculosis (Mtb) and remains a significant health problem worldwide. The success of Mtb as the leading pathogen can largely be attributed to its atypical multilayered cell wall, which is highly impermeable and confers natural protection against attack from the immune system and many antibiotics. Due to its unique character, the mycobacterial cell wall has become the most widely exploited target of anti-TB drugs. AftB is an arabinofuranosyltransferase that functions in the terminal step of cell wall arabinan biosynthesis, catalyzing the transfer of arabinose from the donor decaprenyl-phosphate-arabinose (DPA) onto the cell wall polysaccharides. AftB is essential for Mtb's survival and is deemed a potential new drug target for TB. Here we report the atomic-resolution structures of AftB obtained by cryo-EM in both apo and donor substrate analog bound forms. The apo structure revealed that AftB contains 11 transmembrane helices belonging to the GT-C glycosyltransferase superfamily and a periplasmic segment of a novel fold which might direct the binding of polysaccharides acceptor. AftB forms a large putative substrate-binding cavity, with one side facing the periplasmic domain and the other facing the lipid bilayer. The substrate-bound structure further unmasked the nature of the active site. Using synthetic AftB donor and acceptor substrates, we recapitulated the enzymatic reaction in vitro. And the functional analysis using AftB variants further proved the importance of the active site revealed from our structures. We proposed that AftB catalyzes the glycosylation reaction by utilizing a conserved aspartate residue as the base catalyst. Collectively, our work provides a structural basis for understanding the molecular mechanism of AftB and a blueprint for developing new drugs to combat the increasing threat caused by Mtb.

Enhanced immunogenicity of Mycobacterium bovis BCG through CRISPRi mediated depletion of AftC

Mycobacterium tuberculosis causes the disease tuberculosis and affects a third of the world’s population. The recent COVID-19 pandemic exacerbated the situation with a projected 27% increase in tuberculosis related deaths. M. tuberculosis has an elaborate cell wall consisting of peptidoglycan, arabinogalactan and mycolic acids which shield the bacilli from the toxic bactericidal milieu within phagocytes. Amongst, the numerous glycosyltransferase enzymes involved in mycobacterial cell wall biosynthesis, arabinofuranosyltransferase C (aftC) is responsible for the branching of the arabinan domain in both arabinogalactan and lipoarabinomannan. Using Clustered Regularly Interspaced Short Palindromic Repeats interference (CRISPRi) we have generated aftC knockdowns in Mycobacterium bovis BCG and demonstrated the generation of a truncated, immunogenic lipoarabinomannan within its cell envelope. The aftC depleted BCG mutants were unable to form characteristic mycobacterial pellicular biofilms and elicit a potent immunostimulatory phenotype compared to wild type M. bovis BCG in a THP1 cell line. This study paves the way to further explore novel BCG mutants as promising vaccine boosters in preventing pulmonary tuberculosis.

Development of 6-Methanesulfonyl-8-nitrobenzothiazinone Based Antitubercular Agents.

The 6-trifluoro substituted 8-nitrobenzothiazinones (BTZs) represent a novel type of antitubercular agents, and their high antimycobacterial activity is related to the inhibition of decaprenylphosphoryl-β-d-ribose 2'-oxidase (DprE1), an enzyme essential for the biosynthesis of mycobacterial cell wall. While extraordinary whole-cell activity was reported for the clinically advanced compound PBTZ169, its poor aqueous solubility signals the potential low bioavailability. To ameliorate the BTZ physiochemical property, a series of 6-methanesulfonyl substituted compounds were designed and prepared, and their antitubercular activity and DprE1 inhibition ability were evaluated. Among these compounds, MsPBTZ169 and compounds 2 and 8 exhibited minimum inhibitory concentrations (MICs) of less than 40 nM; moreover, these compounds displayed increased aqueous solubility and acceptable metabolic stability. Taken together, this study suggested that the 6-methanesulfonyl substituted 8-nitrobenzothiazinone derivatives, in combination with side chain modification, might provide BTZ type antitubercular agents with improved drug-like properties.

Design and Synthesis of Highly Active Antimycobacterial Mutual Esters of 2-(2-Isonicotinoylhydrazineylidene)propanoic Acid.

The combination of two active scaffolds into one molecule represents a proven approach in drug design to overcome microbial drug resistance. We designed and synthesized more lipophilic esters of 2-(2-isonicotinoylhydrazineylidene)propanoic acid, obtained from antitubercular drug isoniazid, with various alcohols, phenols and thiols, including several drugs, using carbodiimide-mediated coupling. Nineteen new esters were evaluated as potential antimycobacterial agents against drug-sensitive Mycobacterium tuberculosis (Mtb.) H37Rv, Mycobacterium avium and Mycobacterium kansasii. Selected derivatives were also tested for inhibition of multidrug-resistant (MDR) Mtb., and their mechanism of action was investigated. The esters exhibited high activity against Mtb. (minimum inhibitory concentrations, MIC, from ≤0.125 μM), M. kansasii, M. avium as well as MDR strains (MIC from 0.25, 32 and 8 µM, respectively). The most active mutual derivatives were derived from 4-chloro/phenoxy-phenols, triclosan, quinolin-8-ol, naphthols and terpene alcohols. The experiments identified enoyl-acyl carrier protein reductase (InhA), and thus mycobacterial cell wall biosynthesis, as the main target of the molecules that are activated by KatG, but for some compounds can also be expected adjunctive mechanism(s). Generally, the mutual esters have also avoided cytotoxicity and are promising hits for the discovery of antimycobacterial drugs with improved properties compared to parent isoniazid.

Synthesis of sulfamide analogues of deoxthymidine monophosphate as potential inhibitors of mycobacterial cell wall biosynthesis

The recently discovered enzyme Mycobacterium tuberculosis thymidine monophosphate kinase (TMPKmt), which catalyses the phosphorylation of deoxythymidine monophosphate (dTMP) to give deoxythymidine diphosphate (dTDP), is indispensable for the growth and survival of M. tuberculosis as it plays an essential role in DNA synthesis. Inhibition of TMPKmt is an attractive avenue for the development of novel anti-tuberculosis agents. Based on the premise that sulfamide may be a suitable isostere of phosphate, deoxythymidine analogues comprising various substituted sulfamides at C5′ were modelled in silico into the active site of TMPKmt (PDB accession code: 1N5K) using induced-fit docking methods. A selection of modelled compounds was synthesized, and their activity as inhibitors of TMPKmt was evaluated. Three compounds showed competitive inhibition of TMPKmt in the micromolar range (10–50 μM). Compounds were tested in vitro for anti-mycobacterial activity against M. smegmatis: three compounds showed weak anti-mycobacterial activity (MIC 250 μg/mL).

Cell Wall Associated Factors of Mycobacterium tuberculosis as Major Virulence Determinants: Current Perspectives in Drugs Discovery and Design.

Mycobacteria genus is responsible for deadly diseases like tuberculosis and leprosy. Cell wall of bacteria belonging to this genus is unique in many ways. It plays a major role in the pathogenesis and intracellular survival inside the host. In intracellular pathogens, their cell wall acts as molecular shield and interacts with host cell milieu to modulate host defense responses. In this review, we summarize the factors that participate in the biosynthesis of unique mycobacterial cell wall, understand their potential as drug targets and the recent developments where they have been evaluated as possible drug targets. Several cell wall associated factors that play crucial roles in the synthesis of cell wall components like Antigen 85 complex, Glycosyltransferases (GTs), LM (lipomannan) and LAM (lipoarabinomannan), mAGP Complex, lipolytic enzyme have been categorically documented. Most of the presently used anti TB regimens interrupted cell wall synthesis, but the emergence of drug resistant strains made it mandatory to identify new drug targets. Novel drug candidates which could inhibit the synthesis of cell wall components have been thoroughly studied worldwide. Studies demonstrated that the cell wall components are unique in terms of their contribution in mycobacterium pathogenesis. Targeting these can hamper the growth of M. tuberculosis. In this study, we scrutinize the drugs under trials and the potential candidates screened through in silico findings.

Mannich Ketones as Possible Antimycobacterial Agents.

Twenty-three known unsaturated and fused Mannich ketones and their reduced derivatives (amino alcohols) were selected for an antituberculotic study. They were screened against several mycobacterial strains including Mycobacterium tuberculosis, M. xenopi, and M. gordonae, and minimum inhibitory concentration values were also determined using the standard antituberculotic drug isoniazid (INH) as a reference. Structure-activity relationships were also studied. The mode of action of the test compounds was investigated using transmission electron microscopy, high-performance liquid chromatography, and matrix-assisted desorption/ionization mass spectrometry. Several test substances proved to be as potent as INH, but their antimycobacterial spectra were broader than that of INH. Our findings suggest that their mode of action is probably through the inhibition of mycobacterial cell wall biosynthesis.

Biochemical and structural investigations on phosphoribosylpyrophosphate synthetase from Mycobacterium smegmatis.

Mycobacterium smegmatis represents one model for studying the biology of its pathogenic relative Mycobacterium tuberculosis. The structural characterization of a M. tuberculosis ortholog protein can serve as a valid tool for the development of molecules active against the M. tuberculosis target. In this context, we report the biochemical and structural characterization of M. smegmatis phosphoribosylpyrophosphate synthetase (PrsA), the ortholog of M. tuberculosis PrsA, the unique enzyme responsible for the synthesis of phosphoribosylpyrophosphate (PRPP). PRPP is a key metabolite involved in several biosynthetic pathways including those for histidine, tryptophan, nucleotides and decaprenylphosphoryl-arabinose, an essential precursor for the mycobacterial cell wall biosynthesis. Since M. tuberculosis PrsA has been validated as a drug target for the development of antitubercular agents, the data presented here will add to the knowledge of the mycobacterial enzyme and could contribute to the development of M. tuberculosis PrsA inhibitors of potential pharmacological interest.

Identification of inhibitors targeting Mycobacterium tuberculosis cell wall biosynthesis via dynamic combinatorial chemistry.

In this study, we report a dynamic combinatorial approach along with highly efficient in situ screening to identify inhibitors of UDP-galactopyranose mutase (UGM), an essential enzyme involved in mycobacterial cell wall biosynthesis. These two technologies converged to the identification of a new UGM inhibitor chemotype. Importantly, the best molecule not only displayed high affinity for the target enzyme but also exhibited in vitro growth inhibition against whole Mycobacterium tuberculosis cells. The strategy described here provides an avenue to explore a novel inhibitor class for UGMs and paves the way for further pharmacological studies on tuberculosis treatment.

Mycobacterial cell wall biosynthesis: a multifaceted antibiotic target

Mycobacterium tuberculosis (Mtb), the etiological agent of tuberculosis (TB), is recognized as a global health emergency as promoted by the World Health Organization. Over 1 million deaths per year, along with the emergence of multi- and extensively-drug resistant strains of Mtb, have triggered intensive research into the pathogenicity and biochemistry of this microorganism, guiding the development of anti-TB chemotherapeutic agents. The essential mycobacterial cell wall, sharing some common features with all bacteria, represents an apparent 'Achilles heel' that has been targeted by TB chemotherapy since the advent of TB treatment. This complex structure composed of three distinct layers, peptidoglycan, arabinogalactan and mycolic acids, is vital in supporting cell growth, virulence and providing a barrier to antibiotics. The fundamental nature of cell wall synthesis and assembly has rendered the mycobacterial cell wall as the most widely exploited target of anti-TB drugs. This review provides an overview of the biosynthesis of the prominent cell wall components, highlighting the inhibitory mechanisms of existing clinical drugs and illustrating the potential of other unexploited enzymes as future drug targets.

Synthesis of arabinose glycosyl sulfamides as potential inhibitors of mycobacterial cell wall biosynthesis

A series of arabinose glycosyl sulfamides with varying alkyl chain types and lengths were synthesised as mimics of decaprenolphosphoarabinose (DPA), and as potential inhibitors of mycobacterial cell wall biosynthesis. Unprecedented conversion of the desired furanose to the thermodynamically more stable pyranose form occurred during final de-protection. Biological testing against Mycobacterium smegmatis revealed low to moderate anti-mycobacterial activity with marked dependence on alkyl chain length, which in the case of mono-substituted sulfamides was maximal for a C-10 chain.

Synthesis and Preliminary Screening of O-β-D-Galactopyranosides as Potential Mimic of UDP-Galp against Mycobacterium tuberculosis

O-Galactopyranosides were synthesized as potential mimics of uridine 5c-diphosphate galactopyranose (UDP-Galp), which is the substrate of the mutase enzyme involved in the mycobacterial cell wall biosynthesis by converting UDP-Galp to UDP-Galf. In vitro assay against the Mycobacterium tuberculosis H37Rv strain gave promising results with minimum inhibitory concentration (MIC) values in the range of 3.13 to 25 μg/mL.

Inactivation of tesA Reduces Cell Wall Lipid Production and Increases Drug Susceptibility in Mycobacteria

Phthiocerol dimycocerosates (PDIMs) and phenolic glycolipids (PGLs) are structurally related lipids noncovalently bound to the outer cell wall layer of Mycobacterium tuberculosis, Mycobacterium leprae, and several opportunistic mycobacterial human pathogens. PDIMs and PGLs are important effectors of virulence. Elucidation of the biosynthesis of these complex lipids will not only expand our understanding of mycobacterial cell wall biosynthesis, but it may also illuminate potential routes to novel therapeutics against mycobacterial infections. We report the construction of an in-frame deletion mutant of tesA (encoding a type II thioesterase) in the opportunistic human pathogen Mycobacterium marinum and the characterization of this mutant and its corresponding complemented strain control in terms of PDIM and PGL production. The growth and antibiotic susceptibility of these strains were also probed and compared with the parental wild-type strain. We show that deletion of tesA leads to a mutant that produces only traces of PDIMs and PGLs, has a slight growth yield increase and displays a substantial hypersusceptibility to several antibiotics. We also provide a robust model for the three-dimensional structure of M. marinum TesA (TesAmm) and demonstrate that a Ser-to-Ala substitution in the predicted catalytic Ser of TesAmm renders a mutant that recapitulates the phenotype of the tesA deletion mutant. Overall, our studies demonstrate a critical role for tesA in mycobacterial biology, advance our understanding of the biosynthesis of an important group of polyketide synthase-derived mycobacterial lipids, and suggest that drugs aimed at blocking PDIM and/or PGL production might synergize with antibiotic therapy in the control of mycobacterial infections.

Decaprenylphosphoryl-β-D-Ribose 2′-Epimerase, the Target of Benzothiazinones and Dinitrobenzamides, Is an Essential Enzyme in Mycobacterium smegmatis

BackgroundThe unique cell wall of bacteria of the suborder Corynebacterineae is essential for the growth and survival of significant human pathogens including Mycobacterium tuberculosis and Mycobacterium leprae. Drug resistance in mycobacteria is an increasingly common development, making identification of new antimicrobials a priority. Recent studies have revealed potent anti-mycobacterial compounds, the benzothiazinones and dinitrobenzamides, active against DprE1, a subunit of decaprenylphosphoribose 2′ epimerase which forms decaprenylphosphoryl arabinose, the arabinose donor for mycobacterial cell wall biosynthesis. Despite the exploitation of Mycobacterium smegmatis in the identification of DprE1 as the target of these new antimicrobials and its use in the exploration of mechanisms of resistance, the essentiality of DprE1 in this species has never been examined. Indeed, direct experimental evidence of the essentiality of DprE1 has not been obtained in any species of mycobacterium.Methodology/Principal FindingsIn this study we constructed a conditional gene knockout strain targeting the ortholog of dprE1 in M. smegmatis, MSMEG_6382. Disruption of the chromosomal copy of MSMEG_6382 was only possible in the presence of a plasmid-encoded copy of MSMEG_6382. Curing of this “rescue” plasmid from the bacterial population resulted in a cessation of growth, demonstrating gene essentiality.Conclusions/SignificanceThis study provides the first direct experimental evidence for the essentiality of DprE1 in mycobacteria. The essentiality of DprE1 in M. smegmatis, combined with its conservation in all sequenced mycobacterial genomes, suggests that decaprenylphosphoryl arabinose synthesis is essential in all mycobacteria. Our findings indicate a lack of redundancy in decaprenylphosphoryl arabinose synthesis in M. smegmatis, despite the relatively large coding capacity of this species, and suggest that no alternative arabinose donors for cell wall biosynthesis exist. Overall, this study further validates DprE1 as a promising target for new anti-mycobacterial drugs.

Novel Small-Molecule Inhibitors of Arylamine N-Acetyltransferases: Drug Discovery by High Throughput Screening

Arylamine N-acetyltransferases (NATs) are a family of enzymes found in eukaryotes and prokaryotes. While the precise endogenous function of NAT remains unknown for most organisms, recent evidence has shown that the expression of human NAT1 is up-regulated in estrogen receptor positive breast cancer. Additionally, NAT in mycobacteria is required for mycobacterial cell wall biosynthesis and survival of the organisms within macrophage. It is therefore important to develop small molecule inhibitors of NATs as molecular tools to study the function of NATs in various organisms. Such inhibitors may also prove useful in future drug design, for example in the development of anti tubercular agents. We describe a high-throughput screen of a proprietary library of 5016 drug-like compounds against three prokaryotic NAT enzymes and two eukaryotic NAT enzymes.

Nvestigation of ABC transporter from mycobacterial arabinogalactan biosynthetic cluster

Two genes from the "mycobacterial arabinogalactan biosynthetic cluster" spanning the region from Rv3779 to Rv3809c in the genome of Mycobacterium tuberculosis H37Rv were annotated as possible components of the ATP-binding cassette transporter. Rv3781 encodes a nucleotide-binding domain and Rv3783 determines production of a membrane-spanning domain. We have examined possible roles of these genes in mycobacterial cell wall biosynthesis through inactivation of their respective orthologs in Mycobacterium smegmatis mc(2)155, phenotypic characterization of the mutant strains via metabolic labeling with [U-(14)C]-glucose, cell-free reactions with UDP-[U-(14)C]-galactose monitoring galactan build-up and transcriptional analysis. Several lines of evidence suggest that this ABC transporter is involved in biosynthesis of arabinogalactan, although more investigation is needed to establish its precise role or the transported substrate.

Mycobacterium tuberculosis Phosphoribosylpyrophosphate Synthetase: Biochemical Features of a Crucial Enzyme for Mycobacterial Cell Wall Biosynthesis

The selection and soaring spread of Mycobacterium tuberculosis multidrug-resistant (MDR-TB) and extensively drug-resistant strains (XDR-TB) is a severe public health problem. Currently, there is an urgent need for new drugs for tuberculosis treatment, with novel mechanisms of action and, moreover, the necessity to identify new drug targets. Mycobacterial phosphoribosylpyrophosphate synthetase (MtbPRPPase) is a crucial enzyme involved in the biosynthesis of decaprenylphosphoryl-arabinose, an essential precursor for the mycobacterial cell wall biosynthesis. Moreover, phosphoribosylpyrophosphate, which is the product of the PRPPase catalyzed reaction, is the precursor for the biosynthesis of nucleotides and of some amino acids such as histidine and tryptophan. In this context, the elucidation of the molecular and functional features of MtbPRPPase is mandatory. MtbPRPPase was obtained as a recombinant form, purified to homogeneity and characterized. According to its hexameric form, substrate specificity and requirement of phosphate for activity, the enzyme proved to belong to the class I of PRPPases. Although the sulfate mimicked the phosphate, it was less effective and required higher concentrations for the enzyme activation. MtbPRPPase showed hyperbolic response to ribose 5-phosphate, but sigmoidal behaviour towards Mg-ATP. The enzyme resulted to be allosterically activated by Mg2+ or Mn2+ and inhibited by Ca2+ and Cu2+ but, differently from other characterized PRPPases, it showed a better affinity for the Mn2+ and Cu2+ ions, indicating a different cation binding site geometry. Moreover, the enzyme from M. tuberculosis was allosterically inhibited by ADP, but less sensitive to inhibition by GDP. The characterization of M. tuberculosis PRPPase provides the starting point for the development of inhibitors for antitubercular drug design.

Synthesis, biological activity, and evaluation of the mode of action of novel antitubercular benzofurobenzopyrans substituted on A ring

The 8-, 9-, 10-, and 11-halo, hydroxy, and methoxy derivatives of the antimycobacterial 3,3-dimethyl-3 H-benzofuro[3,2- f][1]benzopyran were synthesized by condensation of the diazonium salts of 2-chloroanilines ( 13– 17) with 1,4-benzoquinone ( 18), reduction of the intermediate phenylbenzoquinones 19– 22 to dihydroxybiphenyls, cyclisation to halo-2-hydroxydibenzofurans 24– 27, and construction of the pyran ring by thermal rearrangement of the corresponding dimethylpropargyl ethers 35– 38. Palladium catalyzed nucleophilic aromatic substitution permitted conversion of the halo to the corresponding hydroxy derivatives which were methylated to methoxy-3,3-dimethyl-3 H-benzofuro[3,2- f][1]benzopyran. All compounds substituted on the A ring were found more potent than the reference compound 1 against Mycobacterium bovis BCG and the virulent strain Mycobacterium tuberculosis H37Rv. The effect of the most active derivatives on mycolate synthesis was explored in order to confirm the preliminary hypothesis of an effect on mycobacterial cell wall biosynthesis. The linear 9-methoxy-2,2-dimethyl-2 H-benzofuro[2,3- g][1]benzopyran ( 46) exhibiting a good antimycobacterial activity and devoid of cytotoxicity appeared to be the most promising compound.