Study of SQ109 analogs binding to mycobacterium MmpL3 transporter using MD simulations and alchemical relative binding free energy calculations.

N-geranyl-N΄-(2-adamantyl)ethane-1,2-diamine (SQ109) is a tuberculosis drug that has high potency against Mycobacterium tuberculosis (Mtb) and may function by blocking cell wall biosynthesis. After the crystal structure of MmpL3 from Mycobacterium smegmatis in complex with SQ109 became available, it was suggested that SQ109 inhibits Mmpl3 mycolic acid transporter. Here, we showed using molecular dynamics (MD) simulations that the binding profile of nine SQ109 analogs with inhibitory potency against Mtb and alkyl or aryl adducts at C-2 or C-1 adamantyl carbon to MmpL3 was consistent with the X-ray structure of MmpL3 - SQ109 complex. We showed that rotation of SQ109 around carbon-carbon bond in the monoprotonated ethylenediamine unit favors two gauche conformations as minima in water and lipophilic solvent using DFT calculations as well as inside the transporter's binding area using MD simulations. The binding assays in micelles suggested that the binding affinity of the SQ109 analogs was increased for the larger, more hydrophobic adducts, which was consistent with our results from MD simulations of the SQ109 analogues suggesting that sizeable C-2 adamantyl adducts of SQ109 can fill a lipophilic region between Y257, Y646, F260 and F649 in MmpL3. This was confirmed quantitatively by our calculations of the relative binding free energies using the thermodynamic integration coupled with MD simulations method with a mean assigned error of 0.74 kcal mol-1 compared to the experimental values.

Synthesis and Testing of Analogs of the Tuberculosis Drug Candidate SQ109 against Bacteria and Protozoa: Identification of Lead Compounds against Mycobacterium abscessus and Malaria Parasites.

SQ109 is a tuberculosis drug candidate that has high potency against Mycobacterium tuberculosis and is thought to function at least in part by blocking cell wall biosynthesis by inhibiting the MmpL3 transporter. It also has activity against bacteria and protozoan parasites that lack MmpL3, where it can act as an uncoupler, targeting lipid membranes and Ca2+ homeostasis. Here, we synthesized 18 analogs of SQ109 and tested them against M. smegmatis, M. tuberculosis, M. abscessus, Bacillus subtilis, and Escherichia coli, as well as against the protozoan parasites Trypanosoma brucei, T. cruzi, Leishmania donovani, L. mexicana, and Plasmodium falciparum. Activity against the mycobacteria was generally less than with SQ109 and was reduced by increasing the size of the alkyl adduct, but two analogs were ∼4-8-fold more active than SQ109 against M. abscessus, including a highly drug-resistant strain harboring an A309P mutation in MmpL3. There was also better activity than found with SQ109 with other bacteria and protozoa. Of particular interest, we found that the adamantyl C-2 ethyl, butyl, phenyl, and benzyl analogs had 4-10× increased activity against P. falciparum asexual blood stages, together with low toxicity to a human HepG2 cell line, making them of interest as new antimalarial drug leads. We also used surface plasmon resonance to investigate the binding of inhibitors to MmpL3 and differential scanning calorimetry to investigate binding to lipid membranes. There was no correlation between MmpL3 binding and M. tuberculosis or M. smegmatis cell activity, suggesting that MmpL3 is not a major target in mycobacteria. However, some of the more active species decreased lipid phase transition temperatures, indicating increased accumulation in membranes, which is expected to lead to enhanced uncoupler activity.

Synthesis and in vitro proof-of-concept studies on bispecific iron oxide magnetic nanoparticles targeting PSMA and GRP receptors for PET/MR imaging of prostate cancer.

Prostate cancer (PCa) is the most common malignancy worldwide in men. This is a proof-of-concept study describing the development of 68Ga-magnetic iron oxide nanoparticles (mNP) targeting prostate specific membrane antigen (PSMA) and gastrin releasing peptide (GRPR) receptors as potential tools for diagnosis of PCa with PET/MRI. Two pharmacophores targeting PSMA, 1, and GRPR, 2, were coupled to mNPs carrying -SH (mNP-S1/2) or -NH2 (mNP-N1/2) groups. The mNP-S1/2 and mNP-N1/2 were characterized for their size, zeta potential, structure, and efficiency of functionalization using dynamic light scattering (DLS), FT-IR and RP-HPLC. A direct 68Ga-labelling procedure was followed, where 68Ga-mNP-N1/2 proved superior to 68Ga-mNP-S1/2 regarding radiolabelling efficiency, and thus were further evaluated in vitro. Toxicity studies in PCa cells (LNCaP, PC-3) showed low toxicity, and minimal hemolysis of red blood cells. In vitro assays in cells expressing PSMA (LNCaP), and GRPR (PC-3), showed specific time-dependent binding (40 min to plateau), high avidity (PC-3: Kd = 28.27 nM, LNCaP: Kd = 11.49 nM) and high internalization rates for 68Ga-mNP-N1/2 in both cell lines.