We have examined the backbone dynamics of two alternating purine-pyrimidine dodecamers. One sequence consists of "pure" GC bases; the other one contains 5-methylcytosines. The effect of the methyl groups on the backbone substates BI/BII was investigated by means of molecular dynamics. The methylation influences, on one hand, the transition barrier between BI and BII and, on the other hand, the state of equilibrium. The kinetic consequences are an increase of the DeltaG of Gp5mC steps by 1.5 kcal/mol and a decrease of the DeltaG of 5mCpG steps by 0.8 kcal/mol (compared with the nonmethylated DNA). Thus, the additive group differentiates between the two occurring dinucleotide steps and renders the phosphate of the 5-methylcytosine more rigid, as proposed by experimental studies. The thermodynamic consequences are an increase of the DeltaG of Gp5mC steps by 1.1 kcal/mol and a decrease of the DeltaG of 5mCpG steps by 0.8 kcal/mol. The reason for this shift in equilibrium is still not completely clear on a molecular basis. But we can conclude that the indirect readout of DNA is influenced by methylation.

Since the first approval of a biosimilar medicinal product in 2006, scientific understanding of the features and development of biosimilar medicines has accumulated. This review scrutinizes public information on development programs and the contribution of the clinical studies for biosimilar approval in the European Union (EU) and/or the United States (US) until November 2019. The retrospective evaluation of the programs that eventually obtained marketing authorization and/or licensure revealed that in 95% (36 out of 38) of all programs, the comparative clinical efficacy studies confirmed similarity. In the remaining 5% (2 out of 38), despite meeting efficacy outcomes, the biosimilar candidates exhibited clinical differences in immunogenicity that required changes to the manufacturing process and additional clinical studies to enable biosimilar approval. Both instances of clinical differences in immunogenicity occurred prior to 2010, and the recurrence of these cases is unlikely today due to state-of-the-art assays and improved control of process-related impurities. Biosimilar candidates that were neither approved in the EU nor in the US were not approved due to reasons other than clinical confirmation of efficacy. This review of the development history of biosimilars allows the proposal of a more efficient and expedited biosimilar development without the routine need for comparative clinical efficacy and/or pharmacodynamic studies and without any compromise in quality, safety, or efficacy. This proposal is scientifically valid, consistent with regulation of all biologics, and maintains robust regulatory standards in the assessment of biosimilar candidates. Note: The findings and conclusion of this paper are limited to biosimilar products developed against the regulatory standards in the EU and the US.

Daunomycin is one of the most important agents used in anticancer chemotherapy. It interacts with DNA through intercalation of its planar chromophore between successive base pairs. The effect of intercalation on structure, dynamics and energetics is the topic of a wealth of scientific studies. In the present study, we report a computational examination of the energetics of the intercalation process. In detail, we concentrate on the energetic penalty that intercalation of daunomycin introduces into DNA by disturbing it from its unbound conformation. For these means, we are analyzing already published molecular dynamics simulations of daunomycin-DNA complexes and present novel simulations of a bisdaunomycin-DNA and a 9-dehydroxydaunomycin-DNA intercalated complex using the MM-GBSA module implemented in the AMBER suite of programs. Using this molecular dynamics based, continuum solvent method we were able to calculate the energy required to form an intercalation site. Consequently, we compare the free energy of the duplex d(CGCGCGATCGCGCG)(2) in the B-form conformation with the respective conformations when intercalated with daunomycin and a bisintercalating analog. Our results show that the introduction of one single intercalation site costs approximately 32 kcal/mol. For double intercalation, or intercalation of the bisintercalator, the respective value for one intercalation site decreases to 27 and 24 kcal/mol, respectively, at a theoretical salt concentration of 0.15 M. This proposes that at least in these cases, a synergistic effect takes place. Although it is well known that intercalation leads to substantial disturbance of the DNA conformation, already performed investigations suggest a lower energetic penalty. Nevertheless to the best of our knowledge the calculations presented here are the most complete ones and consider hydration effects for the first time. The interaction energy between the ligand and the DNA certainly over-compensates this penalty for introducing the intercalation site and thus favors complexation. Such analyses are helpful for the description of allosteric effects in protein ligand binding.

In the present study, we try to determine if the dynamics of the B-DNA backbone phosphates (and especially their interconversions between their two distinct conformations B I and B II ) are fast enough to be sufficiently sampled in the course of molecular dynamics simulations in the nanosecond time range. For this purpose, we performed twelve 10-ns simulations of the Drew-Dickerson dodecamer d(CGCGAATTCGCG) 2 to investigate the dynamics of B I /B II interconversion. We forced the DNA backbone angles and with restraints to values that are characteristic for B I and B II , resulting in DNA double helices with all phosphates in the B I or B II substate. These restraints were removed after 10 ns, and unrestrained simulations at temperatures of 250, 275, 287.5, 300, and 325 K were performed for another 10 ns, which allowed us to analyze the dynamics of relaxation in detail. These simulations were compared to simulations of the undisturbed dodecamer at 250 and 300 K, as a reference for the equilibrated state. We found that the relaxation from the B II state is considerably fast, with high rate constants, and is dependent on temperature. From this temperature dependence of the rate constants, we calculated the activation energy necessary for the B II to B I transition to be 2.5 kcal/mol. Half-life times of the B II state derived from the relaxation process are in the range of 110-370 ps, which indicates that a simulation time of 10 ns is sufficiently long to investigate conformational transitions of the DNA backbone. The structures of the all-B I DNA are more similar to structures found for the DrewDickerson dodecamer by X-ray crystallography than the all-B II DNA. This fact is not astonishing, because the B I conformation has been observed to be privileged. Nevertheless, both structures are quite different from canonical A-or B-DNA. That observation is revealing, because we expected the all-B II DNA to be the transition state to canonical A-DNA or at least structurally very similar. Furthermore, we find that the relaxation of our rather-distorted starting structures is fast and, despite the large difference at the beginning, leads to a similar equilibrium, which, again, is similar to the undisturbed simulation.

Ligands which are able to recognize DNA sequence specifically are of fundamental interest as transcription controlling drugs. Recently a polyamide ligand was developed (ImHpPyPy-beta-Dp) which differentiates in a dimeric arrangement between all four possible base pair steps in the minor groove. This is a landmark for the design of DNA binding drugs because it was believed that such a recognition could only be possible in the major groove of DNA. Although the OH groups of the hydroxypyrrole (Hp) moieties of the ligands are responsible for this sequence discrimination, experiments showed that this OH group also reduces the absolute binding constant. We performed a free energy calculation by means of thermodynamic integration in order to find out the influence of this single hydroxyl on DNA binding. In our simulation, we found that the hydroxyl group reduces binding by about 1.3 kcal/mol, which is in excellent agreement with the experimentally determined value of 1.2 kcal/mol. In further MD simulations, the structural reasons for this reduction was estimated. The results of these simulations qualitatively agree with the X-ray structures, but in contrast, in the simulations both (ImHpPyPy-beta-Dp and ImPyPyPy-beta-Dp) ligand-DNA (d(CCAGTACTGG)(2)) complexes exhibit only slight structural differences. This is consistent with a recently published second pair of similar polyamide DNA crystal structures. Thus, we believe that the explanations resulting from the X-ray structures must be modified. We attribute the large structural differences between the two polyamide DNA complexes to a buffer molecule which binds only in the case of the ImHpPyPy-beta-Dp-DNA complex at the region of interest. We propose that the differential hydration of both ligands in the unbound state is responsible for the reduction of the binding constant. Additionally, we suggest an indirect readout of DNA, because of a lengthening of the Watson-Crick base pairs, which possibly contributes to the differentiation between T.A, A.T from G.C, C.G base pairs.