Isomer-sourced structure iteration methods for in silico development of inhibitors: Inducing GTP-bound NRAS-Q61 oncogenic mutations to an "off-like" state.

The NRAS-mutant subset of melanoma represent some of the most aggressive and deadliest types associated with poor overall survival. Unfortunately, for more than 40 years, no therapeutic agent directly targeting NRAS mutations has been clinically approved. In this work, based on microsecond scale molecular dynamics simulations, the effect of Q61 mutations on NRAS conformational characteristics is revealed at the atomic level. The GTP-bound NRAS-Q61R and Q61K mutations show a specific targetable pocket between Switch-II and α-helix 3 whereas the NRAS-Q61L non-polar mutation category shows a different targetable pocket. Moreover, a new isomer-sourced structure iteration method has been developed for the in silico design of potential inhibitor prototypes for oncogenes. We show the possibility of a designed prototype HM-387 to target activated NRAS-Q61R and that it can gradually induce the transition from the activated NRAS-Q61R to an "off-like" state.

In silico design of a lipid-like compound targeting KRAS4B-G12D through non-covalent bonds.

One of the most common drivers in human cancer is the peripheral membrane protein KRAS4B, able to promote oncogenic signalling. To signal, oncogenic KRAS4B not only requires a sufficient nucleotide exchange, but also needs to recruit effectors by exposing its effector-binding sites while anchoring to the phospholipid bilayer where KRAS4B-mediated signalling events occur. The enzyme phosphodiesterase-δ plays an important role in sequestering KRAS4B from the cytoplasm and targeting it to cellular membranes of different cell species. In this work, we present an in silico design of a lipid-like compound that has the remarkable feature of being able to target both an oncogenic KRAS4B-G12D mutant and the phosphodiesterase-δ enzyme. This double action is accomplished by adding a lipid tail (analogous to the farnesyl group of the KRAS4B protein) to an previously known active compound (2H-1,2,4-benzothiadiazine, 3,4-dihydro-,1,1-dioxide). The proposed lipid-like molecule was found to lock KRAS4B-G12D in its GDP-bound state by adjusting the effector-binding domain to be blocked by the interface of the lipid bilayer. Meanwhile, it can tune GTP-bound KRAS4B-G12D to shift from the active orientation state to the inactive state. The proposed compound is also observed to stably accommodate itself in the prenyl-binding pocket of phosphodiesterase-δ, which impairs KRAS4B enrichment at the lipid bilayer, potentially reducing the proliferation of KRAS4B inside the cytoplasm and its anchoring at the bilayer. In conclusion, we report a potential inhibitor of KRAS4B-G12D with a lipid tail attached to a specific warhead, a compound which has not yet been considered for drugs targeting RAS mutants. Our work provides new ways to target KRAS4B-G12D and can also foster drug discovery efforts for the targeting of oncogenes of the RAS family and beyond.

Cobalt-Catalyzed Effective Access to Quinoxalines with Insights in Annulation of Terminal Alkynes and o-Phenylenediamines.

A novel methodology for the annulation of terminal alkynes and o-phenylenediamines by using a combination of a cobalt catalyst and oxygen as a terminal oxidant is reported. This method shows wide substrate scope and good functional group tolerance and provides a wide range of quinoxalines in good to high yields. The method is demonstrated by its gram-scale and broad potential applications. Furthermore, this protocol serves as a powerful tool for the late-stage functionalization of various complex bioactive molecules and drugs to provide a new class of molecules containing two distinct bioactive molecules directly linked. Detailed mechanistic studies reveal that the current reaction goes through a novel mechanism different from the previously reported glyoxal mechanism.

Discovering and Targeting Dynamic Drugging Pockets of Oncogenic Proteins: The Role of Magnesium in Conformational Changes of the G12D Mutated Kirsten Rat Sarcoma-Guanosine Diphosphate Complex.

KRAS-G12D mutations are the one of most frequent oncogenic drivers in human cancers. Unfortunately, no therapeutic agent directly targeting KRAS-G12D has been clinically approved yet, with such mutated species remaining undrugged. Notably, cofactor Mg2+ is closely related to the function of small GTPases, but no investigation has been conducted yet on Mg2+ when associated with KRAS. Herein, through microsecond scale molecular dynamics simulations, we found that Mg2+ plays a crucial role in the conformational changes of the KRAS-GDP complex. We located two brand new druggable dynamic pockets exclusive to KRAS-G12D. Using the structural characteristics of these two dynamic pockets, we designed in silico the inhibitor DBD15-21-22, which can specifically and tightly target the KRAS-G12D-GDP-Mg2+ ternary complex. Overall, we provide two brand new druggable pockets located on KRAS-G12D and suitable strategies for its inhibition.

In Silico Drug Design of Benzothiadiazine Derivatives Interacting with Phospholipid Cell Membranes.

The use of drugs derived from benzothiadiazine, a bicyclic heterocyclic benzene derivative, has become a widespread treatment for diseases such as hypertension, low blood sugar or the human immunodeficiency virus, among others. In this work we have investigated the interactions of benzothiadiazine and four of its derivatives designed in silico with model zwitterionic cell membranes formed by dioleoylphosphatidylcholine, 1,2-dioleoyl-sn-glycero-3-phosphoserine and cholesterol at the liquid-crystal phase inside aqueous potassium chloride solution. We have elucidated the local structure of benzothiadiazine by means of microsecond molecular dynamics simulations of systems including a benzothiadiazine molecule or one of its derivatives. Such derivatives were obtained by the substitution of a single hydrogen site of benzothiadiazine by two different classes of chemical groups, one of them electron-donating groups (methyl and ethyl) and another one by electron-accepting groups (fluorine and trifluoromethyl). Our data have revealed that benzothiadiazine derivatives have a strong affinity to stay at the cell membrane interface although their solvation characteristics can vary significantly-they can be fully solvated by water in short periods of time or continuously attached to specific lipid sites during intervals of 10-70 ns. Furthermore, benzothiadiazines are able to bind lipids and cholesterol chains by means of single and double hydrogen-bonds of characteristic lengths between 1.6 and 2.1 Å.

Structure of benzothiadiazine at zwitterionic phospholipid cell membranes.

The use of drugs derived from benzothiadiazine, which is a bicyclic heterocyclic benzene derivative, has become a widespread treatment for diseases such as hypertension (treated with diuretics such as bendroflumethiazide or chlorothiazide), low blood sugar (treated with non-diuretic diazoxide), or the human immunodeficiency virus, among others. In this work, we have investigated the interactions of benzothiadiazine with the basic components of cell membranes and solvents, such as phospholipids, cholesterol, ions, and water. The analysis of the mutual microscopic interactions is of central importance to elucidate the local structure of benzothiadiazine as well as the mechanisms responsible for the access of benzothiadiazine to the interior of the cell. We have performed molecular dynamics simulations of benzothiadiazine embedded in three different model zwitterionic bilayer membranes made by dimyristoylphosphatidylcholine, dioleoylphosphatidylcholine, 1,2-dioleoyl-sn-glycero-3-phosphoserine, and cholesterol inside aqueous sodium-chloride solution in order to systematically examine microscopic interactions of benzothiadiazine with the cell membrane at liquid-crystalline phase conditions. From data obtained through radial distribution functions, hydrogen-bonding lengths, and potentials of mean force based on reversible work calculations, we have observed that benzothiadiazine has a strong affinity to stay at the cell membrane interface although it can be fully solvated by water in short periods of time. Furthermore, benzothiadiazine is able to bind lipids and cholesterol chains by means of single and double hydrogen-bonds of different characteristic lengths.

Pd-Catalyzed Intramolecular Chemoselective C(sp2)-H and C(sp3)-H Activation of N-Alkyl- N-arylanthranilic Acids.

A controllable palladium-catalyzed intramolecular C-H activation of N-alkyl- N-arylanthranilic acids has been developed. The methodology allows selective synthesis of 1,2-dihydro-(4 H)-3,1-benzoxazin-4-ones and carbazoles from the same starting materials and palladium catalyst. The selectivity is controlled by the oxidant. Silver oxide promotes C(sp3)-H activation/C-O cyclization to provide 1,2-dihydro-(4 H)-3,1-benzoxazin-4-ones, while copper acetate contributes to C(sp2)-H activation/decarboxylative arylation to afford carbazoles. This protocol is demonstrated by its wide substrate scope and good functional group tolerance.