Non-typeable Haemophilus influenzae (NTHi) is an opportunistic pathogen that causes several respiratory diseases. It employs the Sap (sensitivity to antimicrobial peptide) transporter to achieve resistance against the human antimicrobial peptides (hAMPs). In H. influenzae, the Sap system comprises six subunits, SapABCDFZ, akin to the canonical ATP-binding cassette (ABC) importers. The subunit HiSapA, which plays a role in the substrate binding, has been reported to capture hAMPs such as LL-37 and β-defensins. However, the substrate types and their binding mechanism(s) by HiSapA have not been fully understood. In this study, attempts were made to fill such gaps using in silico approaches. The results of this study suggest that five hAMPs, hBD-2, hBD-3, hNP-1, hNP-4, and hLL-37 can bind to HiSapA with a binding energy ranging from -24.03 to -78.55 kcal mol-1. Further, specific sequence motifs, RRYKQ of hBD-2, PKEEQ of hBD-3, RRYGT of hNP-1, RLVFCR of hNP-4, and LGDFFR of hLL-37, present in the hAMPs, were identified as crucial for HiSapA interaction. In addition, an analysis of the conformational changes and variations in the volumes of the binding-site pocket suggested that there is an increase (from 823 Å3 to 2095 Å3) upon hAMPs binding to HiSapA, indicating its binding mechanism is similar to the previously proposed mechanism, viz. "Venus Flytrap", known for SBPs of ABC importers. In summary, the findings of this study can be utilized for structure-based drug development.

Earlier we have reported two novel dual-target anti-leishmanial drug candidates, ZINC000008876351 and ZINC000253403245. These compounds were designed to target two crucial enzymes in the antioxidant defense system of Leishmania donovani: iron superoxide dismutase (FeSODA) and trypanothione reductase (TryR). The aim was to optimize these dual-target agents within liposomal formulations for the treatment of visceral leishmaniasis. Liposomal formulations were thoroughly characterized to confirm successful drug encapsulation. In vitro assays demonstrated that the liposome-encapsulated drugs, ZINC000253403245-liposome and ZINC000008876351-liposome, exhibited enhanced antiparasitic activity compared to their free drug counterparts. Both formulations showed significantly lower IC50 values, indicating stronger inhibition of L. donovani growth in both promastigote and amastigote stages at low micromolar concentrations. Mechanistic studies revealed that both free and encapsulated forms of the drugs induced apoptosis-like cell death in L. donovani promastigotes, as evidenced by mitochondrial membrane depolarization and phosphatidylserine externalization, assessed via flow cytometry. These results suggest that liposomal encapsulation enhances therapeutic efficacy by inducing mitochondrial dysfunction and promoting parasite death. The promising results of the ZINC000253403245-liposome and ZINC000008876351-liposome formulations highlight their potential as new, effective treatments for visceral leishmaniasis, offering a promising alternative to current therapies and representing a significant advancement in drug development for this neglected tropical disease.

Leishmaniasis, caused by the genus Leishmania remains a significant global health challenge. Current chemotherapeutics have limitations in terms of drug resistance, toxicity, and limited efficacy. As most of the currently used chemotherapeutics are single-target drugs, the chances of drug resistance development are very high. To overcome this challenge, the polypharmacology approach to designing multiple molecular target drugs is becoming a promising approach against leishmaniasis. Parasite Leishmania has a unique redox defense system to maintain cellular oxidative stress that helps it survive inside the host oxidative environment generated by the host immune response. This evolutionary divergence of redox systems of Leishmania from the human host is ideal for therapeutic interventions. This review focuses on the currently used anti-leishmanial therapeutics limitations and considers antioxidant defense system as a potential drug target pathway. By focusing on these phylogenetically distinct and parasite-specific enzymes, polypharmacological drugs can be developed to inhibit multiple molecular targets, simultaneously increasing efficacy and reducing the potential for resistance while minimizing adverse effects on the human host. This paradigm, which moves from single target to multi-target shift, holds promise for more effective treatments.

MlaC is involved in the transportation of phospholipids between the inner and outer membranes of Gram-negative bacteria. This MlaC-mediated transport safeguards the outer membrane asymmetry thereby preserving its integrity and shielding effect against antibiotics, detergents, etc. MlaC is constituted by two domains, viz. nuclear transport factor 2-like and phospholipid-binding protein. These unique structural properties contribute to a novel ligand binding process and a diverse conformational landscape, which still remains a marginally investigated subject. In order to fill this knowledge gap, comprehensive molecular docking and simulation studies were performed. The docking experiments performed using these structures against different phospholipids reveal, for the first time, their preference for certain substrate sizes and organizations of binding planes. The conformational dynamicity of MlaC was studied by simulation using 13 different systems in different liganded states for 1000 ns each. A distinct behavioural pattern was observed between the apo open and holo open states, with the former being conformationally more flexible. A time-dependent study of the changes in binding-pocket volume further substantiated the differences in the protein dynamics. The study further aided in the identification of global and local movements, paving the path for the investigation of coordinated motions. The extensive analyses performed on different liganded systems disclose (anti-)correlated motions that have helped in the understanding of the motions and enigmatic conformational landscape of MlaC. Further, an unanticipated MlaC crystal state that further adds to the understanding of MlaC flexibility.

Illicit transport pertains to the unauthorized entry of molecules into cells through transporters that are initially intended for other physiological substances. Recently, it has been demonstrated that the peptide-based antibiotic negamycin can permeate the cytosolic membrane of Escherichia coli via dipeptide (EcDpp), sensitivity to antimicrobial peptide (EcSap), and oligopeptide (EcOpp) transporters. However, no example of such an illicit transport mechanism for the Haemophilus influenzae Sap (HiSap) transporter has been reported. So, an in-depth in silico study was performed to identify new peptide-based antibiotics showing binding affinities for the substrate-binding proteins EcDppA, EcSapA, and HiSapA. The results indicated that the three target proteins share sequence and structural similarities among them. Moreover, a virtual screening of 230 peptide-based antibiotics against these proteins identified eight compounds with higher binding affinities. Among these, three compounds (1, 6, and 129) demonstrate superior absorption, distribution, metabolism, excretion, and toxicity (ADMET) profiles as well as drug-like characteristics. Comprehensive molecular dynamics (MD) simulation hints towards the conformational stability and favourable binding energy of these compounds with EcDppA, EcSapA, and HiSapA. The probability density function (PDF) and dynamic cross-correlation map (DCCM) underscored the significance of the binding-site loop in ligand dynamics and major domain movements, respectively. In conclusion, the results from this study propose that Compounds 1, 6, and 129 could function as effective broad-spectrum antibiotics against Gram-negative pathogens and can also act as a template for designing more such peptide-based antibiotics for illicit transport across various other pathogens.

Nucleolar essential protein 1 (Nep1; also known as ribosomal RNA small subunit methyltransferase Nep1) is a crucial factor in forming small ribosomal subunits in eukaryotes and archaea. Nep1 possesses an S‐adenosyl‐L‐methionine (SAM)‐dependent SpoU‐TrmD (SPOUT) ribosomal RNA (rRNA) methyltransferase (MTase) fold and catalyzes pseudouridine (Ψ) methylation at specific sites of the small subunit (SSU) rRNA. Mutations in Nep1 proteins result in a severe developmental disorder in humans and reduced growth in yeast, suggesting its role in ribosome biogenesis. In this study, the crystal structures of Nep1 from the archaebacterium Pyrococcus horikoshii ( Ph Nep1), both in its apo and holo (adenosine or 5‐methylthioadenosine bound) forms have been reported. The structural analysis of Ph Nep1 revealed an α/β fold featuring a deep trefoil knot akin to the SPOUT domain, with two novel extensions—a globular loop and a β–α–β extension. Moreover, the cofactor‐binding site of Ph Nep1 exhibits a preformed pocket, topologically similar to that of other SPOUT‐class MTases. Further, structural analysis of Ph Nep1 revealed that it forms a homodimer coordinated by inter‐subunit hydrogen bonds and hydrophobic interactions. Moreover, the results of this study indicate that Ph Nep1 can specifically methylate consensus RNAs, having a pseudouridine (ψ) located at position 926 of helix 35 (h35) of 16S rRNA in P. horikoshii . The stability of the Nep1–RNA complex seems to be primarily assisted by the conserved arginine residues located at the dimeric interface.

The mammalian cell entry (Mce) proteins are critical for the entry and persistence of Mycobacterium tuberculosis (Mtb) within alveolar macrophages. These proteins assemble into an ATP-binding cassette transporter comprising six substrate-binding proteins (MceA-F), two transmembrane domains (YrbEAB), and two nucleotide-binding domains (MceG2). Although Mtb harbours four distinct mce operons, the regulatory mechanisms governing differential operon expression remain insufficiently understood. In this study, we analyzed the genetic organization, regulatory features, and functional roles of operons encoding Mce proteins across two bacterial families, utilizing a comparative sequence-based approach. Our results reveal a conserved operonic arrangement, and the gene neighbourhood trends further establish the involvement of Mce proteins in cholesterol uptake. Notably, the presence of transcriptional regulators, translational coupling mechanisms, conserved gene clusters encoding translation machinery and accessory proteins suggests a complex, multi-layered regulatory network controlling mce operon expression. Thus, this study offers key computational insights into the complex regulatory framework of the Mce system while also outlining potential experimental approaches to validate the proposed hypotheses.

The non-typeable Haemophilus influenzae Sap (sensitivity to antimicrobial peptide) transporter, a member of the ATP-binding cassette (ABC) superfamily, is implicated in the uptake of host-produced antimicrobial peptides (hAMPs) and heme. It comprises a substrate-binding protein (HiSapA), transmembrane domains (HiSapBC), nucleotide-binding domains (HiSapDF), and an accessory protein (HiSapZ). The structural and mechanistic studies of the HiSap transporter are underexplored. In this study, a comprehensive in silico structural analysis using molecular dynamics simulations at both atomistic and coarse-grained levels was performed for a total of ~283 μs to elucidate the conformational dynamics and transport mechanism of the HiSap transporter. The results suggest that HiSapA can govern the complex dynamics through an alternate-access mechanism. Further, stable interactions between HiSapA and its potential ligands, heme and human β-defensin 3 (hBD-3), suggest its direct role in substrate recruitment. In addition, the membrane lipids (POPE, POPG, and cardiolipin) were found to be involved in modulating the conformational dynamics of the HiSap transporter. The hypothetical accessory protein HiSapZ was found to be a stable component of the HiSap transporter complex. Interestingly, only one copy of the HiSapZ, localized in the proximity to HiSapC, was observed to adopt a rigid conformation, revealing its dual role in structural integrity and ion transport. Overall, the findings of this study provide the first-hand structural details, dynamics, and mechanism of the HiSap transporter. Moreover, the study lays a strong groundwork for future structural investigations on other ABC importers across various other Gram-negative pathogens, potentially facilitating the rational structure-based drug design.

In Gram-negative bacteria, the non-canonical ABC transporter, namely, maintenance of lipid asymmetry (Mla) system, ferries phospholipids (PLs) between the inner (IM) and outer (OM) membranes to preserve the PL asymmetry of the OM. The system utilizes three sub-cellular complexes-lipoprotein MlaA-OmpC/F (OM), MlaC (periplasmic), and MlaFEDB complex (IM). The structural studies on the Mla system have primarily been dedicated to its organization in IM and transport mechanisms. The characteristics of the individual components of the Mla system are lacking in the literature. In this study, individual components, namely MlaA, MlaB, MlaE, and MlaF were analyzed using computational tools. This has resulted in the identification of unique features and their characterization, including understanding the dynamicity of the C-terminal extension (CTE) of MlaA, which protrudes into the periplasm and the orientation of the protein, as well as binding patterns. Utilization of artificial intelligence has led to the understanding of the conformational landscape of MlaA and the validation of the macromolecular arrangement of Mla systems. Based on the results obtained, we were able to propose a fascinating mechanism of ligand transport, namely, bait-capture-pull. Our results reveal the poorly understood interfaces of the MlaB-MlaF complex. Furthermore, the results also suggest that MlaE possesses an EQ loop, which helps maintain a unique orientation. Overall, the findings of this study provide a new perspective on non-vesicular PL transport mediated by the enigmatic Mla system, thereby providing a holistic understanding.

The maintenance of the lipid asymmetry (Mla) system plays a critical role in facilitating the transport of phospholipids between the inner and outer membranes of the Gram-negative bacteria. In E. coli, the system consists of six proteins: MlaA-OmpF/C complex (outer membrane), MlaC (periplasm), and MlaFEDB complex (inner membrane). Despite extensive research on the core proteins (MlaFED) of the Mla system, the occurrence of Mla components like MlaA, MlaB, and MlaC in diderm remains uncertain. Therefore, this gap presents a significant opportunity for further investigation, particularly regarding MlaC, which serves as the sole mobile component of the Mla system. This has led to the identification of multiple copies of MlaC in 63 distinct genera of Proteobacteria and related phyla. Interestingly, amongst these genera, the genetic arrangements of the mla operon were observed to be varying and, thus, were further categorized into four distinct groups. The variations among the genetic organization of the mla operons suggest their evolution through various processes, such as duplications, losses, rearrangements, and fusions. Further, the results of this study highlight the MlaC's substrate promiscuity, illuminating new avenues for the Mla system.

The Sap transport system belongs to an ATP-binding cassette importer, which is reported to render resistance against host-produced antimicrobial peptides (AMPs) amongst various Gram-negative bacteria. The Sap system imports the AMPs across the membrane into the cytoplasm, wherein they are cleaved by the proteases. The Sap system comprises five components: a substrate-binding protein (SBP, SapA), two transmembrane domains (TMDs, SapBC), and two nucleotide-binding domains (NBDs, SapDF). Interestingly, the membrane components (SapBCDF) of the Escherichia coli Sap (EcSap) system were suggested to function as a putrescine exporter. On the contrary, recent in silico reports suggested its multifaceted attributes in the uptake of dipeptides, AMPs, and heme. To establish the multifarious nature, extensive molecular dynamics simulations of EcSapA in its apo and holo (bound to dipeptides, AMPs, and heme) forms were performed to gain structural insights into its molecular plasticity. The results of this study suggest that EcSapA possesses a wide and promiscuous binding site that is favorable for accommodating varying lengths of ligands with a ligand-dependent conformational dynamics mechanism. Further, the estimated binding energies of the ligands suggest that EcSapA shows a preferential binding for cationic AMPs, followed by heme and dipeptides. In summary, the study highlights the ligand-binding dynamics within the promiscuous binding site of EcSapA, enlightening a lucrative target for drug development.

Antimicrobial peptides (AMPs) are majorly utilized by the hosts to clear off the invading bacterial pathogens. The AMPs help in the clearance of bacterial pathogens primarily by disrupting their membrane homeostasis. However, most Gram-negative pathogens have developed multiple machineries, enabling them to resist the action of AMPs. One such machinery is the sensitivity to the antimicrobial peptides (Sap) transport system. The Sap system belongs to the ATP-binding cassette (ABC) transporters and consists of five components, viz. SapABCDF. It is reported that it uptakes AMPs inside the cell that are proteolytically degraded by proteases. In contrast, in Escherichia coli, the Sap (EcSap) transport system was suggested as a putrescine exporter. In this study, with the aid of computational biological approaches, the functional prospects of the EcSap transporter were investigated. The results of this study suggest that the protein EcSapA can bind dipeptides having aromatic amino acids. Further, it can bind to oligopeptides, including AMPs. AMPs such as protamine and protegrin-1 show binding to the protein EcSapA. In addition, the molecule heme shows binding affinity toward the protein EcSapA. In summary, EcSapA seems to be involved in the uptake of a wide range of molecules, such as dipeptides, AMPs and heme. The results of this study can be utilized to design inhibitors targeting the protein SapA, as inhibiting this protein may render the bacterial system sensitive to the attacking AMPs, hence allowing the host machinery to clear off the invading pathogen.

Antimicrobial peptides (AMPs) disrupt the integrity of the bacterial membrane, ultimately leading to their death. In counter-defense, pathogens are reported to have developed systems such as the sensitivity to antimicrobial peptides (Sap) transport system that evade the action of AMPs and sequester essential micronutrients. However, recent contrasting reports cloud the functional prospects of the Sap system. Hence, this study aimed to characterize the Escherichia coli Sap (EcSap) transport system using biophysical techniques. The results obtained from various approaches suggested the binding of heme to the substrate-binding component (EcSapA) of the EcSap system. Further, this study suggests the interaction of EcSapA with the AMP protamine. In summary, the findings of this study suggest the dual ligand-binding ability of EcSapA. Impact statement The present study reports the functional prospects of the enigmatic substrate-binding protein SapA of E. coli. This analysis highlights the essentiality of the intra-protein disulfide bonds in maintaining the structural integrity of EcSapA. Further, biophysical studies of EcSapA highlight its dual ligand binding propensity, earmarking it as a drug target.

5-Methyluridine (m5U) rRNA modifications frequently occur at U747 and U1939 (Escherichia coli numbering) in domains II and IV of the 23S rRNA in Gram-negative bacteria, with the help of S-adenosyl-l-methionine (SAM)-dependent rRNA methyltransferases (MTases), RlmC and RlmD, respectively. In contrast, Gram-positive bacteria utilize a single SAM-dependent rRNA MTase, RlmCD, to modify both corresponding sites. Notably, certain archaea, specifically within the Thermococcales group, have been found to possess two genes encoding SAM-dependent archaeal (tRNA and rRNA) m5U (Arm5U) MTases. Among these, a tRNA-specific Arm5U MTase (PabTrmU54) has already been characterized. This study focused on the structural and functional characterization of the rRNA-specific Arm5U MTase from the hyperthermophilic archaeon Pyrococcus horikoshii (PhRlmCD). An in-depth structural examination revealed a dynamic hinge movement induced by the replacement of the iron-sulfur cluster with disulfide bonds, obstructing the substrate-binding site. It revealed distinctive characteristics of PhRlmCD, including elongated positively charged loops in the central domain and rotational variations in the TRAM domain, which influence substrate selectivity. Additionally, the results suggested that two potential mini-rRNA fragments interact in a similar manner with PhRlmCD at a positively charged cleft at the interface of domains and facilitate dual MTase activities akin to the protein RlmCD. Altogether, these observations showed that Arm5U MTases originated from horizontal gene transfer events, most likely from Gram-positive bacteria.

5-Methylcytosine methyltransferases (m(5)C MTases) are known to be involved in the modification of RNA. Although these enzymes have been relatively well characterized in bacteria and eukarya, a complete understanding of the archaeal counterparts is lacking. In this study, the identification and characterization of archaeal RNA m(5)C MTases were performed. As a case study, a hyperthermophilic archaeon, Pyrococcus horikoshii OT3, which possesses five putative RNA m(5)C MTases, was chosen. Among the five putative RNA m(5)C MTases, two proteins (PH0851 and PH1991) have been characterized as homologs of a bacterial rRNA MTase (RsmB) and eukaryal tRNA MTase (NSUN6), respectively. The in-depth characterization of the remaining three putative RNA m(5)C MTases (PH1078, PH1374, and PH1537) in this study suggests the presence of the signature architecture and catalytic residues plausibly involved in the binding of their cognate RNA substrates. Additionally, the results also suggest the existence of two RsmB-like proteins (PH0851 and PH1078) belonging to the same subfamily IV of m(5)C RNA MTase. However, the proteins PH1374 and PH1537 belong to the same subfamily V but bind to different substrates, rRNA and tRNA, respectively. The findings further indicate that archaeal RNA m(5)C MTases link those from bacteria and eukarya.

The dual-target inhibitors (ZINC000008876351 and ZINC000253403245) were identified by utilizing an advanced computational drug discovery method by targeting two critical enzymes such as FeSODA (Iron superoxide dismutase) and TryR (Trypanothione reductase) within the antioxidant defense system of Leishmania donovani (Ld). In vitro enzyme inhibition kinetics reveals that both the compound's ability to inhibit the function of enzyme LdFeSODA and LdTryR with inhibition constant (Ki) value in the low μM range. Flow cytometry analysis, specifically at IC50 and 2X IC50 doses of both the compounds, the intracellular ROS was significantly increased as compared to the untreated control. The compounds ZINC000253403245 and ZINC000008876351 exhibited strong anti-leishmanial activity in a dose-dependent manner against both the promastigote and amastigote stages of the parasite. The data indicate that these molecules hold promise as potential anti-leishmanial agents for developing new treatments against visceral leishmaniasis, specifically targeting the LdFeSODA and LdTryR enzymes. Additionally, the in vitro MTT assay shows that combining these compounds with miltefosine produces a synergistic effect compared to miltefosine alone. This suggests that the compounds can boost miltefosine's effectiveness by synergistically inhibiting the growth of L. donovani promastigotes. Given the emergence of miltefosine resistance in some Leishmania strains, these findings are particularly significant.

Dimethyladenosine transferase 1 (DIMT1), an ortholog of bacterial KsgA is a conserved protein that assists in ribosome biogenesis by modifying two successive adenosine bases near the 3' end of small subunit (SSU) rRNA. Although KsgA/DIMT1 proteins have been characterized in bacteria and eukaryotes, they are yet unexplored in archaea. Also, their dynamics are not well understood. Here, we structurally and functionally characterized the apo and holo forms of archaeal DIMT1 from Pyrococcus horikoshii. Wild-type protein and mutants were analyzed to capture different transition states, including open, closed, and intermediate states. This study reports a unique inter-domain movement that is needed for substrate (RNA) positioning in the catalytic pocket, and is only observed in the presence of the cognate cofactors S-adenosyl-L-methionine (SAM) or S-adenosyl-L-homocysteine (SAH). The binding of the inhibitor sinefungine, an analog of SAM or SAH, to archaeal DIMT1 blocks the catalytic pocket and renders the enzyme inactive.

The membrane-associated solute-binding protein (SBP) MlaD of the maintenance of lipid asymmetry (Mla) system has been reported to help the transport of phospholipids (PLs) between the outer and inner membranes of Gram-negative bacteria. Despite the availability of structural information, the molecular mechanism underlying the transport of PLs and the ancestry of the protein MlaD remain unclear. In this study, we report the crystal structures of the periplasmic region of MlaD from Escherichia coli ( Ec MlaD) at a resolution range of 2.3–3.2 Å. The Ec MlaD protomer consists of two distinct regions, viz. N-terminal β-barrel fold consisting of seven strands (referred to as MlaD domain) and C-terminal α-helical domain (HD). The protein Ec MlaD oligomerizes to give rise to a homo-hexameric ring with a central channel that is hydrophobic and continuous with a variable diameter. Interestingly, the structural analysis revealed that the HD, instead of the MlaD domain, plays a critical role in determining the oligomeric state of the protein. Based on the analysis of available structural information, we propose a working mechanism of PL transport, viz. “asymmetric protomer movement (APM)”. Wherein half of the Ec MlaD hexamer would rise in the periplasmic side along with an outward movement of pore loops, resulting in the change of the central channel geometry. Furthermore, this study highlights that, unlike typical SBPs, Ec MlaD possesses a fold similar to EF/AMT-type beta(6)-barrel and a unique ancestry. Altogether, the findings firmly establish Ec MlaD to be a non-canonical SBP with a unique ligand-transport mechanism.

Human hosts possess a complex network of immune responses against microbial pathogens. The production of antimicrobial peptides (AMPs), which target the pathogen cell membranes and inhibit them from inhabiting the hosts, is one such mechanism. However, pathogens have evolved systems that encounter these host-produced AMPs. The Sap (sensitivity to antimicrobial peptides) transporter uptakes AMPs inside the microbial cell and proteolytically degrades them. The Sap transporters comprise five subunits encoded by genes in an operon. Despite its ubiquitous nature, its subunits are not found to be in tandem with many organisms. In this study, a total of 421 Sap transporters were analyzed for their operonic arrangement. Out of 421, a total of 352 operons were found to be in consensus arrangement, while the remaining 69 show a varying arrangement of genes. The analysis of the intergenic distance between the subunits of the sap operon suggests a signature pattern with sapAB (-4), sapBC (-14), sapCD (-1), and sapDF (-4 to 1). An evolutionary analysis of these operons favors the consensus arrangement of the Sap transporter systems, substantiating its prevalence in most of the Gram-negative pathogens. Overall, this study provides insight into bacterial evolution, favoring the maintenance of the genetic organization of essential pathogenicity factors.

Antioxidant defense mechanisms are important for a parasite to overcome oxidative stress and survive within host macrophage cells. Mitochondrial iron superoxide dismutase A (FeSODA) and trypanothione reductase (TR) are critical enzymes in the antioxidant defense mechanism of Leishmania donovani . FeSODA is responsible for neutralizing reactive oxygen species in mitochondria, while TR is responsible for reducing trypanothione, the molecules that help the parasite fight oxidative stress in Leishmania. In this study, we used multitarget ligands to inhibit both the FeSODA and TR enzymes. We combined structure-based drug design using virtual screening approach to find inhibitors against both the targets. The ZINC15 database of biogenic compounds was utilized to extract drugs-like molecules against leishmaniasis. The compounds were screened by standard precision (SP) and extra precision (XP) docking methods. Two compounds, ZINC000008876351 and ZINC000253403245, were selected based on molecular docking based on the binding affinity for both the targets. The screened molecules ZINC000008876351 and ZINC000253403245 showed strong hydrogen bonding with the target proteins according to the Molecular mechanics with generalised Born and surface area solvation (MM-GBSA) techniques. These two compounds were also experimentally investigated on promastigotes stage of L. donovani . Under in vitro condition, the compounds show inhibitory effects on L. donovani promastigotes with IC 50 values of 24.82 ± 0.61 µM for ZINC000008876351 and 7.52 ± 0.17 µM for ZINC000253403245. Thus, the screened compounds seem to have good potential as therapeutic candidates for leishmaniasis.